KCA3.1 inhibitors and methods thereof

Abstract

The present disclosure relates to Kcnn 4 (i.e.KCa3.1) inhibitors and uses thereof in the treatment and / or prevention of cystic kidney disease such as polycystic kidney disease.

Description

Attorney Docket No. 063697-504001WOKCA3.1 INHIBITORS AND METHODS THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present disclosure claims the benefit of priority from U.S. patent application no. 63 / 697,148, filed September 20, 2024, the contents of which are incorporated herein by reference in their entirety.FIELD

[0002] The present disclosure relates to the field of Kcnn4 (i.e. KCa3.1) inhibitors and cystic kidney disease, and particularly relates to the use of KCa3.1 inhibitors in the treatment of cystic kidney disease such as autosomal dominant polycystic kidney disease (ADPKD).STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0003] This invention was made with government support under grant nos. W81XWH-18-1- 0463 awarded by the United States Army. The government has certain rights in the invention.BACKGROUND

[0004] Cystic kidney diseases (CKD) are a group of diseases that cause abnormal pockets of fluid (cysts) to form in the kidneys. Cystic kidney diseases include polycystic kidney disease (such as autosomal dominant and autosomal recessive), medullary cystic disease, medullary sponge kidney, polycystic liver disease (PLD, which is associated with cysts in the kidneys) and all forms of nephron ophthi sis. The cysts interfere with normal kidney function and may cause kidney failure.

[0005] Human autosomal dominant polycystic kidney disease (ADPKD) is a highly prevalent genetic disorder that affects up to 1 / 500 individuals, thus 60,000 Canadians and 12.5 million subjects worldwide. It accounts for up to 10% of all cases of end-stage renal disease (ESRD). No prevention or cure is available and -50% of patients ultimately require dialysis and / or transplantation. In Canada, ADPKD medical costs exceeded $150M in year 2000. Since onset of renal failure typically is at mid-life (-50 years of age), therapeutic interventions that delay PKD progression by a factor of 2 or 3 are expected to substantially increase both patient lifespan and quality of life, while reducing societal cost of their care. Mutations in the PKD1Attorney Docket No. 063697-504001WO gene are responsible for -85% of ADPKD cases.

[0006] Epithelial cell proliferation and fluid secretion have been proposed to be the two major mechanisms mediating cyst growth and progression of cystic kidney diseases. Cyst enlargement in cystic kidney diseases is thought to be largely driven by channels and transporters that regulate the transepithelial fluid secretion. One of the most widely studied components of fluid transport in ADPKD is the vasopressin V2 receptor (V2R). Plasma vasopressin levels in ADPKD patients are increased probably due to decreased urinary concentrating ability. V2R is believed to be a major source of renal cAMP in tubular epithelial cells and inhibition of V2R by Tolvaptan slows cyst growth and delays loss of renal function. However, Tolvaptan raises concerns about hepatotoxicity and is often poorly tolerated with aquaretic side effects (>4 liters / day). These side effects alter patient’s quality of life and compliance. The significant proportion of non-responder patients is also a major issue.

[0007] Accordingly, there remains a need for developing new effective, non-toxic and well- tolerated drug candidates for cystic kidney disease.SUMMARY

[0008] The present disclosure relates generally to the field of cystic kidney diseases such as polycystic kidney disease including autosomal dominant polycystic kidney disease (ADPKD). In particular, the present disclosure includes methods of treating or preventing, or delaying the development of a cystic kidney disease in a subject in need thereof. The methods of treating or preventing or delaying the development of a cystic kidney disease include administering a therapeutically effective amount of an inhibitor of the calcium-activated potassium channel (KCa3.1) to the subject. KCa3.1 is a calcium-activated potassium channel encoded by the potassium intermediate / small conductance calcium-activated channel, subfamily N, member 4, (KCNN-l) gene. In the context of this disclosure, when the term “Kcnn4” when used in italics generally refers to the Kcnn4 gene. When not italicized, the term “Kcnn4” can be used interchangeably with “KCa3.1” to refer to the gene product. As such, also provided herein are methods of treating, preventing or delaying the development of a cystic kidney disease in a subject in need thereof, the method including administering a therapeutically effective amount of a therapeutic agent to downregulate the expression of the Kcnn4 gene.

[0009] It has been shown herein that the Kcnn4 gene is up regulated in Pkdl-positive cysticAttorney Docket No. 063697-504001WO kidney models. Further, it was demonstrated in greater detail below that genetic inactivation of the Kcnn4 gene and / or the inhibition of the gene product, KCa3.1, led to reduction of number of existing cysts and of cyst area. Inhibition of KCa3.1 or downregulation or inactivation of Kcnn4 gene was also shown herein to prevent development of new cysts. Upon treatment with KCa3.1 inhibitor, cystic kidney disease models were shown to improve or return to normal.

[0010] The present disclosure includes a method of treating, preventing or delaying the development of a cystic kidney disease in a subject in need thereof. The method can include administering a calcium-activated potassium channel (KCa3.1) inhibitor to the subject. The KCa3.1 inhibitors can have a number of effects in the subject treated. For example, the KCa3.1 inhibitor can decrease a number of new cyst growth or enlargement in the subject relative to an untreated subject. The KCa3.1 inhibitor can decrease the number of new cysts in the subject by about 60% to about 95%. The KCa3.1 inhibitor can also decrease a number of cysts, such as cysts already existing, in the subject relative to an untreated subject. For example, the KCa3.1 inhibitor decreases the number of cysts in the subject by about 10% to about 50%. The KCa3.1 inhibitor can decrease an average percent cyst area in the subject relative to an untreated subject. For example, the average percent cyst area is decreased by about 30% to about 70%.

[0011] In some cases, the subject can have kidney fibrosis. The KCa3.1 inhibitor can reduce the kidney fibrosis. For example, the kidney fibrosis can be decreased by about 10% to about 60%.

[0012] The KCa3.1 inhibitor can be administered orally.

[0013] The subject can be treated once daily or multiple times daily. For example, the subject can be treated for about 1 day to about 30 days. Or the subject can be subjected to chronic treatment.

[0014] Any suitable KCa3.1 inhibitor can be used. For example, the KCa3.1 inhibitor can include senicapoc, clotrimazole, TRAM-34, NS6180, nifedipine, 4-Phenyl-4H-pyran or bicyclic hexadiene lactone, or a combination thereof.

[0015] In some cases, the KCa3.1 inhibitor is TRAM-34, senicapoc, or combination thereof. In some cases, the KCa3.1 inhibitor can be senicapoc.

[0016] The KCa3.1 inhibitor can be administered at a dose of about 10 mg / kg to about 150 mg / kg of bodyweight of the subject. The KCa3.1 can be administered at a dose of about 5 mg to about 100 mg.Attorney Docket No. 063697-504001WO

[0017] The KCa3.1 inhibitor can be administered once, twice or three times daily.

[0018] The KCa3.1 inhibitor can be administered in combination with a second therapeutic agent. The second therapeutic agent can be a Cftr inhibitor, a TMEM16a inhibitor, or a combination thereof. For example, the second therapeutic agent can be a Cftr inhibitor. Suitable Cftr inhibitor can include PPQ-102. Suitable TMEM16a inhibitor can be CaCCinh- A01.

[0019] Additionally or alternatively, the second therapeutic agent can be a vasopressin receptor antagonist. Suitable vasopressin receptor antagonist can include unselective antagonists, and selective antagonists such as VIA antagonists, VIB antagonists, V2 antagonists. For example, the vasopressin antagonist can be tolvaptan.

[0020] The KCa3.1 inhibitor can be formulated into a pharmaceutically acceptable composition. The composition can further include a pharmaceutically acceptable excipient.

[0021] The KCa3.1 inhibitor is used treat or delay the development of a cystic kidney disease. The cystic kidney disease can be a polycystic kidney disease such as autosomal dominant polycystic kidney disease (ADPKD). In some cases, the ADPKD is adult on-set and / or moderately progressive ADPKD. In some cases, the subject can be an adult.

[0022] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects, and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0024] FIG. 1 is Analysis of Kcnn4 expression in the kidneys of human ADPKD and Pkdl mouse models. (A) Analysis of human KCNN4 expression in healthy / normal and ADPKD kidney tissues by quantitative PCR. (B) Analysis of mouse Kcnn4 expression in Pkdlcko(P10; black dot), PkcU' '' (P10; yellow dot),SBPkdl (2 months; blue dot) and Pkdlwt(7 months; green dot) kidneys relative to age-matched wildtype (WT; white dot) byAttorney Docket No. 063697-504001WO quantitative PCR. (C) RNA in situ hybridization overview of Kcnn4 expression by RNAscope in kidneys of Pkdlcko(P5) andSBPkdl (4 months) with age-matched WT.*p<0.05, **p<0.01

[0025] FIG. 2 is Expression analysis of key channels and transporters in the epithelial secretory pathway in Pkdl mouse kidneys. Analysis oiNkccl, Nkcc2, Cftr, Aqpl &vA Aqp2 in PIO Pkdlcko(A) and 6-8-month-oldSBPkdl (B) mouse kidneys by quantitative PCR.*p<0.05, **p<0.01

[0026] FIG. 3 is Analysis of Kcnn4 expression in mouse embryonic kidneys.Amplification of Kcnn4 (58bp) and S16 (103bp) cDNA via quantitative PCR using RNA extracted from E14.5 and E16.5 metanephroi showing Kcnn4 is expressed early in embryo.

[0027] FIG. 4 is Analysis of renal cAMP level in orthologous Pkdl mouse models.Quantification of cAMP levels (pmol cAMP per mg of protein) in P10 Pkdlcko(black bar), 5- 6-week-oldSBPkdl (blue bar) and 6-8month-old Pkdlwt(green bar) kidneys in comparison to age-matched WT kidneys (open bar). *p<0.05, ***p<0.001, ****p<0.0001

[0028] FIG. 5 is Analysis of cAMP signaling cascade MAPK / ERK / Myc in distinct orthologous Pkdl mouse models. Representative western blot analysis (left) and quantification (right) of cAMP downstream effectors phospho-ERK, total ERK and c-Myc in P10 Pkdlcko(A), P10 Pkdlv / v(B), 5-6-week-oldSBPkdl kidneys (C), 6-8-month-old Pkdlwt(D), in comparison to age-matched wildtype (white bar). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

[0029] FIG. 6 is Histological analysis of adult Kcnn4 knockout mouse kidneys.Representative H&E histological sections of 7-month-old adult mouse kidneys comparing Kcnn4 knockout (Kcnn4~ / ~') to age-matched wildtype Kcnn4+I+).

[0030] FIG. 7 is Treatment of mouse metanephroi with Kcnn4 activator SKA-111. (A)Schematic representation of the experimental design: isolated E14.5 Pkdl+ / +and Pkdl'7' metanephroi are stimulated with lOOmM cAMP from day 1 until day 4 with or without simultaneous addition of KCa3.1 activator SKA-111. (B) Representative images ofPkdl+ / +metanephroi with (right) or without (left) 30mM SKA-111 upon completion of treatment on day 4. (C) Quantification of percentage cyst area (%) in Pkdl+ / +metanephroi treated with lOmM, 20mM or 30mM SKA-111 in comparison to vehicle (DMSO) is carried out upon completion of experiment. *p<0.05, **p<0.01

[0031] FIG. 8 is Treatment of Pkdl'' metanephroi with SKA-111. (A) Representative images of Pkdl ’ ’metanephroi with (right) or without (left) lOmM SKA-111 upon completionAttorney Docket No. 063697-504001WO of treatment on day 4. Quantification of percentage cyst area (B) and cyst number (C) in Pkdl~ ~ metanephroi treated with lOmM SKA-111 in comparison to vehicle is carried out upon completion of experiment. ****p<0.0001

[0032] FIG. 9 is Analysis of cyst indices in Pkdl'' metanephroi upon Kcnn4 genetic inactivation. (A) Schematic representation of the experimental design: isolated E14.5 Pkdl+ / +, Pkdl~ ~ and Pkdt ~; Kcnn4~ ~ metanephroi are stimulated with lOOmM cAMP from dayl until day 4. (B) Representative images of Pkdl+ / +, PkdP ~ and PkdP ~ ; Kcnn4~ ~ metanephroi stimulated with lOOmM cAMP upon completion of treatment on day 4.Quantification of percentage cyst area (C) and cyst number (D) in Pkdl+ / +, Pkdt7~ and Pkdt7~ ; Kcnn4~ ~ metanephroi stimulated with lOOmM cAMP is carried out upon completion of experiment. **p<0.01, ****p<0.0001

[0033] FIG. 10 is Treatment of Pkdl'7' metanephroi with KCa3.1 inhibitor TRAM-34.(A) Schematic representation of the experimental design: Pkdl7' metanephroi isolated at E14.5 were stimulated with lOOmM cAMP from day 1 until day 4 with or without simultaneous addition of KCa3.1 inhibitor TRAM-34 used at 20mM or 40mM. (B) Representative images of Pkdl7' metanephroi treated with (center and right) or without (left) TRAM-34 upon completion of treatment on day 4. (C) Quantification of percentage cyst area in Pkdl7' metanephroi in comparison to those treated with 20mM or 40mM of TRAM-34 is carried out upon completion of experiment. ***p<0.001, ****p<0.0001

[0034] FIG. 11 is Treatment of Pkdl'7' metanephroi with KCa3.1 inhibitor Senicapoc at time of cyst induction. (A) Schematic representation of the experimental design: Pkdl7' metanephroi isolated at E14.5 were stimulated with lOOmM cAMP on day 1 until day 4 with or without simultaneous addition of KCa3.1 inhibitor Senicapoc used at 20mM. (B) Representative images of A 7 / _metanephroi with (right) or without (left) 20mM Senicapoc upon completion of treatment on day 4. Quantification of percentage cyst area (C) and cyst number (D) in Pkdl7' metanephroi in comparison to vehicle upon completion of treatment on day 4. ****p<0.0001

[0035] FIG. 12 is Treatment of Pkdl'7' metanephroi with Senicapoc 2 days after cyst induction. (A) Schematic representation of the 5-day experimental design: Pkdl7' metanephroi isolated at E14.5 were stimulated with lOOmM cAMP on day 1. On day 3, metanephroi were treated with or without 20mM Senicapoc until day 5. (B) Representative images of Pkdl7' metanephroi with (bottom) or without (top) Senicapoc on day 3, 4, and 5 of the experiment. (C) Quantification of percentage cyst area in Pkdl7' metanephroi with (redAttorney Docket No. 063697-504001WO bar) or without (black bar) Senicapoc relative to Pkdl+ / +(open bar) from day 3 to day 5. (D) Quantification of percentage cyst area in PkdPk~ metanephroi with (red circle) or without (black circle) Senicapoc relative to Pkdl+ / +(open circle) from day 3 to day 5. Each curve follows longitudinal progression of cyst area in individual metanephros over 3 -day period. ***p<0.001

[0036] FIG. 13 is Histomorphometric analysis of Pkdlckoand Pkdlcko; Kcnn4 kidneys.(A) Macroscopic kidney images of PIO WT, Pkdlckoand Pkdlcko; Kcnn4~ ~ and quantification (right) of kidney weight to body weight ratio (KBW) of WT (open bar), Pkdlcko(black bar), Pkdlcko; Kcnn4~ ~ (grey bar) at P5 and PIO (the number of animals analyzed are written within the bars). (B) Representative H&E histologic kidney sections of PIO WT, Pkdlcko, and Pkdlcko; Kcnn ^. Quantification of percentage cyst area (C) and cyst number per mm2of kidney area (D) in Pkdlckoand Pkdlcko; Kcnn4~ ~ at P5 and PIO in comparison to WT.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

[0037] FIG. 14 is Analysis of individual cysts based on tubular lumen size. Cysts in kidney sections of P5 and PIO Pkdlckoand Pkdlcko; Kcnn4~ ~ are listed by size and sorted into corresponding size intervals.

[0038] FIG. 15 is Analysis of cyst tubular origin in Pkdlckoand Pkdlcko; Kcnn4 ' kidneys.Cyst tubular origin in P5 WT, Pkdlckoand Pkdlcko; Kcnn4~ ~ kidneys was analyzed by immunofluorescence using markers specific for proximal tubule (LTL; green), distal tubule (LEL; blue) and collecting duct (DBA; red). No proximal tubular cysts are observed in WT, Pkdlckoor Pkdlcko, Kcnnd kidneys. Quantification of percentage cystic tubules (%) in distal tubule (DT) and collecting duct (CD) of Pkdlcko(black dot) and Pkdlcko, Kcnn4~k~ (grey dot) kidneys is shown on the right. *p<0.05, **p<0.01

[0039] FIG. 16 is Assessment of kidney function and survival in Pkdlckoand Pkdlcko;Kcnn4 / ~. (A) Blood Urea Nitrogen (BUN) analysis of P10 WT (open circle), Pkdlcko(black circle) and Pkdlcko; Kcnn4~ ~ (grey circle) kidneys. (B) Kaplan-Meier survival curve of PkdlckoNQ sus Pkdko; Kcnn ^. *p<0.05, **p<0.01

[0040] FIG. 17 is Analysis of cAMP level and proliferation status of Pkdlckoversus Pkdlcko; Acnn^ 'kidneys. (A) cAMP level is measured by ELISA and is expressed as pmol cAMP per mg of protein. (B) Representative western blot analysis (left) and quantification (right) of cAMP downstream effectors phospho-ERK, total ERK and c-Myc in P10 Pkdlckoversus Pkdlcko; Kcnn4~ ~ kidneys. (C) Quantification of renal epithelial cell proliferation by Ki67 immunohistochemistry in P10 WT, Pkdlckoand Pkdlcko; Kcnn4~ ~ kidneys (right) withAttorney Docket No. 063697-504001WO representative images shown on the left. Proliferation is expressed as the number of ki67 positive cells per mm2kidney surface. *p<0.05, **p<0.01, ***p<0.001

[0041] FIG. 18 is Analysis of kidney fibrosis in Pkdlckoversus Pkdlcko; Kcnn4 ~. Fibrosis is detected by Sirius Red and is expressed as the percentage of renal fibrotic surface (%) in PIO WT, Pkdlckoand Pkdlcko; Kcnn4~ ~ kidneys, (right) Representative images are shown on the left. *p<0.05 **p<0.01

[0042] FIG. 19 is Analysis of primary cilia length and canonical Wnt signaling in Pkdlckoversus Pkdlcko; Kcnn4' / ' kidneys. (A) Quantification of mean primary cilia length in PIO WT, Pkdlckoand Pkdlcko; Kcnn4~ ~ kidneys using immunofluorescent anti-alpha acetylated tubulin marker (right). Representative images are shown on the left with white arrows pointing at individual cilium. (B) Length of all primary cilia found in the entire kidney section were measured and sorted into intervals based on their length. The primary cilia distribution of PIO Pkdlckoshowed a distinct shift towards the right of x-axis in comparison to WT that was shifted slightly towards the left upon genetic inactivation of Kcnn4. (C) Representative western blot analysis (left) and quantification (right) of active b-catenin and total b-catenin in PIO WT versus Pkdlcko(top) and Pkdlckoversus Pkdlcko; Kcnn4~ ~ (bottom) kidneys. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

[0043] FIG. 20 is Western blot analysis of MAPK / ERK / c-Myc and Wnt / b-catenin in Pkdlckoversus Pkdlcko; Kcnn4 ' at P5. (A) Representative western blot analysis (top) and quantification (bottom) of MAPK / ERK (left), Wnt / b-catenin (center) and c-Myc (right) in P5 WT versus Pkdlckokidneys. (B) Representative western blot analysis (top) and quantification (bottom) of MAPK / ERK (left), Wnt / b-catenin (center) and c-Myc (right) in P5 Pkdlckoversus Pkdlcko; Kcnn4~ ~ kidneys. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

[0044] FIG. 21 is Histomorphometric analysis oSBPkdl andSBPkdl; Kcnn4~ ~ kidneys. (A) Macroscopic kidney images of 6-8-month-old WT,SBPkdl andSBPkdl; Kcnn4~ ~ . Quantification of KBW ratio ofSBPkdl (blue bar) andSBPkdl; Kcnn4~ ~ (light blue bar) at 1 and 6 months are shown on the right (the number of animals analyzed are written within the bars). (B) Representative H&E histological kidney sections of 7-month-old WT,SBPkdl andSBPkdl; Kcnn4~ ~ . (C) Quantification of percentage cyst area (%) in 6-8-month-oldSBPkdl with none (Kcnn4+l+partial (heterozygous Kcnn4+ / ~) or total (Kcnnd ^) Kcnn4 inactivation. Analysis of Kcnn4 contribution to kidney cyst area inSBPkdl,SBPkdl; Kcnn4+ / ~ andSBPkdl; Kcnn4~Bsuggest that total annihilation of KCa3.1 is not required for improvement of cystic phenotype. (D) Quantification of cyst number per mm2kidney in 6-8-month-oldSBPkdl,Attorney Docket No. 063697-504001WOSBPkdl; Kcnn4+ / ~ andSBPkdl; Kcnnd '. Contribution of KCa3.1 to the kidney cyst number parallels its involvement in cyst enlargement (cyst area). *p<0.05, **p<0.01, ***p<0.001, ****p<0 0001

[0045] FIG. 22 is Analysis of cyst tubular origin inSBPkdl andSBPkdl; Kcnn4~ kidneys.Cyst tubular origin in 2-month-old WT,SBPkdl andSBPkdl; Kcnn4~ ~ kidneys was analyzed by immunofluorescence using proximal (LTL; green), distal (LEL; blue) and collecting (DBA; red) tubular markers. Quantification of percentage cystic tubules (%) in distal tubule (DT) and collecting duct (CD) oiSBPkdl andSBPkdl; Kcnn4~ ~ kidneys are shown on the right. **p<0.01

[0046] FIG. 23 is Assessment of kidney function and survival inSBPkdl andSBPkdl;Kcnnd'-. (A) BUN analysis of WT (open circle),SBPkdl (blue circle) andSBPkdP, Kcnn4~ ~ (light blue circle) at 6-8 months. (B) Kaplan-Meier survival curve oiSBPkdl versusSBPkdl; Kcnn4 ****p<0.0001

[0047] FIG. 24 is Analysis of cAMP level and proliferation status oSBPkdl versusSBPkdl; A'cn« ' / 'kidneys. (A) cAMP level is measured by ELISA and is expressed as pmol cAMP per mg of protein. (B) Representative western blot analysis (left) and quantification (right) of cAMP downstream effectors phospho-ERK, total ERK and c-Myc in 6-month-oldSBPkdl andSBPkdl; Kcnn4~ ~ kidneys. (C) Quantification of renal epithelial cell proliferation by Ki67 immunohistochemistry in 6-8-month-oldSBPkdl andSBPkdl; Kcnn4~ ~ kidneys (right) with representative images shown on the left. Proliferation is expressed as the number of ki67 positive cells per mm2kidney surface. *p<0.05 **p<0.01

[0048] FIG. 25 is Analysis of kidney fibrosis inSBPkdl versusSBPkdl; Kcnn4 . Fibrosis is detected by Sirius Red and is expressed as the percentage of renal fibrotic surface (%) in 6-8- month-old WT,SBPkdl andSBPkdl; Kcnn4~ ~ kidneys (right). Representative images are shown on the left. **p<0.01, ***p<0.001

[0049] FIG. 26 is Analysis of primary cilia length inSBPkdl versusSBPkdl; Kcnn4 ~ kidneys. (A) Quantification of mean primary cilia length in 2-month-old WT,SBPkdl andSBPkdl; Kcnn4~ / ~kidneys using immunofluorescent anti-alpha acetylated tubulin marker (right). Representative images are shown on the left with white arrows pointing at individual cilium. Mean cilia length was virtually normalized upon loss of Kcnn4. (B) Length of all primary cilia found in the entire kidney section were measured and distributed into intervals based on their length. The primary cilia distribution oiSBPkdl showed a distinct shift towards the right of x-axis in comparison to WT that was completely restored inSBPkdl; Kcnn4~ ~.Attorney Docket No. 063697-504001WO*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

[0050] FIG. 27 is Evaluation of Senicapoc use in adult wildtype mice. Animals are separated into 3 conditions: control vehicle (PEG / cremophor), 30mg Senicapoc / kg of body weight (low dose) or 120mg Senicapoc / kg of body weight (high dose). (A) Schematic representation of the experimental design for wildtype mice by daily oral gavage from 6 weeks to 18 weeks of age. (B) Body weight change show normal weekly weight gain following Senicapoc administration with greater gain in the high dose treatment group. (C) Representative H&E histological kidney sections of wildtype treated with vehicle, low dose, and high dose Senicapoc upon completion of treatment. (D) Quantification of KBW ratio and BUN show no impact upon Senicapoc administration.

[0051] FIG. 28 is Schematic representation of the experimental design for treatment of early-onset models Pkdlckoand Pkdlv / v. Animals are separated into 2 conditions: vehicle (PEG / cremophor) or 120mg Senicapoc / kg of body weight. Senicapoc is administered through daily oral gavage to mothers from P0 to P5 (Pkdlcko) or to P10 (Pkdlv v) in addition to directly feeding into mouth of experimental models from P2 to P5 Pkdlckff) or P10 PkdlvM).

[0052] FIG. 29 is Effect of 5 day Senicapoc treatment to Pkdk". (A) Representative H&E histological kidney sections of Pkdlckotreated with vehicle or 120mg Senicapoc / kg of body weight. (B) 2KW / BW ratios, % cyst area, cyst number in Pkdlckoand Pkdl+ / +pups treated with vehicle or senicapoc; % fibrotic area of kidneys from senicapoc- vs vehicle-treated Pkdlckomice. Pkdlckomice treated with vehicle (grey dots) or with senicapoc (120 mg / kg, by oral gavage to mothers from P0 to P5, and in addition, directly into the mouths of suckling pups from P2 to P5; **, p<0.01; ***, p<0.001, Student’s t-test, one tail.

[0053] FIG. 30 is Effect of 10 day Senicapoc treatment to Pkdlv / v. (A) RepresentativeH&E histological kidney sections of Pkdlv / vtreated with vehicle or 120mg Senicapoc / kg of body weight. (B) Experimental design for oral treatment of Pkdlv / vmice (caramel rim indicative of genotype, open circle) with vehicle (PEG / cremophor) or 120 mg / kg senicapoc (red arrow). Senicapoc or vehicle was administered daily by oral gavage to mothers from P0 to P10 and, in addition, directly into the mouths of suckling pups from P2 to P10. (C) Representative low magnification images of P10 Pkdlv vkidney sections (H&E stained) from mice treated with vehicle (grey circle with caramel rim) or senicapoc (red circle with caramel rim). (D) 2KW / BW ratios, % cyst area, cyst number in Pkdlv vm ce treated with vehicle or senicapoc; % fibrotic area of kidneys from senicapoc- vs vehicle-treated Pkdlv / vmice. **, p<0.01; ***, p<0.001; ****, p<0.0001, Student’s t-test, one tail.Attorney Docket No. 063697-504001WO

[0054] FIG. 31 is Schematic representation of the experimental design for treatment of adult-onset modelsSBPkdl and PkdV' Animals are separated into 3 conditions: vehicle (PEG / cremophor), 30mg Senicapoc / kg of body weight (low dose) or 120mg Senicapoc / kg of body weight (high dose). Senicapoc is administered through daily oral gavage of Senicapoc or vehicle (PEG / cremophor) from 3 weeks of age ^BPkdP) or 6 weeks of age Pkdlwt) for 3 months

[0055] FIG. 32 is Effect of 3 months Senicapoc treatment toSBPkdl. (A) Body weight change show general weekly weight gain inSBPkdl for the duration of Senicapoc treatment except for a short-term weight loss in the high dose group between week 6 to 7. (B) Representative H&E histological kidney sections oiSBPkdl kidneys treated with vehicle, low dose, and high dose Senicapoc upon completion of treatment. (C) Quantification of KBW, percentage cyst area (%), cyst number per mm2of kidney surface and BUN inSBPkdl upon completion of treatment with vehicle (left), 30 mg / kg senicapoc (middle), or 120 mg / kg senicapoc (right). *p<0.05 (D) 2KW / BW ratio, % cyst area and cyst number in kidneys ofSBPkdl mice treated with vehicle or with senicapoc at 120 mg / kg; % fibrotic area of kidneys and blood urea nitrogen inSBPkdl mice treated with vehicle or 120 mg / kg senicapoc. *, p<0.05; **, p<0.01, Student’s t-test, one tail with Welch correction.

[0056] FIG. 33 is Effect of 3 months Senicapoc treatment to Pkdlwt. (A) Body weight change show general weekly weight gain in Pkd l"1for the duration of treatment. (B) Representative H&E histological kidney sections of Pkd l"1kidneys treated with vehicle, low dose, and high dose Senicapoc upon completion of treatment. (C) Quantification of KBW, percentage cyst area (%), cyst number per mm2of kidney surface and BUN in Pkdl"' upon completion of treatment with vehicle (left), 30 mg / kg senicapoc (middle), or 120 mg / kg senicapoc (right). Cyst indices are significantly reduced at low dose and at high dose in comparison to vehicle. Renal function was significantly improved in Pkdl ' kidneys treated with high dose Senicapoc. *p<0.05 **p<0.01 ****p<0.0001 (D) Experimental design for treatment of adult Pkdl ' mice (green rim indicative of genotype, open circle) with control vehicle or with senicapoc (red arrow) at 120mg / kg by daily oral gavage for 12 weeks, starting at 6 weeks of age. (E) Representative low magnification images of kidney sections (H&E stained) from 18 week old Pkdlwtmice treated 12 weeks with vehicle (grey circles, green rim or with senicapoc at 120mg (large red circles, green rim). (F) 2KW / BW ratio, % cyst area and cyst number in kidney sections from 18 week old Pkdl ' mice treated with vehicle (grey circles) or with senicapoc at 120 mg / kg (larger red circles); % fibrotic area in kidney sectionsAttorney Docket No. 063697-504001WO and blood urea nitrogen in Pkdlwtmice treated 12 weeks with vehicle or 120 mg / kg senicapoc. **,p<0.01; ****, p<0.0001, Student’ s t-test, one tail.

[0057] FIG. 34 is Comparison of PkdP7' metanephroi cyst growth between different concentrations of Senicapoc. (A) Schematic representation of the experimental design: PkdP7' metanephroi isolated at E14.5 were stimulated with lOOpM cAMP on day 1 until day 4 with or without simultaneous addition of KCa3.1 inhibitor Senicapoc. (B) Representative images of / / ^metanephroi with or without 5pM, lOpM and 20pM Senicapoc upon completion of treatment on day 4. (C) Quantification of percentage cyst area (%) m PkdP7' metanephroi treated with 5pM, lOpM and 20pM Senicapoc in comparison to the vehicle is carried out upon completion of experiment. ****p<0.0001

[0058] FIG. 35 is Comparison of PkdP7' metanephroi cyst growth between different concentrations of PPQ-102. (A) Schematic representation of the experimental design: PkdP / _metanephroi isolated at E14.5 were stimulated with lOOpM cAMP on day 1 until day 4 with or without simultaneous addition of Cftr inhibitor PPQ-102. (B) Representative images of PkdP' metanephroi with or without IpM, 2pM, 5pM, lOpM and 20pM PPQ-102 upon completion of treatment on day 4. (C) Quantification of percentage cyst area (%) m PkdP7' metanephroi treated with IpM, 2pM, 5pM, lOpM and 20pM PPQ-102 in comparison to the vehicle is carried out upon completion of experiment. ***p<0.001, ****p<0.0001

[0059] FIG. 36 is Comparison of PkdP7' metanephroi cyst growth between different concentrations of CaCCinh-AOl. (A) Schematic representation of the experimental design: PkdP7' metanephroi isolated at E14.5 were stimulated with lOOpM cAMP on day 1 until day 4 with or without simultaneous addition of the Tmeml6a inhibitor CaCCinh-AOl . (B) Representative images of PkdP / - metanephroi with or without lOpM, 20pM, 25 pM, 30pM and 35pM CaCCinh-AOl upon completion of treatment on day 4. (C) Quantification of percentage cyst area (%) in 77 / / ^metanephroi treated with lOpM, 20pM, 25pM, 30pM and 35pM CaCCinh-AOl in comparison to the vehicle is carried out upon completion of experiment. *p<0.05, **p<0.01, ****p<0.0001

[0060] FIG. 37 is Effect of combined Senicapoc and PPQ-102 inhibition versus Senicapoc only inhibition on cyst growth in PkdP7' metanephroi. (A) Schematic representation of the experimental design: PkdP7' metanephroi isolated at E14.5 were stimulated with lOOpM cAMP on day 1 until day 4 with or without simultaneous addition of Senicapoc and PPQ-102. (B) Representative images of PkdP7' metanephroi with single or combined treatment upon completion of treatment on day 4.Attorney Docket No. 063697-504001WO

[0061] FIG. 38 is Comparison of combined Senicapoc and PPQ-102 inhibition versus Senicapoc only inhibition on cyst growth in Pkdl'7' metanephroi. (A) Quantification of percentage cyst area (%) m Pkdl'7' metanephroi comparing Senicapoc / PPQ-102 combination inhibition to 5pM Senicapoc inhibition show synergistic effect. (B) Quantification of percentage cyst area (%) m Pkdl'7' metanephroi comparing Senicapoc / PPQ-102 combination inhibition to lOpM Senicapoc inhibition show synergistic effect. **p<0.01, ***p<0.001, ****p<0.0001

[0062] FIG. 39 is Effect of combined Senicapoc and CaCCinh-AOl inhibition versus Senicapoc only inhibition on cyst growth in Pkdl ' metanephroi. (A) Schematic representation of the experimental design: Pkdl7' metanephroi isolated at E14.5 were stimulated with lOOpM cAMP on day 1 until day 4 with or without simultaneous addition of Senicapoc and CaCCinh-AOl . (B) Representative images oiPkd7' metanephroi with single or combined treatment upon completion of treatment on day 4.

[0063] FIG. 40 is Comparison of combined Senicapoc and CaCCinh-AOinhibition versus Senicapoc only inhibition on cyst growth in Pkdl'7' metanephroi. Quantification of percentage cyst area (%) in Pkdl7' metanephroi comparing Senicapoc / CaCCinh-AOl combination inhibition to 5pM Senicapoc inhibition. **p<0.01, ****p<0.0001DETAILED DESCRIPTION OF THE DISCLOSURE

[0064] It has been shown hereinKcnn4 is up-regulated in Pkdl-positive cystic kidney models. Further, it was demonstrated in greater detail below that genetic inactivation of Kcnn4 and / or inhibition of Kcnn4 gene product Kca3.1 led to reduction of number of existing cysts and of cyst area. Inhibition of KCa3.1 was also shown herein to prevent development of new cysts. Upon treatment with KCa3.1 inhibitor, cystic kidney model was shown to improve or return to normal.

[0065] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0066] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. It is to be understood that the present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognizeAttorney Docket No. 063697-504001WO that there are variations and modifications of the present disclosure, which are encompassed within its scope.

[0067] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0068] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the disclosure can be used in combination with any other unless specifically indicated otherwise.DEFINITIONS

[0069] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0070] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, comprising mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.

[0071] As used herein, a “subject” or an “individual” includes animals, such as human (e.g.,Attorney Docket No. 063697-504001WO human individuals) and non-human animals. In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a health condition of interest (e.g., cancer) and / or one or more symptoms of the health condition. The subject can also be an individual who is diagnosed with a risk of the health condition of interest at the time of diagnosis or later.

[0072] It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising,” particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0073] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[0074] The term “delaying the development of a disease” refers to a decrease in the occurrence of disease symptoms in a subject (e.g., patient). In some cases, the delaying of development of the disease is preventing the occurrence of disease symptoms. The prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely occur absent treatment.

[0075] As used herein, and unless otherwise specified, a “therapeutically effective amount” of an agent is an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease, e.g., cystic kidney disease, or to delay or minimize one or more symptoms associated with the disease. A therapeutically effective amount of a compoundAttorney Docket No. 063697-504001WO means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the disease. The term “therapeutically effective amount” can encompass an amount or number that improves overall therapy of the disease, reduces or avoids symptoms or causes of the disease, or enhances therapeutic efficacy of another therapeutic agent. An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 2010); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (2016); Pickar, Dosage Calculations (2012); and Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Gennaro, Ed., Lippincott, Williams & Wilkins). The term “prevention” or synonyms thereto as used herein can be a reduction of the risk or probability of a subject becoming afflicted with a disease, disorder, or condition, or manifesting one or more symptoms associated with a disease, disorder or condition. For example, prevention can be a reduction of the risk or probability of a subject to develop new cysts or exhibit enlargement or growth of existing cysts. Prevention can additional or alternatively relate to a reduction in risk or probability of a subject to develop a fibrotic disease or experience aggravation of an existing fibrotic disease, such as kidney fibrosis (e.g. cystic kidney fibrosis).

[0076] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.Attorney Docket No. 063697-504001WOMETHODS AND USES OF TREATING CYSTIC KIDNEY DISEASE

[0077] The present disclosure provides methods of treating or preventing cystic kidney diseases in a subject in need thereof with a therapeutically effective amount of a KCa3.1 inhibitor.

[0078] Also provided herein are uses of a therapeutically effective amount of a KCa3.1 inhibitor in the treatment or prevention of cystic kidney disease in a subject in need thereof. The present disclosure also provides uses of a therapeutically effective amount of a KCa3.1 inhibitor in the manufacture of a medicament for the treatment or prevention of cystic kidney disease.

[0079] Also provided herein are KCa3.1 inhibitors or pharmaceutical compositions thereof for use in the treatment or prevention of a cystic kidney disease.I. KCa3.1 Inhibitors

[0080] It is shown herein in greater detail that various KCa3.1 inhibitors were able to reduce cyst number and sizes. As such, it can be appreciated that any suitable KCa3.1 inhibitor can be used with the methods of the present application. For example, suitable KCa3.1 inhibitors include but are not limited to senicapoc, clotrimazole, TRAM-34, NS6180, nifedipine, 4- Phenyl-4H-pyran or bicyclic hexadiene lactone, and a combination thereof. In some instances, the KCa3.1 inhibitor can be senicapoc or TRAM-34. For example, the KCa3.1 inhibitor is senicapoc.

[0081] The KCa3.1 inhibitor can be suitable formulated into a pharmaceutically acceptable composition including the KCa3.1 inhibitor and a pharmaceutically acceptable excipient. The choice of the excipient can be selected based on the mode of administration and dosage of use. Any suitable excipients known in the art can be used.

[0082] Other components can be included in the pharmaceutical composition such as additional therapeutic agents (e.g. a second therapeutic agent) as described herein.A. Dosage and Administration

[0083] The KCa3.1 inhibitor can be administered in any suitable methods of administration. Suitable methods of administration can include for example oral, intramuscular, subcutaneous, or parenteral administration. Parenteral administration can include intravenous or transdermal. For example, the KCa3.1 inhibitor can be administered orally or parenterally. In some instances, the KCa3.1 inhibitor can be administered orally.

[0084] It is contemplated that the subject can be treated for a length of time suitable to theAttorney Docket No. 063697-504001WO conditions of the subject and the subject’s response to the treatment including KCa3.1 inhibitor. For example, the subject can be treated about 1 day to about 30 days, about 1 day to about 20 days, about 1 day to about 15 days, about 2 days to about 7 days, or about 2 days to about 5 days. However, it can also be appreciated that depending on the subject’s need, the treatment can be chronic.

[0085] Since the KCa3.1 inhibitor has been shown herein to reduce or eliminate the appearance of new cysts in subjects predisposed to cystic kidney disease, the KCa3.1 inhibitor can be used to prevent cystic kidney disease or development thereof. Accordingly, the KCa3.1 inhibitor can be administered prior to the appearance of cysts in the subject. Alternatively or additionally, since the KCa3.1 inhibitor is also shown herein to reduce the size and number of existing cysts, the KCa3.1 inhibitor can equally be administered after the appearance of cysts in the subject in need thereof. For example, the KCa3.1 inhibitor can be administered about 1 day to about 5 days after the appearance of cysts.

[0086] The KCa3.1 inhibitor can be administered once daily, twice daily, or three times daily. For example, the KCa3.1 inhibitor can be administered once daily.

[0087] The appropriate dosage can be adjusted according to the needs of the subject. Therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above.

[0088] In some situations, the therapeutic effective amount can be determined based on a reduction or decrease in at least one symptom associated with cystic kidney disease such as reducing the number of existing cysts, reducing the average cyst area, and / or decreasing the number of new cysts.

[0089] For example, a therapeutically effective amount or dose of the KCa3.1 inhibitor can be a dose of about 5 mg to about 100 mg, about 5 mg to about 80 mg, about 5 mg to about 60 mg, about 5 mg to about 50 mg, about 5 mg to about 40 mg, about 5 mg to about 30 mg, about 5 mg to about 20 mg, about 5 mg to about 15 mg, about 5 mg to about 12 mg, about 10 mg to about 50 mg, about 10 mg to about 40 mg, or about 10 mg. In some cases, the dose can be a daily dose.

[0090] When the KCa3.1 inhibitor is senicapoc, the therapeutically effective amount of the KCa3.1 inhibitor can be a dose of about 10 mg / kg to about 1000 mg / kg, about 10 mg / kg toAttorney Docket No. 063697-504001WO about 700 mg / kg, about 10 mg / kg to about 500 mg / kg, about 10 mg / kg to about 150 mg / kg, about 10 mg / kg to about 140 mg / kg, about 20 mg / kg to about 130 mg / kg, about 20 mg / kg to about 120 mg / kg, about 30 mg / kg to about 120 mg / kg, about 20 mg / kg to about 100 mg / kg, about 20 mg / kg to about 800 mg / kg, about 20 mg / kg to about 70 mg / kg, about 20 mg / kg to about 60 mg / kg, about 20 mg / kg to about 50 mg / kg, about 30 mg / kg to about 50 mg / kg, about 40 mg / kg to about 60 mg / kg, about 50 mg / kg to about 150 mg / kg, or about 60 mg / kg to about 120 mg / kg of bodyweight of the subject. For example, the senicapoc can be administered at about 30 mg / kg of bodyweight of the subject. For example, the senicapoc can be administered at about 60 mg / kg of bodyweight of the subject. For example, the senicapoc can be administered at about 90 mg / kg of bodyweight of the subject. For example, the senicapoc can be administered at about 120 mg / kg of bodyweight of the subject.

[0091] Therapeutically effective amounts may vary depending on the condition of the subject to be treated such as species, gender, age, body weight. For example, a therapeutically effect amount of senicapoc in a human subject may be about 2 mg / day to about 70 mg / day, about 5 mg / day to about 70 mg / day, about 7 mg / day to about 60 mg / day, or about 10 mg / day to about 40 mg / day. For example, a therapeutically effect amount of senicapoc in a human subject may be about 2 mg / day, about 5 mg / day, about 10 mg / day, about 15 mg / day, about 20 mg / day, about 25 mg / day, about 30 mg / day, about 35 mg / day, about 40 mg / day, or about 45 mg / day.II. Cystic Kidney Disease

[0092] KCa3.1 inhibitors have been shown herein to be effective in cystic kidney diseases, such as Pkdl gene-positive cystic kidney diseases. Cystic kidney diseases include polycystic kidney disease (such as autosomal dominant and autosomal recessive), medullary cystic disease, medullary sponge kidney, polycystic liver disease (PLD, which is associated with cysts in the kidneys) and all forms of nephronophthisis. In some cases, cystic kidney diseases can be polycystic kidney disease, such as autosomal dominant polycystic kidney disease. In some instances, the cystic kidney disease is associated with a mutation in the polycystin 1 (Pkdl) gene.

[0093] The KCa3.1 inhibitor can be used to treat subjects of all ages. However, it has been shown herein in greater detail that the treatment may be more effective and / or better tolerated in adult-onset cystic kidney disease. As such, in some cases, the cystic kidney disease is adult-on set polycystic disease. Accordingly, in some instances, the subject is an adult.Attorney Docket No. 063697-504001WOA. Reduction and Prevention of Cysts Formation

[0094] KCa3.1 Inhibitors were shown herein in greater detail to be effective to reduce the number and size of existing cysts and to prevent the appearance of new cysts.1. Reduction of existing cysts

[0095] Upon treatment with KCa3.1 inhibitors, the number and the size of existing cysts decreased compared to a subject untreated with KCa3.1 inhibitors. Accordingly, the present disclosure also provides methods of reducing a size of a cyst in a subject in need thereof. The methods can include administering to the subject a therapeutically effective amount of a Kca3.1 inhibitor. The present disclosure also provides uses of a therapeutically effective amount of a Kca3.1 inhibitor in the reduction of a size of a cyst in a subject need thereof. Similarly, the present disclosure includes uses of a therapeutically effective amount of a Kca3.1 inhibitor in the manufacture of a medicament for the reduction of a size of a cyst. The present disclosure further provides KCa3.1 inhibitors or pharmaceutical compositions thereof for use in the reduction of a size of a cyst in a subject in need thereof.

[0096] In some cases, the KCa3.1 inhibitor can decrease a number of cysts in the subject relative to an untreated subject. For example, the KCa3.1 inhibitor can decrease the number of cysts in the subject by about 10% to about 50%, about 10% to about 40%, about 20% to about 40%, about 25% to about 40%, about 25% to about 35%, or about 20%, or about 30%, relative to the untreated subject. For example, the KCa3.1 inhibitor (e.g. senicapoc) can decrease the number of cysts in the subject by about 20% to about 60%, about 25% to about 55%, about 30% to about 55%, about 35% to about 55%, or about 38% or about 51% relative to the untreated subject.

[0097] The KCa3.1 inhibitor can decrease an average percent cyst area in the subject relative to an untreated subject. For example, the average percent cyst area can be decreased by about 15% to about 70%, about 40% to about 60%, or about 50% to about 60%, about 50% or about 61% relative to the untreated subject. For example, the average percent cyst area can be decreased by about 10% to about 40%, about 15% to about 30%, or about 20% to about 30%, or about 25% relative to the untreated subject.2. Reduction of appearance of new cysts

[0098] The KCa3.1 inhibitors were also shown to delay or prevent the development of new cysts. Accordingly, the present disclosure also provides methods of delaying or preventing the development of a cyst in a subject in need thereof. The methods can include administeringAttorney Docket No. 063697-504001WO to the subject a therapeutically effective amount of a Kca3.1 inhibitor. The present disclosure also provides uses of a therapeutically effective amount of a Kca3.1 inhibitor in the delay or prevention of the development of a cyst in a subject need thereof. Similarly, the present disclosure includes uses of a therapeutically effective amount of a Kca3.1 inhibitor in the manufacture of a medicament for the delay or prevention of the development of a cyst. The present disclosure further provides KCa3.1 inhibitors or pharmaceutical compositions thereof for use in the delay or prevention of the development of a cyst in a subject in need thereof.

[0099] In some cases, the KCa3.1 inhibitor can decrease a number of new cyst growth in the subject relative to an untreated subject. For example, the KCa3.1 inhibitor can decrease the number of new cyst growth in the subject by about 60% to about 95%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%, or about 85% or about 87% relative to the untreated subject. For example, the KCa3.1 inhibitor, such as senicapoc, can decrease the number of new cyst growth in the subject by about 10% to about 40%, about 10% to about 35%, about 15% to about 30%, about % relative to the untreated subj ect.3. Reduction of kidney fibrosis

[0100] In subjects with kidney fibrosis, treatment with KCa3.1 inhibitors was shown herein to reduce fibrosis. Therefore, in some instances, when the subject has kidney fibrosis and the KCa3.1 inhibitor can reduce the kidney fibrosis. In some instances, the KCa3.1 inhibitor (e.g. senicapoc) can reduce renal fibrotic area by about 30% to about 70%, about 35% to about 60%, about 40% to about 55%, or about 50%, optionally as indicated by sirius red staining intensity. In some instances, the KCa3.1 inhibitor (e.g. senicapoc) can reduce renal fibrotic area by about 60% to about 90%, about 65% to about 90%, about 70% to about 90%, about 75% to about 85%, or about 85%, optionally as indicated by sirius red staining intensity. For example, the KCa3.1 inhibitor (e.g. senicapoc) can reduce blood urea nitrogen (BUN) by about 20% to about 60%, about 25% to about 50%, about 30% to about 45%, about 35% to about 45%, or about 40% relative to an untreated subject.

[0101] Accordingly, the present disclosure also provides methods of treating kidney fibrosis in a subject in need thereof. The methods can include administering to the subject a therapeutically effective amount of a Kca3.1 inhibitor. The present disclosure also provides uses of a therapeutically effective amount of a Kca3.1 inhibitor in the treatment of kidney fibrosis in a subject need thereof. Similarly, the present disclosure includes uses of aAttorney Docket No. 063697-504001WO therapeutically effective amount of a Kca3.1 inhibitor in the manufacture of a medicament for the treatment or kidney fibrosis. The present disclosure further provides KCa3.1 inhibitors or pharmaceutical compositions thereof for use in the treatment of kidney fibrosis in a subject in need thereof.III. Combination Therapy

[0102] The KCa3.1 inhibitors have been shown here to be effective in the methods of treating or preventing cystic kidney disease when administered in combination with other therapeutic agents. As such, the KCa3.1 inhibitors can be administered in combination with a second therapeutic agent. In particular, it has been shown herein that the KCa3.1 inhibitors can be administered with TMEM16a inhibitor and / or Cftr inhibitor while maintaining usefulness. The TMEM16a inhibitor can be CaCCinh-AOl.

[0103] In particular, it has been shown herein that when KCa3.1 inhibitor and a Cftr inhibitor are administered in combination, a synergistic effect was observed such that a lower dose for the KCa3.1 inhibitor and / or the Cftr inhibitor was needed to achieve the same level of therapeutic effect. Therefore, in some instances, the second therapeutic agent can be Cftr inhibitor. For example, the Cftr inhibitor can be PPQ-102.

[0104] Suitable second therapeutic agents can also include vasopressin receptor antagonists. Suitable vasopressin receptor antagonist can include unselective antagonists, and selective antagonists such as VIA antagonists, VIB antagonists, V2 antagonists. For example, the vasopressin antagonist can be V2 antagonists, such as lixivaptan, mozavaptan, satavaptan, and tolvaptan. For example, the second therapeutic agent can be tolvaptan.

[0105] Accordingly, also provided herein are pharmaceutical compositions comprising a KCa3.1 inhibitor and a second therapeutic agent. The present disclosure also includes said pharmaceutical composition for use in the treatment of fibrotic diseases such as cystic kidney disease.

[0106] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.EXAMPLES

[0107] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art.Attorney Docket No. 063697-504001WOSuch techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY : Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

[0108] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.

[0109] General methodologies used in Examples 1 to 5 are described in Example 7.EXAMPLE 1 : Characterization of AL / w- / -related pathways in two orthologous Pkdl mouse modelsA. Kcnn4 expression is selectively upregulated in ADPKD kidneys

[0110] RNA analysis of kidneys surgically removed from human ADPKD patients revealed a marked 23-fold elevation in Kcnn4 expression compared to healthy human kidneys (Fig. 1A). KCcnn4 expression in healthy human kidneys showed small deviation between individuals. In contrast, the range was much wider in ADPKD patients and varied from 2- to 51 -fold increase as compared to the mean of healthy kidneys. Despite the wide range, all ADPKD individuals consistently expressed greater renal Kcnn4 expression than healthy individuals. [OHl] Like in human ADPKD kidneys, consistent upregulation of renal Kcnn4 RNAAttorney Docket No. 063697-504001WO expression by 2- to 6-fold was found across all analyzed Pkdl orthologous mouse models of different disease onset and progressivity: early-onset rapidly progressive Pkdlckoand Pkdlvand adult-onset slowly progressiveSBPkdl and Pkdlwin comparison to age-matched wildtype animals (Fig. IB). Renal Kcnn4 expression was elevated by 1.9±0.3-fold in PIO Pkdlckoand by 1.7±0.2-fold in PIO Pkdlv / vcompared to wildtype. Comparison between the two early-onset models indicated that the more rapidly progressive Pkdlckoexperienced a greater increase in renal Kcnn4 expression. Likewise, the two adult-onset models expressed a similar fold increase in renal Kcnn4 expression, but the relatively more rapidly progressiveSBPkdl experienced a greater increase than the PkdPPSBPkdl kidneys at 2 months showed 6.2±1.3-fold increase while Pkdlwtat 7 months showed a 5.5±1.3-fold increase n Kcnn4 expression.SBPkdl kidneys were also analyzed at a later stage of disease (8 months). A small decrease in Kcnn4 expression was observed in comparison to 2 months but was nevertheless still highly elevated by 4.5± 1.1- fold compared to wildtype. Another method, in situ hybridization assay, was used to detect and confirm upregulation of Kcnn4 expression in FFPE PkdlckoandSBPkdl kidneys (Fig. 1C). Each white dot indicated binding of the Kcnn4 probe to a corresponding mRNA transcript. To visualize the RNA signals, threshold was first set using the negative control probe so that no signal is detected. With this threshold, no signal was detected in the wildtype samples, consistent with low Kcnn4 expression after development in healthy tissues. In contrast, distinct signals representing binding of Kcnn4 mRNA transcript were easily detectable in PkdlckoandSBPkdl kidneys.

[0112] mRNA expression, was analyzed, of other known channels and transporters of the fluid transport pathway driving cyst fluid secretion which promotes cyst growth: Nkccl, Nkcc2, Cftr, Aqpl an Aqp2 in PIO Pkdlcko(Fig. 2A) and 4-6-month-oldSBPkdl kidneys (Fig. 2B). The mRNA expression of these members did not increase and even reduced compared to wildtype controls.B. Kcnn4 is expressed early in embryonic kidneys

[0113] Kcnn4 expression during early kidney development had not previously been determined. Thus, to investigate modulation of Pkdl^ cysts by Kcnn4, we first needed to confirm that Kcnn4 is expressed at the age at which the metanephroi studies were commenced, E14.5. RNA was extracted from embryonic kidneys at E14.5 and E16.5, reverse transcribed to cDNA and then performed PCR reactions using specific primers for Kcnn4 and S16. Kcnn4 was successfully amplified in all samples and found Aa_ Kcnn4 is expressed as early as E14.5Attorney Docket No. 063697-504001WO(Fig. 3).C. Highly elevated renal cAMP stimulates proliferation through MAPK / ERK pathway in vivo

[0114] cAMP levels were measured in kidneys of Pkdl orthologous mouse models Pkdlcko,SBPkdl and Pkdl"' and found a significant increase in comparison to age-matched wildtype controls (Fig. 4). At baseline, in wildtype kidneys, renal cAMP level varied from ~5 to ~14 pmol / mg protein between different ages, however, individual values within each age group were highly consistent. 225±23, 19±3 and 27±4 pmol cAMP / mg protein was found in PIO Pkdlcko, 5- 6-week-oldSBPkdl and 6-8-month-old Pkdl '. respectively. Although variations exist between individual values in these Pkdl mouse models, all values were highly elevated in comparison to wildtype kidneys. The fold increase in renal cAMP when compared to age- matched wildtype controls was greater in the early-onset Pkdlckokidneys (16-fold) than in the adult-onsetSBPkdl (3.8-fold) and Pkdlwt(2.7-fold).

[0115] As cAMP is known to stimulate proliferation through MAPK / ERK signaling, protein levels of phospho-ERK, total ERK and downstream effector c-Myc were measured in mouse kidneys. Both early- and adult-onset Pkdl mouse kidneys showed significant hyperactivation of MAPK / ERK (Fig. 5A-D). In PIO Pkdlcko, the protein levels of phospho-ERK, phospho- ERK / total ERK and c-Myc increased by 4.7±0.8, 5.4±0.6 and 10.9±0.8 fold compared to controls, respectively (Fig. 5 A). While the total amount of ERK protein was unchanged, the proportion of the activated form of ERK was highly increased, likely contributing to increases in c-Myc level. Likewise, in PIO Pkdlv / v, phospho-ERK, phospho-ERK / total ERK and c-Myc protein levels were increased by 3.5±0.4, 4.6±0.6 and 2.6±0.4 fold, respectively (Fig. 5B). Similar activation was observed in the adult-onset modelsSBPkdl and Pkdlwt, where greater activation was found in the relatively more rapidly progressive modelSBPkdl; phospho-ERK, phospho-ERK / total ERK and c-Myc protein levels increased by 6.5±1.3, 9.6±1.1 and 6.0±0.9 fold compared to wildtype measured at 5-6 weeks of age (Fig. 5C). In 6-8-month-old Pkdl'P we found 2.5±0.4, 6.3±0.7 and 6.5±0.8-fold increase in phospho-ERK, phospho-ERK / total ERK and c-Myc protein levels, respectively (Fig. 5D). This is the only model where a change in total ERK levels were found compared to controls. Indeed, total ERK was reduced by over 50% and may in part contribute to the elevation in phospho-ERK.D. Kcnn4 knockout mice are healthy

[0116] The consistent elevation in Kcnn4 expression across all analyzed mouse modelsAttorney Docket No. 063697-504001WO provided a strong basis for targeting Kcnn4 and / or KCa3.1 for treatment of ADPKD. Before proceeding with genetic inactivation or pharmacologic inhibition of KCa3.1, Kcnn4 knockout mice was analyzed to confirm KCa3.1 is safe to target. The Kcnn4 knockout mice were indistinguishable from the wildtype, with no discernible phenotypes in the kidneys (Fig. 6) or any other organs. In addition, they had normal lifespan and fertility. As the major phenotype of ADPKD is found in the kidneys, we also evaluated kidney function by measuring blood urea nitrogen (not shown) level and found no alterations.EXAMPLE 2: Modulation of KCa3,l in the embryonic kidney modelA. A KCa3.1 agonist exacerbates cysts in Pkdl- / - metanephroi and induces cysts in Pkdl+ / +

[0117] As renal cysts in Pkdl ^ mice usually initiate around El 5.5, to study metanephroi cysts in culture, metanephroi needed to be isolated from E14.5 embryos and cultured for 4 days in humidified 37°C incubator with CO2 (Fig. 7A). Once in culture, Pkd ~ metanephroi required 8-Br-cAMP to be added on day 1 for cyst induction and had to be replaced in fresh media every two days. Any regimens to be tested were simultaneously added with 8-Br-cAMP. In contrary to the inherent ability of Pkdl ’ ’metanephroi to form cysts, Pkdl+ / +metanephroi were generally non-cystic with rare sporadic tubular dilations and unresponsive to 8-Br-cAMP (Fig. 7B). The KCa3.1 agonist SKA-111 was introduced to Pkdl+ / +at 3 concentrations (10, 20 or 30mM) to test if cysts could be induced. Indeed, cysts were detectable within 1-2 days post treatment. Quantification of percent cyst area revealed dose-dependent increase (Fig. 7C). Concentrations up to 30mM were tolerable as demonstrated by unchanged kidney volume and normal metanephroi development.

[0118] E14.5 Pkdl ^ metanephroi were highly responsive to 8-Br-cAMP; cysts were detectable as early as 1 day post treatment, the earliest timepoint we imaged metanephroi. Different concentrations of DMSO (0.1-1%) were tested in Pkdl ^ metanephroi to determine the optimal concentration for use without inducing an additive effect on cyst formation. Results (not shown) indicated that up to 0.25% was acceptable, but the lowest concentration tested (0.1% DMSO) was selected for all ex vivo experiments to minimize potential unwanted additive effects on cyst formation. Pkdl ^ metanephroi subjected to lOmM of KCa3.1 agonist SKA-111 appeared progressively more cystic with time than those treated with vehicle (DMSO) while there was no apparent toxicity on metanephroi development (Fig. 8A). Quantification upon completion of treatment on day 4 showed that SKA-111 led to a marked increase in percentAttorney Docket No. 063697-504001WO cyst surface area and cyst number by 34 % and 44% in comparison to vehicle, respectively (Fig. 8B-C).B. Pkdl- / -; Kcnn4- / - metanephroi are less responsive to cAMP

[0119] To investigate whether Kcnn4 genetic inactivation can influence PkdP^ cystogenesis, Pkdl+ / ~; Kcnn4~ ~ breeders were setup to generate PkdP ~; Kcnn4~ ~ embryos for metanephroi isolation, which were compared to PkdP^ metanephroi cyst growth. 8-Br-cAMP was added to the media on day 1 and replaced on day 3 (Fig. 9A). Differences in severity of cysts between PkdP ~ and upon loss of Kcnn4 in PkdP ~; Kcnn4~ ~ metanephroi were evident from day 1 and became progressively more prominent. A macroscopic comparison on day 4 revealed obvious differences in the severity of cysts (Fig. 9B). Cysts in PkdP ~; Kcnn4~ ~ metanephroi were smaller in size and fewer in number than in the PkdP ~ metanephroi. Indeed, the relative cyst surface area, as well as cyst number were reduced by 38% and 31%, respectively, upon Kcnn4 genetic inactivation when compared to PkdP ~ metanephroi (Fig. 9C-D). Importantly, Kcnn4 inactivation did not alter metanephroi development; PkdP ~; Kcnn4~ ~ metanephroi were healthy with no reduction in kidney volume.C. KCa3.1 pharmacologic inhibition prevents cyst growth and regresses pre-formed cysts

[0120] To understand if KCa3.1 pharmacologic inhibition can prevent or delay PkdP^ cyst growth, two KCa3.1 inhibitors of different potency were introduced into the metanephroi culture media on day 1 simultaneously with 8-Br-cAMP (Fig. 10A). As Pkdl+ / +metanephroi tolerated well 30pM of SKA-111, 20pM or 40pM of TRAM-34 were applied to PkdP ~ metanephroi (Fig. 10B-C). Both concentrations were tolerable; metanephroi showed normal growth. The difference in percent cyst area between PkdP^ metanephroi and those treated with 20pM or 40pM TRAM-34 led to similar reduction (26% versus 28%).

[0121] PkdP^ metanephroi subjected to 20pM of a more potent KCa3.1 inhibitor, Senicapoc, were substantially less cystic compared to vehicle treated metanephroi (Fig. 1 IB). When Senicapoc was given simultaneously with 8-Br-cAMP, both percent cyst area and cyst number greatly reduced by 91% and 87% upon completion of experiment on day 4, respectively (Fig. 11C-D). This striking reduction resulted in nearly indistinguishable tissue morphology from wildtype.

[0122] It was then tested if Senicapoc can be effective towards established PkdP' cysts by adding Senicapoc on day 3, which was 2 days after cyst induction (Fig. 12A). On the followingAttorney Docket No. 063697-504001WO day (day 4), a clear difference was readily detected; those treated with Senicapoc were much less cystic. Cysts diminished in size and number progressively (Fig. 12B). Percent cyst area was plotted in the form of a bar graph (Fig. 12C) and a line graph (Fig. 12D) to show that when compared to PkdP / - metanephroi where cyst area progressively increased from Day 3 to Day 5, cyst growth in those treated with Senicapoc were markedly restricted, and even progressively reduced established cysts. On day 4, percent cyst area was reduced by 50% and by 61% on day 5. To test if cyst modulation was reversible, Senicapoc was removed and cysts re-developed (not shown).EXAMPLE 3 : KCNN4 GENETIC INACTIVATION CAUSES DELAY IN DISEASE PROGRESSION IN 2 PKDI MOUSE MODELS OF DIFFERENT DISEASE ONSET AND SEVERITYA. Kcnn4 genetic inactivation improves cyst indices and ultimately reduces kidney size in early-onset rapid progressive model Pkdlcko

[0123] Renal cysts in the early-onset rapid progressive model Pkdlckowere detectable as early as P2 and progressively increased in number and size until end of life around 3 weeks of age. Pkdlckowas analyzed at P5 and PIO, representing early and mid-stage of disease progression, respectively. The abdomen of Pkdlckomice were extended due to enlarged cystic kidneys and became more prominent with disease progression. At PIO, isolated kidneys were macroscopically larger in size and paler in colour than wildtype kidneys (Fig. 13 A). KBW of cystic P5 Pkdlckokidneys increased by 3.8-fold over wildtype and progressively enlarged with age, reaching 11.4-fold by PIO. In comparison, Pkdlcko; Kcnn4~ ~ had smaller abdomens and isolated kidneys were smaller than Pkdlckokidneys. Accordingly, KBW reduced by 29% at P5 and by 20% at PIO. Kidney sections were stained with H&E to quantify cysts. PIO Pkdlckokidney was highly cystic with little intact tissue remaining compared to the wildtype, while genetic Kcnn4 inactivation helped to preserve more healthy tissue (Fig. 13B). Percent cyst area increased by 16.5-fold in P5 Pkdlckoand by 24.2-fold in PIO Pkdlckocompared to wildtype (Fig. 13C). In just 5 days, a 40% increase was found in percent cyst area in PIO Pkdlckocompared to P5 Pkdlcko. Kcnn4 inactivation reduced percent cyst area equally at both ages, by 20% in P5 Pkdlckoand by 18% in P10 Pkdlcko. Contrary to the significant 40% increase in percent cyst area between P5 and P10 Pkdlcko, overall cyst number did not increase at the same rate and only increased by 14% (Fig. 13D). Genetic Kcnn4 inactivation effectively reduced cyst number at both ages and was significantly greater at P5 (28%) than at P10 (16%).Attorney Docket No. 063697-504001WO

[0124] To determine which factor contributed more to the reduced cyst indices, individual cysts were sorted by size and the distribution of Pkdlckoand Pkdlcko; KcnnP' was plotted at P5 and at PIO (Fig. 14). Overall, PIO animals had bigger cysts and greater proportion of larger cysts compared to P5. Comparison between Pkdlckoand Pkdlcko; Kcnn4~ ~ at P5 or at PIO showed that cyst distribution was unchanged upon genetic inactivation of Kcnn4.B. Genetic Kcnn4 inactivation reduces cysts from distal tubules and collecting ducts but is insufficient to improve kidney function or extend lifespan in Pkdlcko

[0125] Cyst tubular origins of Pkdlckowas analyzed to determine which nephron segments were affected by Kcnn4 inactivation by labeling P5 FFPE kidney sections with tubular segment specific lectin markers (Fig. 15). The proximal, distal, and collecting tubules were coloured in green, blue, and red, respectively. Based on the tubular diameter threshold determined using the wildtype control (See Example 7.C.2), no cysts of proximal tubular origin were detected. All cysts were found exclusively in distal tubules and collecting ducts, consistent with localized expression of Ksp-cadherin. There were fewer large cysts in Pkdlcko; Kcnn^' based on our images. The proportion of cystic tubules within distal and collecting segments were quantified and 1.9-fold greater proportion of cysts were found in the collecting duct. Kcnn4 inactivation effectively reduced the proportion of cysts in both tubular segments at P5 in the Pkdlcko. Cysts in the distal tubules were reduced by 31%, while those in the collecting ducts were reduced by 30%.

[0126] Serum BUN levels were determined and plotted a Kaplan-Meier curve to determine if the improvement in tissue morphology and cyst indices observed were reflected by an improvement in kidney function and survival. The BUN level increased by 2.7-fold in P5 Pkdlckoand by 4.4-fold in PIO Pkdlckocompared to wildtype (Fig. 16A). However, Kcnn4 inactivation did not reduce the BUN levels at any measured timepoint. In both cases, there were large variations between individual samples, but no correlation was found between BUN levels and the size of cystic kidneys. Survival was also monitored in Pkdlckoand Pkdlcko; Kcnn4~ ~ and found no apparent extension of lifespan upon genetic Kcnn4 inactivation. Median survival was 18 days in Pkdlckoand 16 days in Pkdlcko; Kcnn4~ ~ (Fig. 16B).C. Reduction in renal cAMP levels dampens proliferation, improves kidney fibrosis and lowers primary cilia length in Pkdlcko

[0127] As cAMP was consistently upregulated in the mouse models used and is known toAttorney Docket No. 063697-504001WO associate closely with major processes required for cyst progression in ADPKD, including proliferation, fibrosis, and primary cilia elongation, cAMP levels were measured in PIO Pkdlcko; Kcnn4~ ~ and found a significant reduction by 37% compared to PIO Pkdlckoupon loss o Kcnn4 (Fig. 17A). As cAMP is known to stimulate proliferation through MAPK / ERK activation, protein levels of phospho-ERK, total ERK and downstream effector c-Myc were measured (Fig. 17B). While total ERK remained stable, striking reductions were found in the other components. Phospho-ERK and the phospho-ERK to total ERK ratio were almost restored to baseline, while c-Myc was reduced by more than half. Proliferation in kidney epithelial cells were next measured in PIO Pkdlckousing the proliferation marker ki67 (Fig. 17C). In comparison to wildtype controls, which rarely had more than one ki67-positive epithelial cell in a renal tubule, PIO Pkdlckotubular epithelia was highly proliferative with many ki67-positive epithelial cells frequently detected in the same renal tubule. The number of ki67-positive nuclei correlated with cyst size: the larger the cyst, the more ki67-positive epithelial tubular cells. In line with these observations, the number of ki67-positive cells per pm2kidney was increased by 3.5-fold in PIO Pkdlckocompared to wildtype. The number of highly proliferative cystic tubular cells in Pkdlckowas greatly reduced by 43% upon genetic Kcnn4 inactivation.

[0128] Since cAMP signaling also contributes to fibrosis, kidney sections were stained with Sirius Red, a marker for collagen I and IV, and found Pkdlckokidneys were highly fibrotic with many regions of intense red staining signifying collagen accumulation (Fig. 18). The relative fibrotic surface in PIO Pkdlckowas increased by 4.8-fold compared to wildtype. Kcnn4 inactivation reduced percent fibrosis by 39%.

[0129] Additionally, as primary cilia length can also be modulated by cAMP levels, primary cilia were stained with anti-acetylated-tubulin and measured the length of each primary cilia in the kidney sections (Fig. 19A). The average primary cilia length was increased by 1.9-fold in PIO Pkdlcko. Primary cilia were sorted based on their length and plotted their overall distribution (Fig. 19B). About 70% of all the primary cilia found in PIO wildtype were below 3 pm in length. In contrast, the ciliary distribution of PIO Pkdlckowas reversed; nearly 80% of primary cilia were above 3 pm in length. The distribution of Pkdlckocilia lengths was shifted to the right of the x-axis, with a strikingly greater proportion of longer primary cilia. A slight shift of ciliary distribution was found in Pkdlcko; Kcnn4~ ~ to the left of the x-axis. Accordingly, the increased average primary cilia length found in Pkdlckowas reduced by 26%. As Wnt / b-catenin signaling is an important pathway in the primary cilia, protein levelsAttorney Docket No. 063697-504001WO of active and total b-catenin were analyzed using western blot (Fig. 19C). In comparison to wildtype, Wnt signaling was highly activated in PIO Pkdlckoas shown by the significant elevation in active, total, and active / total b-catenin levels by 9.1-fold, 3.7-fold, and 2.4-fold, respectively, in line with results previous reported by the lab (Parrot et al., 2019). However, Kcnn4 inactivation did not significantly contribute to Wnt signaling. Although there was a trend that active and total b-catenin protein levels were reduced by Kcnn4 inactivation, the differences were not significant.

[0130] All western blot analyses were done in parallel at an earlier stage (P5) to monitor if the activation or rescue would be more prominent earlier in the disease. Representative images and quantifications analyzing MAPK / ERK, Wnt / b-catenin and c-Myc in P5 Pkdlckokidneys in comparison to wildtype are shown in Fig. 20A. Fig. 20B compares protein levels of the markers of those signaling pathways in P5 Pkdlcko; Kcnn4~ ~ kidneys in comparison to Pkdlcko. Signaling analysis at P5 resembled that of PIO.

[0131] The fold increase between Pkdlckoand wildtype was listed as well as percent change in Pkdlcko; Kcnn4~ ~ compared to Pkdlckoat P5 and PIO in Fig. 20 for direct numerical comparison between signaling pathway activation at P5 and P10. We found that the activation of MAPK / ERK in the Pkdlckomodel, as well as the rescue by genetic Kcnn4 inactivation were highly consistent between P5 and P10. Although c-Myc hyperactivation was more prominent at P10 than at P5, Kcnn4 inactivation reduced c-Myc by half at both ages (Table 1). The pattern of Wnt / b-catenin activation and rescue by Kcnn4 inactivation was less conclusive as the pattern was less consistent between P5 and P10 as shown in Fig. 20. For instance, there was greater increase of active b-catenin and total b-catenin, but less active / total b-catenin at P10 versus P5 Pkdlcko. At P5, Kcnn4 inactivation did not show tendency to influence active b-catenin levels, but since total b-catenin levels was elevated, the active / total b-catenin ratio was reduced. At P10, although both active and total b-catenin were reduced, total b-catenin was reduced to a greater extent, hence increased the active / total b- catenin ratio. The observed large variations between individual values may explain in part the inconsistency between P5 and P10 upon Kcnn4 inactivation.Table 1. Multistage comparison of signaling activation between P5 and P10 PkdlckOand PkdlckO; Kcnn4 ' kidneys. Numerical comparison of MAPK / ERK, Wnt / b-catenin and c-Myc signaling between P5 and P10 kidneys. Values are displayed as fold increase from WT to Pkdlcko(column 1 and 2) and percentage change (%) in Pkdlcko; Kcnn4~ ~ from Pkdlcko(column 3 and 4).Attorney Docket No. 063697-504001WOD. Heterozygous Kcnn4 knock-out is sufficient to improve cyst indices and kidney morphology in adult-onset slow-progressive modelSBPkdl

[0132] At 6 months, contrary to the enlarged abdomen observed in Pkdlcko, bothSBPkdl andSBPkdl; Kcririd had smaller body size when compared to age-matched wildtype, hence isolated kidneys were smaller as well. Macroscopic examinations showed thatSBPkdl kidneys were paler in colour compared to wildtype, whereas the colour oiSBPkdl; Kcririd kidneys were pinker, suggestive of less cystic kidneys (Fig. 21 A). KBW was calculated at 1 and 6 months of age in this model and found KBW was reduced following genetic Kcnn4 inactivation at both ages, however, the reduction was only significant at 6 months. From 1 to 6 months, KBW oSBPkdl increased by 1.4-fold, while the KBW oSBPkdl ; Kcnn4~ ~ was maintained stable with disease progression. When compared toSBPkdl, genetic Kcnn4 inactivation led to a 10% and 41% reduction in KBW ratio at 1 month and 6 months of age, respectively. Improvements in kidney morphology upon Kcnn4 inactivation was evident in 7- month-old H&E stained histological kidney sections;SBPkdl; Kcnn4~ ~ kidneys were significantly less cystic and had more intact tissues thanSBPkdl kidneys (Fig. 2 IB). This reduction was reflected in the reduced relative cyst surface area (%) and cyst number (Fig. 25C-D). For this analysis, heterozygous Kcnn4 knockoutBPkdl; Kcnn4+ / ~) samples were also included. Interestingly, heterozygous Kcnn4 was sufficient to significantly lower the relative cyst surface area and cyst number to similar level as those quantified inSBPkdl;Kcnn4~Bkidneys. Relative cyst surface area was reduced by 49% and 51%, while cyst number was reduced by 36% and 44% inSBPkdl; Kcnn4+ / ~ andSBPkdl; Kcnn4~ ~ kidneys, respectively. Kcnn4 inactivation had greater impact on the relative cyst surface area than on the cyst number, consistent with what was observed in Pkdlckoand / A rnetanephroi studies.

[0133] Further analyses of the tubular origin of cysts revealed that cysts can be found inAttorney Docket No. 063697-504001WO proximal tubules, distal tubules and collecting ducts (Fig. 22). Genetic Kcnn4 inactivation inSBPkdl reduced cysts by 61% in distal tubules and by 50% in collecting ducts, whereas cysts in proximal tubules were unaffected (quantification not shown).E. Kidney function and lifespan are significantly improved inSBPkdl upon genetic Kcnn4 inactivation

[0134] Kidney function was evaluated by BUN (Fig. 23 A). BUN in 6-8-month-oldSBPkdl increased by 10.1-fold in comparison to wildtype animals, and, strikingly, reduced by 60% upon Kcnn4 inactivation. This likely contributed significantly to the observed extended lifespan oiSBPkdl upon genetic Kcnn4 inactivation. Compared to the median survival of 182 days inSBPkdl, the median survival oiSBPkdl; Kcnn4~ ~ animals was markedly extended to 250 days, equivalent to a 37% increase in lifespan (Fig. 23B). NoSBPkdl animals were found surviving past one year of age, but about 20% oiSBPkdl; Kcnn4~Blived beyond one year. The Kaplan-Meier curve oiSBPkdl; Kcnn4~ ~ was significantly shifted to the right of the x-axis, indicative of better survival.F. Marked reduction in renal cAMP partially or completely attenuates cAMP- driven cystogenic cellular processes inSBPkdl

[0135] cAMP levels inSBPkdl; Kcnn4~ ~ kidneys were reduced by 45% when compared toSBPkdl, and led to reduced MAPK / ERK signaling, as indicated by reduced protein levels of phospho-ERK, phospho ERK / total ERK and c-Myc (Fig. 24A-B). Although the reduction in MAPK / ERK signaling was not significant, there was a trend for MAPK / ERK downregulation that probably contributed in part to the significant reduction in c-Myc levels by 44% due to genetic Kcnn4 inactivation. ki67 was used as a marker to evaluate the proliferative status of kidneys (Fig. 24C). 6-8-month-old wildtype controls showed only low proliferation levels as only a few tubules were ki67 positive, and in those tubules that expressed ki67, only one cell per tubule was positively stained. In contrast, ki67 signals inSBPkdl kidneys were more prevalent and often more than one ki67 positive cell was detected per tubule. Quantification of ki67 positive cells per pm2of kidney surface showed that proliferation increased by 4.3- fold inSBPkdl tubular epithelia when compared to wildtype. Genetic Kcnn4 inactivation significantly reduced proliferation and led to 60% reduction in ki67 signals, partially normalizing the proliferative status oiSBPkdl kidneys.

[0136] At 6-8 months of age,SBPkdl kidneys were highly fibrotic, many areas showed intense Sirius Red staining representing collagen deposition (Fig. 25). Relative renal fibroticAttorney Docket No. 063697-504001WO surface (%) inSBPkdl increased by 13.7-fold compared to wildtype and reduced by 45% upon Kcnn4 inactivation.

[0137] Upon analysis of renal primary cilia length, a 1.3-fold increase was found in average primary cilia length in 2-month-oldSBPkdl in comparison to wildtype, which was remarkably rescued and completely normalized upon Kcnn4 inactivation (Fig. 26A). When the primary ciliary distribution was plotted, about 60% of all primary cilia in wildtype kidneys were shorter than 3pm, like PIO wildtype analyzed earlier (See Example 3.C). In fact, the distribution of primary ciliary length in 2-month-old versus PIO wildtype were almost indistinguishable. The distribution oSBPkdl was less strikingly different from wildtype than the difference between Pkdlckoand age-matched wildtype, as 40% of all primary cilia were shorter than 3 pm, while the remaining 60% evenly distributed within the longer cilia length categories (Fig. 26B). Remarkably, the plot oiSBPkdl; Kcnn4~ ~ primary ciliary distribution completely overlapped with that of the wildtype, supporting primary cilia length was indeed normalized following Kcnn4 inactivation.EXAMPLE 4: ADMINISTRATION OF KCa3 , 1 PHARMACOLOGIC INHIBITOR SENICAPOC IN 4 PKDI MOUSE MODELS INDUCES AN IMPROVEMENT IN DISEASE STATUSA. Oral administration of Senicapoc is safe in wildtype mice

[0138] Initially Senicapoc was dissolved in DMSO solvent and administered to animals by daily intraperitoneal or subcutaneous injections. However, all animals including C57BL / 6 wildtype mice lost -20% in weight immediately after the first injection and did not regain weight or survive beyond 1 week of treatment. Since other groups who have administered Senicapoc in vivo mostly utilized oral gavage or through diet (Jin et al., 2019; Paka et al., 2017; Tubman et al., 2016), the effect of daily Senicapoc administration through oral gavage was evaluated at low dose (30mg Senicapoc / kg of body weight) or high dose (120mg Senicapoc / kg of body weight) in PEG / Cremophor / water solvent to 6-week-old wildtype mice for 3 months (Fig. 27A). Their health was monitored daily and all animals were weighed weekly. It was found that all wildtype mice completed the 3 months of treatment with no appreciable loss in body weight or deterioration in health status. Senicapoc promoted weight gain throughout the course of treatment (Fig. 27B). By 6 weeks post-treatment, mice in the high dose group doubled their weight when compared to before treatment and continued to gain weight. At the end of the treatment period, the kidneys and stained kidney sections with H&E were isolated (Fig. 27C). Kidneys from Senicapoc-treated animals wereAttorney Docket No. 063697-504001WO indistinguishable from vehicle-treated mice. Senicapoc did not alter the KBW or BUN in wildtype animals (Fig. 27C-D). Senicapoc administration to early- and adult-onset Pkdl mouse models were then assessed.B. Senicapoc treatment delays cyst growth in early-onset models

[0139] Senicapoc was given to 2 early-onset rapidly progressive Pkdl mouse models, Pkdlckoand Pkdlv / v, from P0 until P5 or PIO through two routes (Figs. 28, 30B). First, 120 mg Senicapoc / kg of body weight was given daily to mothers from the day of delivery (day 0) for 5 or 10 days. Senicapoc was also administrated daily to the newborn Pkdlckoand Pkdlv / vpups directly in the comer of their mouth from P2 until the end of treatment at a concentration and volume that is equivalent to 120mg Senicapoc / kg of body weight. Administration was initiated as early as possible given their rapid disease progression and short window for treatment.

[0140] All animals tolerated Senicapoc well with no mortality by the end of treatment. Kidneys were isolated at P5 for Pkdlckoand at P10 for Pkdlv / vand processed for H&E analysis. Kidneys from Senicapoc-treated Pkdlckowere slightly enlarged, with no significant change in cyst area (Fig. 29A) but with significantly reduced cyst number per unit area in Pkdlckofollowing Senicapoc treatment (about 20%) (Fig. 29B). Despite the reduction in cyst numbers in Pkdlc'" the effect was not sufficient to delay cyst enlargement or cause a global improvement in delaying cyst growth over the short time period of treatment of this rapidly progressive disease model. Nonetheless, senicapoc treatment was associated with a 50% reduction in fibrotic area, as indicated by Sirius Red staining intensity. (Fig. 29B)

[0141] The less rapidly progressive early-onset Pkdlv / vwere more responsive to Senicapoc treatment as illustrated by H&E-stained histological kidney sections (Fig. 30A). Kidneys from Pkdlv / vtreated with Senicapoc were smaller in size, less cystic and retained much less fibrotic tissues compared to kidneys from vehicle-treated animals. Although cyst number did not significantly differ after senicapoc treatment, KBW ratio and % cyst area were both significantly reduced by -25%, , with a highly significant 85% reduction in renal fibrosis as indicated by Sirius Red staining intensity (Fig. 30C and D).C. Senicapoc treatment delays kidney cyst growth and improves kidney function in the adult-onset models

[0142] The 3 months of Senicapoc daily oral administration for adult-onset models began at 3 weeks of age forSBPkdl and at 6 weeks of age for Pkdl"' (Fig. 31). These ages were chosenAttorney Docket No. 063697-504001WO such that treatment began just before or around the time of kidney cysts initiation.

[0143] The weekly body weight change of low dose Senicapoc treatedSBPkdl animals was similar to vehicle treated animals of the same genotype (Fig. 32A). The high dose group experienced a small decrease of body weight in the middle of Senicapoc treatment during week 6 and 7, but quickly regained weight afterwards, surpassing that of low dose and vehicle groups. Examination of kidney cysts using H&E-stained kidney sections showed that the cystic kidneys in the low dose Senicapoc-treated group were less severe compared to vehicle-treated animals. However, in both groups, cysts can be found throughout the kidney, while the high dose Senicapoc-treated mice displayed a shift in cyst localization to the renal cortex, significantly reducing cyst presence in the medulla (Fig. 32B). To understand how Senicapoc treatment affects renal indices, KBW, percent cyst surface area, cyst number and BUN were analyzed (Fig. 32C and D). KBW lowered by 33% and 31% following low dose and high dose Senicapoc treatment, respectively. Although the reduction in relative renal cyst surface area was not significant, a clear tendency was observed for cyst surface area to reduce upon Senicapoc treatment, notably by 27% (low dose) and 36% (high dose). While cyst numbers did not reduce in the low dose group, and even increased by 11%, the high dose group displayed a reduction of cyst numbers by 43%. Although low dose Senicapoc improved some disease parameters, overall, the high dose had greater effect on cyst indices. Senicapoc also led to a 50% decrease in renal fibrotic area. However, senicapoc treatment did not decrease BUN. (Fig. 32D)

[0144] Next, the effect of high and low dose Senicapoc administration was examined in another moderately progressive adult-onset model, Pkdl"'. The weekly body weight change of low and high dose Senicapoc treated Pkdl"' was similar to vehicle-treated animals (Fig. 33A). Senicapoc did not induce excess body weight gain. Kidneys from Senicapoc treated animals removed after completion of treatment were pinker in colour macroscopically, indicative of more healthy renal tissue. Furthermore, H&E-stained kidney sections were observed and it was found that kidneys from Pkdl ' treated with Senicapoc were less cystic, with a greater effect in the high dose treatment group (Fig. 33B and E). KBW ratio, relative cyst surface area (%), cyst number and BUN were analyzed (Fig. 33C and F). Although an increase in KBW was observed by 2% and 14% in low and high dose Senicapoc treatment groups, respectively, all other parameters significantly improved after Senicapoc treatment. A 50% and 61% reduction was found in percent cyst area, 38% and 51% reduction in cyst number, 60% reduction in renal fibrotic area, and 40% improvement in kidney function asAttorney Docket No. 063697-504001WO indicated through serum BUN analysis. (Fig. 33C and F)EXAMPLE 5: EVA UATION OF COMBINED CHANNEL INHIBITION VERSUS SINGLE TREATMENT ONA. Targeting PkdP' cysts with single treatment

[0145] The level of rescue of PkdP7~ cysts was established by targeting each channel individually with different concentrations of specific inhibitors, then compared the results to those obtained through combined inhibition to find the optimal inhibitor combination for potential use.1. KCa3.1 inhibitor Senicapoc

[0146] Pkdt7~ metanephroi were treated with 5, 10, and 20pM of Senicapoc added simultaneously with cAMP on day 1 and replaced on day 3 (Fig. 34A). Representative images of metanephroi after 4 days of culture showed increasing cystic phenotypic rescue as inhibitor dosage increased (Fig. 34B). There appeared to be a dose dependent reduction in cyst area from 5 to lOpM, causing 11% and 19% reduction in cyst area (Fig. 34C). However, at 20pM, cyst area drastically reduced by 91%.2. Cftr inhibitor PPQ- 102

[0147] Pkdt ~ metanephroi were subjected to 1, 2, 5, 10, and 20pM of PPQ- 102 on day 1 with cAMP in fresh media and replaced on day 3 (Fig. 35 A). Representative images of metanephroi upon completion of the treatment showed a gradual reduction of the cystic phenotype with increasing dosage of PPQ- 102 (Fig. 35B). This trend was also demonstrated by the reduction in cyst area with increasing PPQ- 102 concentrations (Fig. 35C). While PPQ- 102 has a higher IC50 compared to Senicapoc (90nM versus 1 InM), it was found that PPQ- 102 treatment induced a more potent effect at low doses (below 20pM) in the metanephroi culture conditions. For instance, 2pM of PPQ- 102 had the same rescue effect (determined as cyst area reduction) as lOpM of Senicapoc. At higher dose (20pM), the two inhibitors led to similar rescue effect.3. Tmem 16a inhibitor CaCCinh- A01

[0148] Pkdt ~ metanephroi were treated with 10, 20, 25, 30 and 35pM of CaCCinh-AOl on day 1 at the same time as cAMP in fresh media and replaced on day 3 (Fig. 36 A). Representative images of metanephroi upon completion of treatment showed very small differences between different doses, except for 35pM where the rescue was more prominentAttorney Docket No. 063697-504001WO(Fig. 36B). There was an overall downward trend in percent cyst area with increasing doses of CaCCinh-AOl but was less consistent than the effect observed with the two previously described inhibitors (Fig. 36C). The only concentration that had a significant impact on cyst reduction was at 35pM, where cyst area lowered by 56%. The higher IC50 (2.1pM) explains why a much greater concentration is needed to induce an effect. Indeed, 25 or 30pM of CaCCinh-AOl was required to achieve the same reduction in cyst area as 2pM of PPQ-102 or lOpM of Senicapoc. Comparison between the three inhibitors showed that Senicapoc or PPQ- 102 would potentially be better options when treatment efficiency is considered the only factor as they produced a larger rescue effect with smaller concentrations.B. Targeting Pkdl ' cysts with combined treatment

[0149] There is an increasing interest in combination therapy, its main advantage being the potential to achieve a synergistic effect with overall lower dosages to reduce toxicity. Several in vivo studies have been done by others to test different treatment combinations in ADPKD models (Kanhai et al., 2020; Leonhard et al., 2019). The ex vivo Pkdl ’ ’metanephroi was used to screen two inhibitor combinations targeting channels or transporters that could be interacting partners in driving ADPKD fluid secretion based on preliminary data and observations.1. Targeting KCa3.1 and Cftr results in synergistic effect

[0150] Pkdl ^ metanephroi were treated with 5 or lOpM of Senicapoc along with 2, 5 or lOpM of PPQ-102 on day 1 and replaced on day 3 (Fig. 37A). Representative images of Pkd l "metanephroi on day 4 comparing single and combination treatment clearly showed augmented rescue of cystic phenotypes through combination inhibition (Fig. 37B).

[0151] The combinations of 5pM Senicapoc with different concentrations of PPQ-102 were quantified in Fig. 38 A. Combinations with lOpM Senicapoc were quantified in Fig. 38B. Overall, both graphs showed a downward slope in percent cyst area by combining with increasing concentrations of PPQ-102. All combinations except the lowest one (5pM Senicapoc & 2pM PPQ-102 combination seems to be additive) suggest there is synergistic rescue of A / ’^cysts, where lower total concentrations achieved the same rescue effect as a higher dosage single inhibitor treatment.2. Targeting KCa3.1 and Tmeml6a is less effective

[0152] Pkdt7' metanephroi were treated with 5pM of Senicapoc along with 10, 20, 25 or 30pM of CaCCinh-AOl on day 1 and replaced on day 3 (Fig. 39A). Representative images ofAttomey Docket No. 063697-504001WO A 7 / _metanephroi on day 4 submitted to the different treatment regimens are shown in Fig. 39B.

[0153] The difference between single and combination treatment is less obvious here compared to the combination between Senicapoc and PPQ-102. In some cases, addition of CaCCinh-AOl did not cause greater reduction in cysts than using Senicapoc alone (Fig. 39). In fact, the combination of 5pM Senicapoc with 10 or 25 pM CaCCinh-AOl even led to more cystic metanephroi. The only combination that showed synergy was 5pM Senicapoc with 30pM CaCCinh-AOl . However, the degree of rescue gained from targeting these two channels can be achieved through the combination of PPQ-102 and Senicapoc at much lower concentrations, making the latter a more ideal combination to realize.3. Targeting KCa3.1 and Vasopressin receptor

[0154] Tolvaptan is an approved therapeutic in the treatment of ADPKD. However, tolvaptan is known to induce a number of side effects including polyuria, polydipsia, and hepatotoxicity. Combination of tolvaptan and senicapoc can reduce risk of side effects and improving hepatotoxicity.EXAMPLE 6: DISCUSSIONA. The significance of Kcnn4 upregulation in ADPKD

[0155] Consistent Kcnn4 hyperactivation as measured in various Pkdl mouse kidneys, and more importantly in human ADPKD kidneys, strongly suggests that Kcnn4 and its gene product KCa3.1 is implicated in the progression of ADPKD. The present in vivo results build on the in vitro work by Albaqumi el al., where the investigators reported activation of Kcnn4 in cells derived from human ADPKD kidney cysts. The present studies were designed to elucidate the role of Kcnn4 and KCa3.1 in ADPKD progression using various mouse models, which also allowed analysis whether this channel may represent an attractive therapeutic target for ADPKD treatment. In the following discussion,, Kcnn4 and KCa3.1’s involvement in ADPKD pathogenesis is described.B. Xcn« / KCa3.1 may be hyperactivated and sustained to maintain the cyst fluid secretory pathway

[0156] The current accepted model of cyst fluid secretion requires interaction between CFTR, NKCC1 and Na+-K+-ATPase to drive net fluid transport through apical AQP2 into the cyst lumen, hence progressively enlarging cysts (Jouret & Devuyst, 2020). One component that is not fully understood is how the excess potassium ions brought in by Nkccl and Na+-Attorney Docket No. 063697-504001WOK+-ATPase are recycled. This step is critical as potassium efflux can cause hyperpolarization which is required to drive continual chloride secretion through Cftr. The need for chloride secretion through Cftr to drive cyst growth is well documented by Magenheimer et al. ex vivo and by Yang et al. in vivo. It was hypothesized that KCa3.1 could be a potential candidate to fulfill this role due to its PKD-relevant properties demonstrated in other pathophysiological conditions, and based on our previous identification of Kcnn4 as a downstream target of c- Myc (Pang et al., 2012), which is an inducer of PKD. Various components of the cyst fluid secretory pathway are generally studied independently and rarely altogether. Here we analyzed major components of the secretory pathway, Nkccl, Cftr an Aq[)2 with isomers Nkcc2 and Aqpl, in parallel with Kcnn4. Hyperactivated Nkccl, Cftr and Aqp2 were expected to be found, but instead, selective upregulation of Kcnn4 was observed. Expression levels of Nkccl, Cftr and Aqp2 reduced, and were in fact indistinguishable from Nkcc2 and Aqpl which are not directly implicated in ADPKD, suggesting Cftr, Nkccl and Aqp2 may be inactive at this time. This may be in part due to differential gene regulation with disease progression. As we had conducted this analysis during mid-life in Pkdlcko(PIO) and end stage inSBPkdl (6-8-month-old), the results only illustrate the expression levels at these timepoints, which may not reflect the expression levels at earlier stages. Based on the data, it is suggested that Cftr and Nkccl may be activated during the early phase of the disease to activate the fluid secretory pathway, then Kcnn4 expression rises to sustain this pathway. Once KCa3.1 begins to recycle the excess potassium ions and establishes a hyperpolarized membrane, persistent hyperactivation of Cftr and Nkccl are perhaps no longer needed. Instead, the upregulated Kcnn4 expression may be sustained throughout life in ADPKD (as seen in 2 months versus 8 monthsSBPkdl kidneys) and is sufficient to maintain and drive the fluid secretory pathway. To test this hypothesis and to better understand the interactions between various components of the pathway as disease progresses, an analysis of the expression of those channels should be realized at different timepoints. As has been seen for fibrosis in ADPKD, Kcnn4fKCa3.V s role in ADPKD progression transitions from initially protective to later disease driven (Fragiadaki et al., 2020). Likewise, components of the fluid secretory pathway could experience changes in expression to fulfill the need of each component in maintaining the pathway as disease progresses. Furthermore, as channel activity or protein levels were not measured due to technical constraints, it is unknown if channel expression is reflective of channel activity. Therefore, perhaps a thorough analysis of channel expression, protein level and activity of major channels and transporters of the fluidAttorney Docket No. 063697-504001WO secretory pathway at different phases of disease progression could help us understand better how the different components of the secretory pathway cooperatively mediate cyst fluid secretion with ADPKD progression. Early versus later stages were addressed for analysis of Pkdlckoby comparing P5 versus PIO, but perhaps a more meticulous approach would be to include pre-cystic age (~P1). As we had seen in Senicapoc-treated PkdPRmetanephroi, those who received Senicapoc at the time of cyst induction had greater impairment in cyst formation and growth than those who received Senicapoc two days after cyst induction. Based on this observation, it could be more efficient to take preventative measures than to treat after disease is established.C. Elevation in cAMP may reflect severity of cystic phenotypes

[0157] Elevated cAMP levels represent a central mechanism mediating cyst growth through stimulation of fluid secretion and proliferation (Calvet, 2015). Increased renal cAMP levels have been reported in several non-orthologous Pkdl models (Wang et al., 2005; Yamaguchi et al., 1997), while reducing renal cAMP levels through vasopressin receptor V2 inhibition is the target of the only FDA approved drug for ADPKD, Tolvaptan. However, apart from PkdlRC / RCwhere renal cAMP has also been found highly elevated (Hopp et al., 2012), cAMP has not been examined in other orthologous Pkdl models. It was measured and confirmed herein that renal cAMP is consistently upregulated in all Pkdl orthologous mouse models analyzed. cAMP is thought to stimulate proliferation by MAPKZERK activation, as demonstrated by in vitro studies conducted by Yamaguchi et al. phospho-ERK and ERK levels were analyzed and found consistent activation in both, early- and adult-onset Pkdl mouse kidneys, consistent with proliferation being one of the major drivers of cyst growth, even in adult-onset models where proliferation is normally maintained at baseline in healthy animals. The greater fold increase in cAMP in PkdlckoversusSBPkdl did not stimulate higher proliferation measured by MAPK / ERK activation or ki67 positive cells per kidney surface area in this model, indeed, both models encountered similar stimulation in proliferation. The greater proliferation in Pkdlckoat baseline compared toSBPkdl is potentially due to differences in age, as seen in the wildtype where higher rate of proliferation is observed in P10 versus in adult. In fact, most of the analysis we conducted reveal a similar fold increase in PkdlckoandSBPkdl when compared to wildtype, which nicely illustrates that key features of ADPKD are reproducible, regardless of the underlying genetic cause. Upon examination of the different parameters analyzed, fold increases in cAMP levels appear to associate moreAttorney Docket No. 063697-504001WO closely with disease progressivity as defined by severity of cysts (quantified by percent cyst surface area). Indeed, greater cyst surface areas were observed in PkdlckoversusSBPkdl, which also reflects the proportional difference in cAMP levels. Aside from differences in cAMP levels, we also detected greater c-Myc activation in Pkdlckokidneys, while inSBPkdl we observed more fibrotic kidneys with greater functional loss. Greater activation of c-Myc in Pkdlckokidneys can be explained in part by the higher renal cAMP level that stimulate greater activation of downstream effector c-Myc to sustain proliferation of renal epithelial cells. As cAMP is thought to differentially regulate proliferation (activation versus inhibition) depending on the pattern of B-Raf protein, analyzing B-Raf levels may be helpful to understand the different elevation in cAMP we observed between rapid versus slow progressive mouse models. Fibrotic kidneys, on the other hand, require longer time to develop and exacerbate, therefore greater collagen accumulation inSBPkdl may be expected given the longer lifespan. It is intriguing how greater decline in kidney function is found inSBPkdl given the vastly different lifespan and cyst severity at the time of analysis. AsSBPkdl and Pkdlckoare generated differently with opposing Pkdl levels and differ in cyst distribution (no proximal tubular cysts in Pkdlcko), both factors could play a role. More importantly, BUN in Pkdlckowas analyzed around early- to mid-stages of disease progression, whereasSBPkdl samples were collected near end stage. Understandably, greater kidney functional loss would be expected inSBPkdl.D. Kcnn4 genetic inactivation causes greater rescue of MAPK / ERK signaling in the early-onset Pkdlckomouse model

[0158] Analyses of cyst-associated processes show a similar improvement in PkdlckoandSBPkdl kidneys upon genetic Kcnn4 inactivation, although the rescue in terms of kidney function parameters and lifespan was overall more effective in theSBPkdl mouse model. The first phenomenon we had observed was the strikingly limited enlargement oiSBPkdl; Kcnn4~ ~ kidneys, which also remained stable with age whileSBPkdl kidneys continued to enlarge.This effect was not as pronounced in Pkdlcko; Kcnn4~ ~ kidneys, which is likely due to their rapid progressivity. The only analysis where we observe a greater rescue upon genetic Kcnn4 inactivation in the rapid progressive model (Pkdlcko; Kcnn4~ / ~) than in the slow progressive model (^BPkdl; Kcnn4~ / ~) was in MAPK / ERK activation, where phospho-ERK, ERK, phospho-ERK / ERK are restored and downstream effector c-Myc is reduced by over half. This significant rescue led to -43% reduction in proliferation measured by ki67 staining,Attorney Docket No. 063697-504001WO suggesting c-Myc activation maybe more indicative of downstream events such as proliferation. As there are numerous signaling pathways upstream of c-Myc, such as Wnt, the partial normalization of c-Myc despite complete restoration of MAPK / ERK signaling could be attributed to other signaling pathways unaffected by Kcnn4 inactivation. As canonical Wnt signaling is hyperactivated in ADPKD, implicated in primary cilia assembly / disassembly, and more importantly, it is completely normalized in polycystin / 7 / tSS double knockout mice (Lancaster et al., 2011; Parrot et al., 2019; Shao et al., 2020), active and total b-catenin levels were analyzed in Pkdlcko; Kcnn4 compared to Pkdlckoand found overall no significant rescue of the increased levels observed. In fact, Wnt / b-catenin activation even showed a tendency to increase upon Kcnn4 inactivation (as indicated by greater total b-catenin levels), in part explaining why c-Myc is only partially normalized. To address the signaling status in Pkdlckoat an earlier age, the level of activation and rescue by Kcnn4 inactivation of signaling pathways at P5 was analyzed and compared. It was expected thar greater activation and rescue would be found at P5 compared to PIO, since the proliferative status of renal epithelial cells reduces with age. However, identical hyperactivation of MAPK / ERK was found at both ages despite the marked greater increase in kidney size and cyst indices between PIO and wildtype, while greater fold increase of c-Myc reflects the more severe PIO Pkdlckokidneys. This suggests that MAPK / ERK activation is sustained between P5 and PIO despite the supposed difference in proliferative status due to increasing age. It would be interesting to examine whether the activation of MAPK / ERK is maintained throughout the disease and if it translates into a sustained rate of increase in cyst indices. The normalized MAPK / ERK and reduced c-Myc by half at both ages upon Kcnn4 inactivation indicate that targeting Kcnn4 is equally effective in downregulating MAPK / ERK and effector c-Myc at early versus midstage in this model. This rescue not only shows dna Kcnn4 is tightly associated with MAPK / ERK signaling in Pkdlcko, but it also indicates that the renal epithelial cells remain highly plastic in this rapid progressive model, such that targeting Kcnn4 restores MAPK / ERK equally at P5 and PIO. In contrast, the effect of Kcnn4 inactivation on Wnt signaling is less conclusive in the Pkdlckomodel. Upon genetic Kcnn4 inactivation, Wnt / b-catenin activation in the kidneys appears to be reduced at P5, but tend to elevate at PIO, although no significance is found at either age. This contrasting effect may be in part due to large deviations between individual samples. These results suggest that KCa3.1 may not be a major regulator of canonical Wnt signaling in ADPKD. Therefore, we did not analyze this pathway further inSBPkdP InSBPkdl, sustained hyperactivation of MAPK / ERK was found even at 6Attorney Docket No. 063697-504001WO months. It would be interesting to analyze and compare MAPKZERK in theSBPkdl at earlier ages, to better understand how MAPKZERK signaling progresses with disease. Although no significant downregulation of MAPKZERK was found inSBPkdl; Kcnn4 the 44% reduction in c-Myc levels upon genetic Kcnn4 inactivation suggests that other pathways upstream of c- Myc may be more relevant for KCa3.1 mediated signal transduction stimulating proliferation in this moderately progressive mouse model. Analysis of signaling pathways reveal that the rescue of Pkdlckoby genetic Kcnn4 inactivation appears more efficient than inSBPkdl , where phospho-ERKZERK is not significantly reduced but is more efficient at rescuing kidney function and extending lifespan.E. Kcnn4 / n3.\ is implicated in cyst initiation and enlargement ex vivo and in vivo

[0159] To determine whether changes in KCa3.1 level may directly contribute to cystogenesis, the effects of a KCa3.1 activator was studied on an ex vivo system of cystogenesis using PkdPBmetanephroi. Unlike previous metanephroi studies, where the gene of interest is frequently targeted by genetic inactivation or pharmacologic inhibition, both approaches were used, as well as a pharmacologic activator of KCa3.1 to validate the channel’s implication in cyst growth. Results using all three approaches complement each other and converge to show that KCa3.1 modulates PkdrBcyst growth. Indeed, the present ex vivo observations suggest that KCa3.1 is implicated in both, cyst enlargement and cyst initiation, as shown by comparable changes in cyst area and cyst number using the different approaches.

[0160] First, KCa3.1 activator SKA-111 was introduced to Z / ’metanephroi, which are inherently highly responsive to cyst induction by cAMP. Pkdl ’ ’metanephroi appear more severely cystic upon treatment with SKA-111, accompanied by increased cyst number and cyst area, therefore suggesting that an increase in KCa3.1 activity significantly accelerates cyst progression by promoting both cyst initiation and enlargement. More importantly, when added to the normally cAMP unresponsive Pkdl+ / +metanephroi, cysts remarkably developed, implying that upon cAMP stimulation, KCa3.1 can induce cysts ex vivo.

[0161] Consequently, the hyperactivated Kcnn4 in Pkdl mouse models are likely to favour cyst formation and growth. Following this hypothesis, the PKD phenotype of this model would thus be ameliorated by dampening KCa3.1 through genetic inactivation or pharmacologic inhibition, which could represent an attractive strategy for treatment of human ADPKD. This was investigated in the metanephroi model. When introducing pharmacologicAttorney Docket No. 063697-504001WO inhibitors ex vivo, two inhibitors of different potency and selectivity were selected. When / / ^metanephroi were subjected to TRAM-34 and cAMP, a mild reduction in cyst area was detected, despite utilizing high concentrations of the inhibitor (20 and 40mM). In contrast, 20mM of the more potent and selective Senicapoc led to a major reduction in cystic parameters when added simultaneously with cyst induction through cAMP treatment, virtually restoring A 7 / _metanephroi phenotype to normal. This finding showed that Senicapoc may be preventative whereas currently there is no drug that can prevent ADPKD.

[0162] The rapid progressivity of Pkdlckowas monitored when identifying the age of cyst onset. Tubule dilations and few cysts are detectable from P2. While P3 Pkdlckokidneys still retained the normal pink to red colour as seen in wildtype, P5 kidneys lost all colour and completely turned pale. This drastic transition is remarkable and highly indicative of the rapid progressivity of Pkdlcko. Although P5 kidneys still retained some intact renal tissue, it is insufficient to retain a normal appearance. PIO kidneys lost even more intact tissue and are filled with cysts. To compare the severity and degree of rescue by Kcnn4 inactivation at early- versus mid-stage of the disease, cysts in P5 and PIO Pkdlckokidneys were quantified. From P5 to PIO, additional cysts formed (14%), fluid was actively secreted into the lumen of both new and existing cysts, greatly expanding cyst surface by 40%, culminating in larger kidneys shown by the 3.8-fold increase in KBW. Unlike the parallel increase in cyst number and cyst area observed ex vivo in PkdPBmetanephroi treated with SKA-111, a proportionally greater increase was observed in cyst surface in the Pkdlckomodel, suggesting cyst enlargement has a greater contribution to cyst growth than initiation of new cysts in vivo. Alternatively, without wishing to be bound by theory, cyst initiation could be a more gradual event and only a small subset is required to drive cyst growth. Doing a similar analysis examining cysts at two ages in the slowly progressiveSBPkdl would help clarify. KBW was compared at two ages and it was found it remains stable and only increased by 1.4-fold from 1 month to 6 months of age in the slow progressive model. This showed that the robust machinery mediating cyst growth adapted to both fast pace in Pkdlckoor slow pace inSBPkdl regardless of the underlying genetics.

[0163] Reduction in cyst indices in both rapidly and slowly progressive models due to Kcnn4 inactivation correlates well with the present ex vivo findings and supports implication of Kcnn4 in both cyst initiation and enlargement. As both indices tend to reduce in parallel upon loss oiKcnn4, it is difficult to decipher in which process KCa3.1 is more involved. To address this question, individual cysts were sorted in P5 and PIO Pkdlckoand Pkdlcko; Kcnn4~Attorney Docket No. 063697-504001WO / _by size and found that at both ages, the distribution of cysts was unchanged between Pkdlckoand Pkdlcko; Kcnn4~ ~ . Kcnn4 inactivation did not appear to influence the proportion of cysts within each size interval, suggesting the less severe cystic kidneys observed in Pkdlcko; Kcnn4~ ~ are likely due to KCa3.1 acting on cyst initiation. Therefore, the present data suggested strongly that the improvement of the cystic phenotype in Pkdlcko; Kcnnf'' animals is a consequence of Kcnn4 genetic inactivation limiting cyst initiation in the Pkdlckomodel and consequently restricted the amount of cyst fluid secretion that is normally found in Pkdlckokidneys. Furthermore, it was found that genetic Kcnn4 inactivation induces a more significant reduction in KBW and cyst indices at P5 versus PIO, suggesting interventions during early phases of disease may be more effective. This is in line with the findings by Dong et al. where reversal of cysts in Pkdl or Pkd2 knockout mice, upon re-expression of Pkdl or Pkd2, was more rapid in animals at earlier stages of disease (Dong et al., 2021). Additionally, when analyzing cysts inSBPkdl, it was discovered that the Kcnn4 heterozygous knockout animalsBPkdl; Kcriri4 ) have similar cyst area and cyst number as their homozygousSBPkdl; Kcnn4~Blittermates. This leads to suggest that a complete knockout of Kcnn4 is perhaps not essential to delay cyst growth, and that therefore a reduction of activity could be sufficient when disease progression is gradual, like in human ADPKD. This finding may have implications when planning the dosage to be given to patients as total blockage of KCa3.1 may not be necessary.F. Apparent renal plasticity is demonstrated in metanephroi

[0164] The rescue of the cystic phenotype by Senicapoc treatment suggests Senicapoc could rescue established cysts ex vivo. Senicapoc was added to the metanephroi culture two days after cyst induction by cAMP, which allowed us to observe the evolution of individual cysts. When the same cyst was followed before and after introduction of Senicapoc, it was found that cyst size was reduced immediately on the following day, and the cysts were almost completely resorbed in just two days. Administration of Senicapoc thus not only prevented cyst growth, but also induced regression in cyst size of preformed cysts. It has previously been reported by Dong et al. using a genetic approach of Pkd re-expression that, a high degree of renal plasticity is retained by ADPKD kidneys in vivo. In line with these results, renal plasticity was observed here in an ex vivo context using a pharmacologic KCa3.1 channel inhibitor. This implies that the treatment of ADPKD patients with Senicapoc after the disease is established could still be beneficial and may lead to regression of pre-existingAttorney Docket No. 063697-504001WO cysts. Senicapoc may thus be effective not only as a preventative measure to delay cyst growth but also as a treatment for established cysts.G. cAMP levels are modulated by KCa3.1

[0165] We continued our investigation in vivo to identify other cyst-mediating processes KCa3.1 may be implicated in. Given the critical requirement of cAMP for biological processes and the prevalence of KCa3.1 in many pathophysiological conditions, the reduction in renal cAMP in both PkdlckoandSBPkdl upon loss of Kcnn4 is intriguing and worth investigating in the future as this could reveal a new mechanism by which cAMP levels may be modulated. cAMP levels are generally believed to be regulated by the balance between activities of two enzymes: adenylyl cyclase (AC) and cyclic nucleotide phosphodiesterase (PDE). Although there appears to be no direct link between the activity of an ion channel and the modulation of a second messenger, cAMP and KCa3.1 in fact share common features that may be facilitating this process. Firstly, both closely associate with MAPKZERK signaling. AC and PDE activities are known to be regulated by numerous signaling pathways, including MAPKZERK (Sassone-Corsi, 2012). Our data also show that genetic Kcnn4 inactivation can dampen MAPKZERK signaling. Inactivation of Kcnn4 can thus reduce AC and PDE levels through downregulation of MAPKZERK signaling and, consequently lower cAMP levels. In this case, AC and PDE levels would most likely be reduced in parallel since AC6 mediated-cAMP activation of PKA in turn can stimulate PDE4 (Agarwal et al., 2019). Secondly, both KCa3.1 and cAMP are tightly associated with calcium. KCa3.1 drives calcium entry, therefore facilitating calcium signaling by sustaining hyperpolarization, while AC6 and PDE4 levels are also regulated by calcium (Halls & Cooper, 2011; Wang et al., 2010). Lowered calcium signaling due to KCa3.1 inactivation could consequently modulate levels of AC6 and PDE4 such that cAMP production may be reduced. Finally, cAMP has been described by Sassone-Corsi to interact with ion channels. cAMP can bind to and modulate the function of a family of cyclic-nucleotide-gated ion channels permeable to calcium, potassium, and sodium. The calcium that is permitted to flow can in turn modulates cAMP production by regulating activity of ACs and PDEs. Since both V2R and Kcnn4 are localized on the basolateral membrane (Klein et al., 2009; Robben et al., 2004), it is also possible that they interact, and through this interaction KCa3.1 may modulate cAMP levels.Attorney Docket No. 063697-504001WOH. KCa3.1 is involved in renal proliferation, fibrosis, primary cilia elongation and influences kidney function and survival

[0166] As KCa3.1 has demonstrated to be pro-proliferative and profibrotic in other contexts (Anumanthan et al., 2018; Bi et al., 2013; Cruse et al., 2006; Huang et al., 2014; Paka et al., 2017; Yu et al., 2013), and as another potassium channel from the same family can cause elongation of primary cilia (Nam et al., 2022) in vitro, those parameters were evaluated in the present murine models of ADPKD. The elevation in proliferation, fibrosis, and primary cilia length observed in PkdlckoandSBPkdl were indeed partially, and sometimes even completely, normalized, upon genetically inactivating Kcnn4. Although ciliary length modulation is a dynamic process, the high overlap of renal primary ciliary distribution of P10 versus 2-month-old wildtype suggest that the primary cilia length is stably maintained in healthy conditions. While the functional role of primary cilia and its length is still being investigated, based on the findings of Shao et al. where reduction of primary cilia length impeded cyst formation and that the length in small cysts of human ADPKD kidneys correlated with cyst size (Shao et al., 2020), the observed shortened primary cilia following loss of Kcnn4 in our mouse models very likely contributed to the amelioration of cystic phenotypes. More importantly, a new mechanism was revealed in which a non-IFT machinery component can modulate primary cilia length, supporting the recent report by Nam et al. where they showed KCa2.3 channel can increase primary cilia length (Nam et al., 2022). These results clearly demonstrated that KCa3.1 is strongly implicated in these cystmediating processes. Although Kcnn4 inactivation was insufficient to limit kidney functional decline or improve survival in Pkdlckogiven the rapid progressivity of the disease, Kcnn4 inactivation even partially rescued kidney function and extended lifespan by almost 40% in the much more gradual progressiveSBPkdl model.I. Other potassium channels could be compensating for loss of Kcnn4

[0167] Treating PkdP ~; Kcnn4~ ~ metanephroi with cAMP did not completely abolish cysts like it has been observed in PkdPB; Cftr ^ metanephroi in a study carried out by Magenheimer et al. This suggests that other potassium channels may be compensating for the absence of KCa3.1. The less effective rescue m ' PkdP ~; Kcnn4~ ~ metanephroi may indicate that KCa3.1 is downstream of Cftr and provides the driving force for continuous chloride transport through Cftr, but is probably not the only source, since inactivation does not completely prevent cyst formation. The partial rescue of various cyst-mediating events byAttorney Docket No. 063697-504001WOKcnn4 inactivation in Pkdlcko; Kcnn4~ ~ andSBPkdl; Kcnn4~ ~ models also support involvement of other potassium channels. Indeed, there is evidence for a compensatory mechanism between KCa3.1 and Kvl.3 in T cells (Chiang et al., 2017).J. Delivering Senicapoc in DMSO solvent by intraperitoneal or subcutaneous injection is lethal for young and aged mice

[0168] Senicapoc was administered to both early-onset and adult-onset mouse models hoping that targeting KCa3.1 would be effective for both models. Other in vivo studies utilizing Senicapoc generally administer through diet and sometimes oral gavage. The study began with intraperitoneal or subcutaneous injection, as this method would be most convenient, and the exact volume injected into the animals can easily be controlled. Unfortunately, neither the adult nor the young animals tolerated the injections well and most died within 1 week after treatment initiation. Administration was then attempted through oral gavage. Animals tolerated oral gavage much better and were able to complete the treatment. In contrary to injections, where animals lost weight immediately after the first administration, animals gained weight when treated orally, and in most cases were comparable to or exceeded those treated with the vehicle solution only.

[0169] Due to the smaller window for treatment it was decided to introduce Senicapoc as early as possible for the early-onset rapid progressive models. It was first attempted delivering Senicapoc by subcutaneous injection. However, like adults, young animals did not tolerate this route of administration, perhaps due to the solvent (DMSO). Importantly, a previous in vivo study utilized DMSO solvent for short term subcutaneous injection of Cftr inhibitor into newborn mice but did not report any issues (Yang et al., 2008). Therefore, the lethality in the models might be caused by the combination of Senicapoc in DMSO solvent.

[0170] Since direct oral gavage is not possible in the newborns, Senicapoc was administered to the mothers as soon as pups are born. l-2mL of very concentrated Senicapoc (15mg / mL) that is equivalent to 120mg Senicapoc / kg of bodyweight / day was also administered twice daily, directly in the comer of their mouth, which they ingested from P2 onwards. Senicapoc ingestion was found tolerable for newborn animals with no body weight loss when compared to littermates, and all animals completed treatment.K. Senicapoc is more effective in adult-onset models

[0171] Aside from the kidneys, no macroscopic anomalies were found in any organs after completion of treatment in bothSBPkdl and Pkdlwtanimals.SBPkdl kidneys wereAttorney Docket No. 063697-504001WO indistinguishable between the different treatment groups. In contrast, Pkdlwtkidneys treated with Senicapoc appeared pinker in colour which may suggest improved morphology and function, as was seen in the geneticSBPkdl; Kcnn4~ ~ model. Based on experiences examining kidneys at different disease stages in various mouse models differing in rate of progressivity, more intense kidney color tends to associate with healthier blood supply and more intact tissues. The observation of an increased colour intensity of the kidney appears to be paralleled by the significant improvements in cyst indices leading to partial normalization of kidney function. Although, those improvements are less pronounced in theSBPkdl model, KBW is reduced upon Senicapoc treatment and there is a tendency for cyst indices to be decreased. Comparison between treatedSBPkdl and Pkdl"' animals shows that Senicapoc is more effective towards in the more moderate progressive Pkdl"' model. The effect of Senicapoc appears to be gradual, as an effect is often only observed after daily long-term administration in other in vivo studies and in clinical trials (Ataga et al., 2011; Ataga et al., 2008; Jin et al., 2019; Paka et al., 2017). This frequent treatment regime is imposed by the molecule’s short half-life. Therefore, it makes sense that a bigger effect was found in Pkdl ' where the phenotypes develop more slowly during its longer lifespan. Likewise, it was expected therefore to observe a better improvement of kidney parameters in the relatively more moderate of the two early-onset group models, the Pkdlv / vmodel.

[0172] It was postulated that in the very rapidly progressive Pkdlcko, it may be too late to observe any beneficial effect from Senicapoc treatment if analyzed at P10, so the treatment schedule was modified to end at P5 for the Pkdlckoanimals. Pkdlv / vmice were treated until P10 to allow cysts to develop, as in this model tubule dilations are observed to only occur from P4. In line with the present hypothesis, it was indeed observed a more pronounced improvement in the more moderate Pkdlv / vmodel. Pkdlv / vkidneys appeared smaller macroscopically and had smaller KBW after Senicapoc treatment. However, Pkdlckokidneys treated with Senicapoc even increased in size and slightly higher KBW ratios were observed than for the vehicle-treated animals. Several groups had reported PEG and Cremophor could increase kidney weight in rats, but none had studied this effect in the mice before (Hermansky et al., 1995; Ikeda et al., 2006; Stokes et al., 2013). Here it is reported that mouse kidneys can also increase in weight after PEG and Cremophor administration, since heavier kidneys were observed in vehicle treated animals, when compared to untreated ones (data not shown). Those who received the vehicle solution had heavier kidneys compared to those without vehicle. Remarkedly, Pkdlv / v~ animals treated with Senicapoc overcame thisAttorney Docket No. 063697-504001WO increase in weight induced by the vehicle solution and even reduced KBW. Images of H&E- stained kidney sections show that Senicapoc treatment not only reduced Pkdlv / vkidneys in size, but the kidneys also appeared notably less cystic than those of vehicle treated mice. The improvement in cyst indices is more prominent in the Pkdlv / vmodel than in the Pkdlckomodel. Using the currently available tools, better rescue was found by Senicapoc in the adult- than in the early-onset models. Measuring plasma Senicapoc levels using techniques such as UHPLC-ESI-MS / MS reported by Sorensen el al. (Sorensen et al. 2021) would help understand if the route of administration or the short half-life of Senicapoc played a role in the observation that Senicapoc treatment is more efficient in reducing cyst parameters in the adult- than in the early-onset models of ADPKD. Having such data would also facilitate optimization of the administration protocols. Another option to try to increase the efficient dose of Senicapoc delivered to the kidneys would be to administer higher doses. However, in our hands Senicapoc is insoluble in concentrations exceeding 15mg / mL in the vehicle solution. One way to overcome this barrier could be to administer Senicapoc using kidney- targeted nanoparticles, such as used by the Chung lab (Cox et al., 2023), that has become popular in recent years for more efficient drug delivery.L. Genetic inactivation of Kcnn4 is more efficient than KCa3.1 pharmacologic inhibition

[0173] It is generally expected to observe a greater rescue by genetically targeting Kcnn4 than by use of the pharmacologic approach, as the former is considered to block the target gene more effectively and with fewer off-target effects. However, metanephroi culture, which removes kidneys out of the in vivo context, may not behave the same way as in vivo, which may contribute to their contrasting behaviour. When the conventional usage of lOmicroM of Senicapoc or below was used, an approximately 2-fold greater rescue was observed in cyst indices by genetic Kcnn4 inactivation than by pharmacologic inhibition. However, when a greater dosage of Senicapoc (20microM) was introduced, i.e. more drug was exposed to the metanephroi and allowed to penetrate the culture, the opposite effect is observed, where Senicapoc treatment results in almost complete abolishment of cysts. As Senicapoc, at nanomolar concentrations, is highly specific to KCa3.1 over other potassium channels (Stocker et al., 2003), it can be ruled out that the possibility that Senicapoc is non-specifically blocking other potassium channels simultaneously. Another explanation may be that Senicapoc is inhibiting other processes, such as proliferation as observed in animal studies ofAttorney Docket No. 063697-504001WOAlzheimer’s (Jin et al., 2019), in addition to blocking the KCa3.1 channel pore. This is relevant especially in the isolated metanephroi culture where other factors normally found in vivo are absent. This could be tested by analyzing cyst epithelial cell proliferation levels in Pkdl / -; Kcnn4~ ~ metanephroi and comparing them to PkdP ~ metanephroi treated with Senicapoc.

[0174] A more relevant in vivo comparison would be to examine kidneys of P5 Pkdlcko; Kcnn4~ ~?Lmm3i s with kidneys from P5 Pkdlckomice treated with Senicapoc. Compared to the small tendency for rescue from Senicapoc treatment this rapid progressive model in vivo, the improvements in kidney morphology and cyst indices in double transgenic Pkdlcko; Kcnn4~ ~ animals are significantly more pronounced. This is consistent with the general consideration that genetic inactivation is more efficient than pharmacological inhibition. The lack of improvement observed upon pharmacological Senicapoc treatment may be attributed to ineffective absorption of Senicapoc or an insufficient fraction of the molecule reaching the kidneys due to its short half-life and the high metabolic rate in newborn mice. In the adultonset murine models, a higher improvement in disease parameters was equally observed in double transgenicSBPkdl; Kcnn4~ ~ mice, than inSBPkdl animals treated with Senicapoc. Cyst analysis, KBW and kidney functional assay consistently improved to larger extent by genetically targeting Kcnn4. However, the difference in efficiency between the two approaches is less prominent in the late-onset models, than in the rapid progressive Pkdlckomodel. This suggests that Senicapoc may be more effective as a long-term treatment for a moderate progressive disease phenotype, as its effects appear gradual and therefore would be more appropriate for treatment of slowly progressing cases of the disease.M. Dual inhibition of KCa3.1 and Cftr cause synergistic rescue of cysts ex vivo

[0175] Combination therapy may help to achieve higher beneficial effects with lower absolute treatment doses. There have been several attempts to find promising partner therapeutics to delay disease progression in vivo (Kanhai et al., 2020; Leonhard et al., 2019). Given time constraints, potential partners were evaluated for combination therapy ex vivo that could be tested in vivo in the future. PkdPBmetanephroi was first treated with Senicapoc, PPQ-102 (Cftr inhibitor), CaCCinh-AOl (Tmeml6a inhibitor) individually. Senicapoc was then tested with each of the latter two compounds (i.e. in combination with each) at different concentrations. While carrying out this experiment, it was found, contrary to previous metanephroi studies in which drugs were generally tested at maximal concentrations of 10 microM (Magenheimer etAttorney Docket No. 063697-504001WO al., 2006; Snyder et al., 2011; Yang et al., 2008), that higher concentrations of tested drugs were well-tolerated without additive or toxic effect if prepared in higher stock concentration such as 20mM to achieve final concentration of 20microM when used at 0.1% dilution in vehicle. Synergy was demonstrated in PkdP2' metanephroi treated with Senicapoc and PPQ- 102, such that simultaneous treatment with low concentrations of each compound resulted in a higher degree of phenotypic rescue than the sum of the degree of rescue by individual drugs at the same concentrations. This observation suggests that KCa3.1 and Cftr may be part of the same pathway such that dual inhibition enhances rescue of cyst growth and / or number. One advantage of synergistic inhibition is, that it allows lower total dosages to be given and thus reduces likelihood of adverse events. As ADPKD patients may likely require long term treatment, limiting toxicity will improve safety, a highly important feature of any treatment.EXAMPLE 7: GENERAL METHODSA. Mouse Models1. Mouse lines and genotyping

[0176] Mouse lines used in this study were generated previously by our lab and by others. All animal manipulations were performed in line with all ethical regulations and conducted with the approval by the Institut de Recherches Cliniques Animal Care Committee, in compliance with the Canadian Council of Animal Care ethical guidelines for animal experiments.

[0177] Endogenous Pkdl mutant models: The homozygous Pkdl knockout mutant mouse model PkdltmJSom(Pkdl2) contains a selectable neo cassette inserted into exon 1 of the Pkdl gene, producing no detectable transcript or partial protein products (Wu et al., 2002). The renal Pkdl conditional knockout mouse model C57BL / 6-Tg(Cdhl6-cre)91Igr; Pkdltm2Ggg(Pkdlcko) was produced by homologous recombination of a neomycin cassette flanked by FRT sites where two loxP sites were inserted at intron 1 and at intron 4 of the Pkdl gene, causing the deletion of exon 2-4 upon expression of Cre recombinase (Piontek et al., 2004). The non-cleavable knockin Pkdl mutant PkdltmJJFqJ (Pkdlv / v) model contains a T3041V mutation that prevents cleavage of the PCI protein at its GPS domain (Yu et al., 2007).

[0178] Pkdl transgenic models: In the renal Pkdl dosage increase murine model C57BL / 6J- Tg(Pkdl*)39MtruBPkdl), the endogenous Pkdl promoter was replaced with the “SB” renal-specific promoter, inserted upstream of xe. Pkdl initiation codon. This randomly inserted transgene gives rise to a 9- to 15-fold increase in renal Pkdl levels (Thivierge et al., 2006). The global Pkdl dosage increase model C57BL / 6J-Tg(Pkdl)26Mtru (Pkdlwt) wasAttorney Docket No. 063697-504001WO generated similarly, from a modified Pkdl- AC, in this case containing all WiQ Pkdl endogenous proximal regulatory elements, to promote sustained increased wildtype Pkdl expression within the native tissue following its endogenous temporal regulation. In this model, Pkdl expression is also increased by 9- to 15-fold in the kidneys and the animals show both renal and extrarenal anomalies (Kurbegovic et al., 2010). Both the renal -targeted and the systemic Pkdl dosage increase models carry a silent tag (G2355A) introduced via homologous recombination into exon 10 of the Pkdl transgene, giving rise to a new EcoRI restriction site.

[0179] To generate double Pkdlcko; Kcnn4~ ~ andSBPkdl; Kcnn4~ ~ mutant mouse models, PkdlckoandSBPkdl were first outcrossed with Kcnn4~ ~. Kcnn4~ ~ mice Kcnn4tmlJemn) were generated by deletion of exon 1 upon insertion of a neo cassette replacing 1.7kb surrounding and including exon 1. Mice show normal appearance and fertility (Begenisich et al., 2004). Fl animals were then intercrossed, and the offspring were analyzed by PCR to select the genotype of interest.

[0180] For all employed models, to prepare samples for PCR genotyping, a piece of tail from each mouse was digested overnight with pronase (20mg / mL; Roche, Ref.: 11459643001) at 55°C and DNA extraction was carried out the next day. Briefly, the digested tails were centrifuged for 5 minutes at 13,000 rpm and the supernatant was transferred to a fresh tube. 35pL of sodium acetate (3M, pH 6.9) and 450pL of 100% ethanol were added and the tubes were vigorously agitated. The precipitated DNA was then pelleted 5 minutes at 13,000 rpm and the pellet was washed with 200pL of 70% ethanol. The DNA pellet was then left to air dry and subsequently resuspended in 50pL of TE buffer (lOmM Tris-Cl pH 8.0, ImM EDTA pH 8.0). Primers used for PCR genotyping are provided in Table 2. To determine tagged transgene presence when relevant, an EcoRI digestion was carried out following the PCR reaction.Table 2 - List of Primers used for PCR GenotypingAttorney Docket No. 063697-504001WO2. Sample collection and analysis

[0181] Mouse kidney and blood samples were collected at two ages that serve to represent early and later stages of the disease: P5 or PIO for Pkdlckoand 1-2 months or 6-8 months forSBPkdl. Samples from Pkdlcko; Kcnn4~ ~,SBPkdl; Kcnn4~ ~, and age-matched wildtype controls were collected in parallel. Kidney and body mass were measured for the calculation of the kidney weight to body weight ratio (KBW). Each isolated kidney was sectioned in half. Two halves were snap-frozen in liquid nitrogen and stored at -80°C for western blot analysis, cyclic Adenosine Monophosphate (cAMP) quantification, and RNA analysis. The remaining two halves were fixed in 4% formalin overnight and then processed in the Citadel Tissue Processor for embedding in paraffin blocks. Kidney paraffin blocks were sectioned at 4mm thickness using the microtome (Leica RM2145) for subsequent hematoxylin and eosin (H&E) staining, Kcnn4 in situ hybridization, and immunohistochemistry. Blood samples were collected at experimental endpoints, centrifuged for isolation of plasma, and stored at -80°C for blood urea nitrogen (BUN) assessment. A separate group of PIO Pkdlckoand Pkdlcko; Kcnn4~ ~, together with 2-month-oldSBPkdl andSBPkdl; Kcnn4~ ~ were anesthetized for kidney perfusion, after which kidneys were cut in half and stored in Tissue-Tek optimal cutting temperature (OCT) compound (Sakura Finetek, Ref.: 2580274) at -80°C for primary cilia staining and analysis. For quantification of renal Kcnn4 expression and protein levels of key components of the mitogen-activated protein kinases / extracellular signal-regulated kinases (MAPK / ERK) pathway, a group of PIO Pkdlv / vand 6-8-month-old Pkdl'!kidneys were also processed as described above. All image analyses were performed blinded with respect to the genotypes.3. Metanephroi culture and treatment plan

[0182] To generate PkdPBmetanephroi, Pkdl+ / ~ breeders were set up and checked daily for the presence of a vaginal plug. Any presence of a vaginal plug was noted as E0.5. Two weeks later, E14.5 embryos were isolated from pregnant Pkdl+ / ~ females. A piece of tail was removed from each embryo for DNA extraction as described above. During PCR genotyping, embryos were stored individually in cold phosphate buffered saline (PBS) on ice. Pkdl+ / +and Pkdt^ embryos were selected and metanephroi were isolated under the dissection microscope and then placed on 0.4mm Falcon cell culture inserts (Fisher, Ref.: 08-770).Attorney Docket No. 063697-504001WOInserts carrying metanephroi were then put into 12 well tissue culture plates. Media (400mL) consisting of DMEM:F12 (Wisent, Ref.: 319-075-CL) supplemented with sodium bicarbonate (Sigma, Ref.: S5761-500G), HEPES (Fisher, Ref.: 15630106), ITS universal culture supplement (Wisent, 315-080-XL), prostaglandin E2 (Sigma-Aldrich, P5640), penicillin and streptomycin (ThermoFisher, Ref.: 15070063) were added below all inserts into the wells. E14.5 was denoted as day 0 of metanephroi culture. On day 1, the media was replaced with fresh media containing 8-Br-cAMP (Sigma, Ref.: B7880-25MG) and supplemented with or without the following reagents: KCa3.1 activator SKA-111 (Aobious, Ref.: AOB6599), KCa3.1 inhibitors Senicapoc (BOC Sciences, Ref.: B2693-089474) and TRAM-34 (Tocris, Ref.: 2946), Cftr inhibitor PPQ- 102 (Tocris, Ref.: 4303) and Tmeml6a inhibitor CaCCinh-AOl (Sigma, Ref.: 208293). The media was replaced again on day 3 with the supplemented reagents. Metanephroi culture was maintained in a 37°C humidified CO2 incubator at 5% CO2 until day 4. Pictures were taken daily with 4X objective in brightfield using Zeiss Axi overt S100TV microscope. Cyst number per metanephros and percent cyst area were quantified after completion of the experiment using the Volocity software version 6.0 (Quorum Technologies Inc., Ontario, Canada).

[0183] To determine if Kcnn4 genetic inactivation alters PkdP ~ cyst growth, PkdP ~; Kcnn4~ ~ metanephroi were generated from Pkdl+ / ~; Kcnn4~ ~ breeders. Metanephroi were cultured and treated with 8-Br-cAMP as described above. To determine the response of established PkdP' cysts to Senicapoc, Senicapoc was added on day 3 instead of day 1 and cultured until day 5.B. Expression analysis1. RNA isolation and RT-qPCR

[0184] Human ADPKD kidneys were surgically removed from patients and stored at -80°C. Total RNA from human and mouse kidneys were extracted using the TRIzol (Fisher, Ref.: 15596026) - chloroform method (Rio et al., 2010). Extracted RNA were checked for quality on agarose gel, then reverse transcribed using Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Ref.: M1701) to cDNA for real-time quantitative PCR. Reactions were carried out in triplicate using the Applied Biosystems PowerUp SYBR Green master mix (Fisher, Ref.: A25741) to determine the expression of KCNN4, Kcnn4, Aqpl, Aqp2, Nkccl, Nkcc2, and Cftr. Analysis was completed using the QuantStudio Real-Time PCR Software. Results were expressed as fold increase from healthy human kidneys or age- matched wildtype mouse kidneys. RNA analysis was normalized to mouse ribosomal proteinAttorney Docket No. 063697-504001WO subunit 16 (SI 6) for quantification of mouse genes. KCNN4 expression in human ADPKD kidneys was normalized to HPRT. The primers used for determining RNA expression are listed in Table 3.Table 3. List of primers used for qPCR.2. RNA In situ hybridization

[0185] P5 Pkdlckoand 4-6-month-oldSBPkdl kidneys with age-matched wildtype kidneys were isolated and fixed overnight in formalin, embedded in paraffin, sectioned in 4mm thickness, and placed onto positively charged glass slides. RNA In situ hybridization was conducted using the Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, Ref.: 323100) following manufacturer’s instructions. Briefly, kidney sections were deparaffinized in xylene, re-hydrated in graded alcohols, and permeabilized with H2O2 to block endogenous peroxidase activity to allow the probe to access its target RNA. Samples were then hybridized with murine Kcnn4 probe (Advanced Cell Diagnostics, Ref.: 569381) to bind its target RNA. Hybridization signals were amplified with amplifier reagents followed by dye-labeled probes to facilitate the visualization of fluorescent signals. Images were acquired with Leica DM6 fluorescent microscope at 20X. Each punctate fluorescent white dot represents a single mRNA transcript.C. Kidney cyst characterization1. Cyst quantification

[0186] Formalin-fixed paraffin-embedded (FFPE) kidneys were sectioned at 4mm thickness and coloured with H&E for cyst quantification. Briefly, kidney slides were baked at 55°C forAttorney Docket No. 063697-504001WO30 minutes prior to deparaffinization in xylene. Slides were processed in serial graded alcohols to rehydrate samples, followed by incubation in hematoxylin solution (Fisher, Ref.: SH26-500D). To remove excess hematoxylin, slides were placed under running tap water, followed by incubation with eosin (Sigma, Ref.: E511-25). Excess eosin was removed by 95% ethanol followed by a quick dip in water. Slides were dehydrated under serial graded alcohols and then xylene before placing coverslips using Permount mounting medium (Fisher, Ref.: SP15-500). Images were taken with a Leica MZ12 microscope in brightfield mode. Quantifications of percent cyst surface area and cyst number per pm2of kidney surface were carried out with the Northern Eclipse software (Empix Imaging Inc., Ontario, Canada).2. Cyst tubular origin

[0187] FFPE kidneys were sectioned in 4mm thickness, deparaffinized in xylene, re-hydrated in graded alcohols, and washed in PBS. A heat-induced epitope retrieval step was carried out to break methylene bridges that formed during the fixation step, allowing exposure of antigenic sites for antibody binding. The slides were submerged in a preheated solution containing citric acid (Sigma, Ref.: C0759) and sodium citrate (EMD, Ref.: SX0445-3) and heated in a pressure cooker for a total of 15 minutes. Heated slides were cooled down under running tap water for 30 minutes, then incubated for 30 minutes in fresh 0. l%NaBH4 (Fisher, Ref.: 5678-10) solution to remove autofluorescence. The slides were washed with PBS between each step. Before proceeding to incubation with primary antibodies, samples were incubated for 20 minutes in blocking solution containing 1% bovine serum albumin (BSA) (Sigma-Aldrich, Ref.: A3059-100G), 10% normal goat serum (Vector Labs, Ref.: S-1000), and 0.1 mM CaC12 (Sigma-Aldrich, Ref.: C4901-100G) in PBS. The following antibodies were diluted in blocking solution and incubated overnight with samples at 4°C in the dark: Lotus tetragonolobus lectin (LTL, 1 :200; Vector Laboratories, Ref.: L-1320-5), Lycopersicon esculentum lectin (LEL, 1 : 100; Vector Laboratories, Ref.: L-l 170-2) and Dolichos biflorus agglutinin (DBA, 1 :50; Vector Laboratories, Ref.: RL-1032-2), which are specific markers for proximal tubule (green), distal tubule (blue) and collecting duct (red), respectively. On the following day, antibody solutions were removed, and the slides were washed with PBS.Lycopersicon esculentum lectin required an additional 2-hour incubation at room temperature in the dark with the secondary antibody AMCA Streptavidin diluted in blocking solution (1 :100; Vector Laboratories, Ref.: SA-5008-1). The slides were washed before adding ProLong Gold Antifade Mountant (ThermoFisher, Ref.: P36930) to seal the samples withAttorney Docket No. 063697-504001WO coverslips. Images were acquired using Leica DM5 fluorescent microscope. To quantify the percentage of cystic tubules within each nephron segment, wildtype kidneys were examined to determine the diameter threshold of non-cystic tubules within each segment using Volocity software by manual tracing. Any tubular diameter surpassing the threshold of wildtype tubules (40mm) was considered cystic. Results were expressed as the percentage of cystic tubules within each nephron segment.D. Protein Analysis1. cAMP measurement

[0188] cAMP levels in kidney lysates were detected using the Direct cAMP ELISA Kit from Enzo life sciences (Ref.: ADI-901-066 A) following the manufacturer’s instructions. Frozen mouse kidneys stored at -80°C were ground into fine powder under liquid nitrogen, weighed, and homogenized in 10 volumes of 0.1M HC1 to release cAMP, then centrifuged at 600 x g for 10 minutes to pellet the debris. The supernatant was used for the assay. To prepare the cAMP standards, the stock solution (at 2000 pmol / mL) was used for serial dilutions to obtain standards at 20, 5, 1.25, 0.312, and 0.078 pmol / mL concentrations. Apart from the 5 wells reserved for the standards, additional wells were dedicated for total activity (TA), blank, nonspecific binding (NSB), and 0 pmol / mL standard (Bo). To prepare the assay plate, 50uL of Neutralizing Reagent was first added to all wells except Blank and TA. lOOmL of 0. IM HC1 was then added into NSB and Bo wells. lOOmL of standards or samples were loaded to corresponding wells, followed by 50mL blue conjugate to all wells except Blank and TA. 50mL yellow antibody was added next to all wells except Blank, TA, and NSB. The plate was sealed upon completion of sample loading and incubated for 2 hours at room temperature at low speed (~500rpm). After incubation, all contents were emptied, and wells were washed. For conjugate-substrate reaction, 5mL of blue conjugate was added to the TA well, 200mL substrate solution was added to all wells and the plate was incubated again for 1.5 hours at room temperature with shaking. To stop the reaction, 50mL stop solution was added to all wells. The plate was read at 405nm. The average net OD of each well was calculated using the equation: “Average Net OD = Average OD - Average NSB OD”. The standard curve was plotted using the calculated average net OD. The concentration of cAMP in each sample was then extrapolated using the equation of the standard curve. To normalize for protein content, protein concentrations of all samples were measured using bicinchoninic acid (BCA) protein assay (ThermoFisher, Ref.: 23228). The final values were expressed as pmol cAMP per mgAttorney Docket No. 063697-504001WO of total protein, calculated by dividing cAMP concertation by the total protein concentration. 2. Protein extraction and western blot

[0189] Frozen kidney tissues were homogenized in 500pL ice-cold RIPA buffer (20mM Tris pH 7.5, 2mM EDTA pH 8, 150mM NaCl, and 0.5% Triton) supplemented with Phenylmethanesulfonyl fluoride (Sigma-Aldrich, Ref.: P7626) and protease inhibitor cocktail (Sigma- Aldrich, Ref.: P8340) using an electric Polytron homogenizer. The homogenates were centrifuged at 16,000 x g for 15 minutes at 4°C to pellet the debris. Supernatants were collected and stored at -80°C in aliquots. Protein concentration was quantified using BCA protein assay (ThermoFisher, Ref.: 23228) according to the manufacturer’s instructions. To initiate electrophoresis, equal amounts of protein extracts (40-45mg) were mixed with Laemmli sample buffer containing P-mercaptoethanol and denatured for 10 minutes at 95°C. Denatured samples were loaded onto hand-casted 8, 10, or 12% SDS-polyacrylamide gels along with a molecular weight marker and separated at ~80V until the 37kDa marker reaches about 1cm before the bottom of the gel. Proteins in the gel were transferred onto PVDF or nitrocellulose membranes overnight at 4°C using ~20V and blocked for a minimum of 6 hours at 4°C in PBS containing 0.1% Tween-20 (Sigma-Aldrich, Ref.: P1379) and 5% milk. Immunoblots were hybridized overnight with primary antibodies: rabbit anti-cellular myelocytomatosis oncogene (c-Myc) antibody(l : 1000; Abeam, Ref.: ab32072), mouse anti- phospho-p44 / 42 MAPK (Erkl / 2) antibody (1 : 1000; Cell signaling, Ref.: 9106), rabbit anti- p44 / 42 MAPK (Erkl / 2) antibody (1 : 1000; Cell signaling, Ref.: 4695), mouse anti-active b- catenin antibody (1 :2000; Millipore, Ref.: 05-665), anti-b-catenin antibody (1 :2000;Millipore, Ref.: 06-734), or mouse anti-GAPDH antibody (1 : 10000; Abeam, Ref.: ab8245), followed by 3 washes with PBS containing 0.1% Tween-20. Next, immunoblots were incubated with corresponding secondary antibodies: rabbit anti-mouse IgG (1 :5000; Sigma- Aldrich, Ref.: A9044) or goat anti-rabbit IgG (1 :2000; Sigma- Aldrich, Ref.: A0545) for 4 hours at 4°C and washed again. For signal development, immunoblots were incubated with Amersham ECL Prime western blotting detection reagent (Fisher, Ref.: 12316992) for 5 minutes and covered in transparent plastic wrap. Images were acquired in the darkroom using Kodak films. Quantification and analysis of protein levels were executed with the Imaged software (Schneider et al., 2012).E. ImmunohistochemistryAttorney Docket No. 063697-504001WO1. Proliferation assay

[0190] 4mm thick FFPE kidney sections were deparaffinized with xylene, re-hydrated with a series of graded alcohols, and washed with water. The samples were permeabilized with 10- minute incubation of 2% H2O2 and then washed in PBS. Heat-induced epitope retrieval was necessary to facilitate antibody binding, where slides immersed in pre-heated retrieval solution containing citric acid (Sigma, Ref.: C0759) and sodium citrate (EMD, Ref.: SX0445- 3) were heated in a pressure cooker for 15 minutes, then cooled under running tap water for 30 minutes. Slides were washed in PBS containing 0.02% Tween-20 (Sigma- Aldrich, Ref.: P1379) and then samples were blocked in PBS solution containing 0.02% Tween-20, 10% normal goat serum (Vector Labs, Ref.: S-1000), and 1% BSA (Sigma-Aldrich, Ref.: A3059- 100G) for 45 minutes in preparation for overnight 4°C incubation with the primary antibody, prepared by diluting anti-Ki67 antibody in blocking solution (1 : 100; Leica, Ref.: NCL- Ki67p). On the following day, the primary antibody was washed, and samples were incubated with biotinylated anti-rabbit antibody in blocking solution (1 :300; Vector Laboratories, Ref. : BA-1000-1.5) for 90 minutes at room temperature. Following this incubation, samples were incubated with Vectastain ABC-HRP reagent (Vector Laboratories, Ref.: PK-7100) for 30 minutes at room temperature. The slides were washed with PBS, then colored with DAB substrate kit (Vector Laboratories, Ref.: SK-4100) for detection of Ki67 signals. To complete the experiment, slides were dehydrated in a series of graded alcohols followed by xylene and finally covered with coverslips using Permount mounting medium (Fisher, Ref.: SP 15-500). Images were acquired with Leica MZ12 microscope in brightfield. Proliferation was quantified by manually counting the number of tubules with two or more Ki67-positive epithelial cells. The final values were expressed as the number of Ki67-positive nuclei per mm2of kidney surface, the latter was determined using the Northern Eclipse software.2. Kidney fibrosis

[0191] 4pm thick FFPE kidney sections were de-paraffinized in xylene, then re-hydrated in a series of graded alcohols. Sections were washed in distilled water for 5 minutes before 30 minutes of incubation with Sirius Red (Sigma- Aldrich, Ref.: 365548) dissolved in picric acid (0.1%; Sigma, Ref.: P-6744-1GA) prepared 24 hours in advance. After incubation with Sirius Red for 45 minutes, slides were rinsed with tap water for 3 minutes followed by graded alcohols for dehydration. Finally, slides were immersed in xylene and covered with coverslips using Permount mounting medium (Fisher, Ref.: SP 15-500). Images were takenAttorney Docket No. 063697-504001WO with a Leica MZ 12 microscope in brightfield mode. Sirius red binds to collagen I and IV and appears dark red compared to pink non-fibrotic surfaces. Kidney fibrosis was quantified using the Northern Eclipse software and expressed as the percent of collagen area within the entire kidney surface.3. Kidney perfusion and primary cilia immunostaining

[0192] To perfuse the kidneys, the mouse needed to be anesthetized with Avertin (2.5%) given by intraperitoneal injection at 0.0165 pL per g of mouse weight. Once anesthetized, the mouse was stabilized onto a platform to open the abdomen cavity and cut the diaphragm and rib cage to fully expose the heart. A 26G1 / 2 needle was inserted into the left ventricle of the heart and the following fluids (made in PBS) were perfused: 20mL PBS (IX), 50mL Paraformaldehyde (4%), and 20mL sucrose (10%). The heart should continue to pump throughout the perfusion procedure, while the color of the kidneys should gradually turn pale, being an indicator of perfusion efficiency and blood wash-out. After completion, kidneys were cut in half to check for proper perfusion, if successful the interior should be whitecolored. Kidneys were then placed in 10% sucrose for 1 hour at 4°C, then immersed in 30% sucrose for 3 hours at 4°C. Finally, the perfused kidneys were stored in OCT compound at - 80°C.

[0193] Perfused kidneys stored in OCT compound were sectioned at low temperature using a cryostat-microtome (Leica CM3050S) and stored at -80°C. Tris buffered saline (TBS) wash solution (0. IM Tris pH 7.5, 150mM NaCl) was prepared one day before the experiment using fresh IM Tris-HCl pH 7.5. Slides were allowed to thaw to room temperature prior to immunostaining (~30 minutes) and then washed in TBS followed by 30 minutes incubation in 0.1% NaBH4 (Fisher, Ref.: 5678-10) solution. Slides were washed again to remove residual NaBH4 and incubated in 1% SDS for 5 minutes before another wash. Blocking solution incubation followed and lasted for 45 minutes at 4°C in a sealed box. The blocking solution was prepared by diluting unconjugated goat anti-mouse IgG (1 :5; Jackson Immunoresearch, Ref.: 115-005-166) in a TBS solution consisting of 10% normal goat serum (Vector Labs, Ref.: S1000) and 1% BSA (Sigma-Aldrich, Ref.: A3059-100G). The slides were washed and incubated overnight (ideally for 14 hours) at 4°C with the primary antibody in a sealed box. The primary antibody was prepared by diluting mouse monoclonal antiacetylated tubulin antibody (1 : 1000; Sigma-Aldrich, Ref.: T7451) in the blocking solution. On the following day, slides were washed with TBS incubated with secondary antibody for 1Attorney Docket No. 063697-504001WO hour at 4°C in a sealed dark box. The secondary antibody was prepared by diluting goat antimouse IgG conjugated to Alexa Fluor 555 (1 :500; Invitrogen, Ref.: A28180) in the blocking solution. From this step onwards, slides must be protected from light. The slides were washed with TBS containing 0.02% Tween-20 (Sigma-Aldrich, Ref.: P1379) followed by TBS wash. Nuclei were colored with DAPI for 3 minutes at room temperature, washed with TBS and a rapid dip in water, and finally ProLong Gold Antifade Mountant (ThermoFisher, Ref.: P36930) was added to seal samples with coverslips. Images were acquired with Leica DM5 fluorescent microscope. To analyze primary cilia length, each primary cilia was manually traced using the Volocity software and sorted by length into corresponding intervals. The distribution of primary cilia length was plotted based on the percentage of primary cilia within each interval. The average primary cilia length was also displayed as a bar graph.F. Blood urea nitrogen measurement

[0194] Blood urea nitrogen (BUN) in mouse serum was measured using the QuantiChrom™ Urea Assay Kit from BioAssay Systems (Ref.: DIUR-100) following manufacturer’s instructions. Before assaying samples, a quick test was done to determine the dilution factor that should be used so that all values fall within the detection range. A dilution factor of 10 was sufficient, therefore, all serum samples were diluted 1 / 10 in distilled water prior to the assay. 5pL of samples, water blank and standard were loaded in duplicates in a 96 well clear bottom plate. The working reagent was prepared just prior to use by mixing equal volumes of reagent A and B so that 200pL can be loaded to each well. The plate was incubated for 20 minutes at room temperature with low speed shaking and was read at 520nm. Urea concentration was calculated with the equation below:BUN was converted from urea:BUN (mg / dL) = [Urea] 2.14.G. In vivo drug administration1. Treatment plan and sample collection

[0195] For treatment of the adult-onset Pkdl mouse models, histological kidney sections at different ages were examined to determine the age of treatment onset. Treatment initiated at 3 weeks inSBPkdl and at 6 weeks in Pkdl"' and C57. These times were chosen so that treatment began around the time of cyst initiation. Animals were separated into 3 treatment groups: vehicle (PEG / Cremophor), 30mg Senicapoc / kg of body weight in vehicle solutionAttorney Docket No. 063697-504001WO(low dose), and 120mg Senicapoc / kg of body weight in vehicle solution (high dose). Senicapoc was freshly prepared each week in the vehicle solution at 80: 10: 10 (Senicapoc:PEG:Cremophor) and administered daily to all animals by oral gavage for 3 months. Mice were monitored daily for health status and weighed weekly to monitor body weight change.

[0196] The early-onset Pkdl mouse models Pkdlckoand Pkdlv / vwere divided into 2 treatment groups: vehicle and 120mg Senicapoc / kg of body weight in vehicle solution. Animals received Senicapoc directly into the comer of the mouth using a pipette soon after birth (P2) and through milk from P0 until P5 for Pkdlckoor until PIO for Pkdlv / v. For direct oral administration, l-2pL was given twice daily equivalent to 120mg Senicapoc / kg of body weight / day. For Senicapoc administration through milk, mothers were given 120mg Senicapoc / kg of body weight once daily by oral gavage from the day Pkdlckoand Pkdlv / vwere born (P0).

[0197] All animals were sacrificed at the experimental endpoint.2. Sample Analysis

[0198] Upon completion of treatment at the experimental endpoints, all organs were examined macroscopically for adverse phenotypes. Blood samples were collected and processed for isolation of serum which was stored at -80°C. Due to reagent availability, only the high dose group in the two adult-onset models were assessed for BUN. The body and kidney mass were measured. Kidneys were half formalin-fixed and half snap-frozen and stored as described above.3. Statistical Analysis

[0199] All values and graphs are expressed as mean ± standard error of the mean (SEM). Comparison of data sets were conducted with the unpaired Student’s T Test except for the Kaplan-Meier curve which was analyzed by the log-rank test. All statistical analysis was carried out using GraphPad Prism version 8 (GraphPad Software, San Diego, California USA). The significance level was set to a P value of <0.05.

[0200] While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.Attorney Docket No. 063697-504001WOREFERENCESAgarwal, S. R., Fiore, C., Miyashiro, K., Ostrom, R. S., & Harvey, R. D. (2019). Effect of Adenylyl Cyclase Type 6 on Localized Production of cAMP by beta-2 Adrenoceptors in Human Airway Smooth-Muscle Cells. J Pharmacol Exp Ther, 370(1), 104-110. https: / / doi.Org / 10.l 124 / j pet.119,256594Albaqumi, M., Srivastava, S., Li, Z., Zhdnova, O., Wulff, H., Itani, O., Wallace, D. P., & Skolnik, E. Y. (2008). KCa3.1 potassium channels are critical for cAMP-dependent chloride secretion and cyst growth in autosomal-dominant polycystic kidney disease. 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V., Zhou, J. X., Magenheimer, B. S., Reif, G. A., Wallace, D. P., Georg, G.1., Jakkaraj, S. R., Tash, J. S., Yu, A. S. L., Li, X., & Calvet, J. P. (2022). The lonidamine derivative H2-gamendazole reduces cyst formation in polycystic kidney disease. Am J Physiol Renal Physiol, 323(4), F492-F506. https: / / doi.0rg / lO.l 152 / aiprenal.00095, 2022Swenson-Fields, K. I., Vivian, C. J., Salah, S. M., Peda, J. D., Davis, B. M., van Rooijen, N., Wallace, P., & Fields, T. A. (2013). Macrophages promote polycystic kidneyAttorney Docket No. 063697-504001WO disease progression. Kidney Int, 83(5), 855-864. https: / / doi.org / 10.1038 / ki.2Q12.446Thivierge, C., Kurbegovic, A., Couillard, M., Guillaume, R., Cote, O., & Trudel, M. (2006). Overexpression ofPKDl causes polycystic kidney disease. Mol Cell Biol, 26(4), 1538- 1548. https: / / doi.Org / 10.1128 / MCB.26.4.1538-1548.2006Tubman, V. N., Mejia, P., Shmukler, B. E., Bei, A. K., Alper, S. L., Mitchell, J. R., Brugnara, C., & Duraisingh, M. T. (2016). The Clinically Tested Gardos Channel Inhibitor Senicapoc Exhibits Antimalarial Activity. Antimicrob Agents Chemother, 60(1 ), 613-616. https: / / doi.org / 10.1128 / AAC.01668-15University, A., Aarhus University, H., Odense University, H., Aalborg University, H., & Hvidovre University, H. (2021, April 24). Senicapoc in COVID-19 Patients With Severe Respiratory Insufficiency . https: / / ClinicalTrials.gov / show / NCT04594668University of California, D. (2024, December). Senicapoc in Alzheimer's Disease. https: / / ClinicalTrials.gov / show / NCT048Q4241Wang, X., Gattone, V., 2nd, Harris, P. C., & Torres, V. E. (2005). Effectiveness of vasopressin V2 receptor antagonists OPC-31260 and OPC-41061 on polycystic kidney disease development in the PCK rat. J Am Soc Nephrol, 16(4), 846-851. https: / / doi.org / 10.1681 / ASN.200412109QWang, X., Ward, C. J., Harris, P. C., & Torres, V. E. (2010). Cyclic nucleotide signaling in polycystic kidney disease. Kidney Int, 77(2), 129-140. https: / / doi.org / 10.1038 / ki.20Q9.438Ward, C. J., Turley, H., Ong, A. C., Comley, M., Biddolph, S., Chetty, R., Ratcliffe, P. J., Gattner, K., & Harris, P. C. (1996). Polycystin, the polycystic kidney disease 1 protein, is expressed by epithelial cells in fetal, adult, and polycystic kidney. Proc Natl Acad Sci USA, 93(4),Wu, G., Tian, X., Nishimura, S., Markowitz, G. S., D'Agati, V., Park, J. H., Yao, L., Li, L., Geng, L., Zhao, H., Edelmann, W ., & Somlo, S. (2002). Trans-heterozygous Pkdl and Pkd2 mutations modify expression of polycystic kidney disease. Hum Mol Genet, 11(16), 1845-1854. https: / / doi.org / 10.1093 / hmg / l l.16.1845Wulfmeyer, V. C., Auber, B., Haller, H., & Schmitt, R. (2019). Comparison of Different Selectionstrategies for Tolvaptan Eligibility among Autosomal Dominant Polycystic Kidney Disease Patients. 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Claims

1. Attorney Docket No. 063697-504001WOWHAT IS CLAIMED IS:

1. A method of treating or preventing a cystic kidney disease in a subj ect in need thereof, the method comprising administrating a therapeutically effective amount of an inhibitor of calcium-activated potassium channel (KCa3.1 inhibitor) to the subject.

2. The method of claim 1, wherein the KCa3.1 inhibitor decreases a number of new cyst growth in the subject relative to an untreated subject.

3. The method of claim 2, wherein the KCa3.1 inhibitor decreases the number of new cyst growth in the subject by about 60% to about 95%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%, or about 85% relative to the untreated subject.

4. The method of any one of claims 1 to 3, wherein the KCa3.1 inhibitor decreases a number of cysts in the subject relative to an untreated subject.

5. The method of claim 4, wherein the KCa3.1 inhibitor decreases the number of cysts in the subject by about 10% to about 50%, about 10% to about 40%, about 20% to about 40%, about 25% to about 40%, about 25% to about 35%, or about 30%, relative to the untreated subj ect.

6. The method of any one of claims 1 to 5, wherein the KCa3.1 inhibitor decreases an average percent cyst area in the subject relative to an untreated subject.

7. The method of claim 6, wherein the average percent cyst area is decreased by about 30% to about 70%, about 40% to about 60%, or about 50% to about 60%, relative to the untreated subject.

8. The method of any one of claims 1 to 7, wherein the subject has kidney fibrosis and the KCa3.1 inhibitor reduces the kidney fibrosis.

9. The method of claim 8, wherein the kidney fibrosis is decreased by about 10% to about 60%, about 15% to about 55%, about 20% to about 45%, about 25% to about 40%, or about 30% to about 40%.

10. The method of any one of claims 1 to 9, wherein the KCa3.1 inhibitor is administered orally.

11. The method of any one of claims 1 to 10, wherein the subject is treated for about 1Attorney Docket No. 063697-504001WO day to about 30 days, about 1 day to about 20 days, about 1 day to about 15 days, about 2 days to about 7 days, or about 2 days to about 5 days.

12. The method of any one of claims 1 to 10, wherein the KCa3.1 inhibitor is administered chronically.

13. The method of any one of claims 1 to 12, wherein the KCa3.1 inhibitor is selected from senicapoc, clotrimazole, TRAM-34, NS6180, nifedipine, 4-Phenyl-4H-pyran or bicyclic hexadiene lactone, and a combination thereof.

14. The method of claim 13, where the KCa3.1 inhibitor is senicapoc.

15. The method of claim 13 or 14, where the senicapoc is administered at a dose of about 10 mg / kg to about 150 mg / kg, about 10 mg / kg to about 140 mg / kg, about 20 mg / kg to about 130 mg / kg, about 20 mg / kg to about 120 mg / kg, about 30 mg / kg to about 120 mg / kg, about 20 mg / kg to about 100 mg / kg, about 20 mg / kg to about 800 mg / kg, about 20 mg / kg to about 70 mg / kg, about 20 mg / kg to about 60 mg / kg, about 20 mg / kg to about 50 mg / kg, about 30 mg / kg to about 50 mg / kg, about 40 mg / kg to about 60 mg / kg, about 50 mg / kg to about 150 mg / kg, or about 60 mg / kg to about 120 mg / kg of body weight of the subject.

16. The method of any one of claims 1 to 14, wherein the KCa3.1 inhibitor is administered at a dose of about 5 mg to about 100 mg, about 5 mg to about 80 mg, about 5 mg to about 60 mg, about 5 mg to about 50 mg, about 5 mg to about 40 mg, about 5 mg to about 30 mg, about 5 mg to about 20 mg, about 5 mg to about 15 mg, about 5 mg to about 12 mg, or about 10 mg.

17. The method of any one of claims 1 to 16, wherein the KCa3.1 inhibitor is administered one daily, twice daily, or three times daily.

18. The method of any one of claims 1 to 17, wherein the KCa3.1 inhibitor is administered in combination with a second therapeutic agent.

19. The method of claim 18, wherein the second therapeutic agent is a Cftr inhibitor, a TMEM16a inhibitor, a vasopressin receptor antagonist or a combination thereof.

20. The method of claim 19, wherein the Cftr inhibitor is PPQ-102.

21. The method of claim 19 or 20, wherein the TMEM16a inhibitor is CaCCinh-AOl.Attorney Docket No. 063697-504001WO22. The method of any one of claims 19 to 21, wherein the vasopressin receptor antagonist is tolvaptan.

23. The method of any one of claims 1 to 22, wherein the KCa3.1 inhibitor is comprised in a pharmaceutical composition, the pharmaceutical composition comprising the Kcnn4 inhibitor and a pharmaceutically acceptable excipient.

24. The method of any one of claims 1 to 23, wherein the cystic kidney disease is polycystic kidney disease (PKD).

25. The method of claim 24, wherein the PKD is associated with a mutation in the polycystic-1 (PKDl) gene.

26. The method of claim 24 or 25, wherein the PKD is autosomal dominant polycystic kidney disease (ADPKD).

27. The method of claim 26, wherein the ADPKD is moderately progressive adult-onset ADPKD.

28. The method of any one of claims 1 to 27, wherein the subject is an adult.

29. Use of a therapeutically effective amount of a KCa3.1 inhibitor in the treatment or prevention of cystic kidney disease in a subject in need thereof.

30. Use of a therapeutically effective amount of a KCa3.1 inhibitor in the manufacture of a medicament for the treatment or prevention of cystic kidney disease.

31. The use of claim 29 or 30, wherein the KCa3.1 inhibitor is selected from senicapoc, clotrimazole, TRAM-34, NS6180, nifedipine, 4-Phenyl-4H-pyran or bicyclic hexadiene lactone, and a combination thereof.

32. The use of claim 31, wherein the KCa3.1 inhibitor is senicapoc.

33. A KCa3.1 inhibitor for use in the treatment or prevention of a cystic kidney disease.

34. The KCa3.1 inhibitor for use of claim 33, wherein the KCa3.1 inhibitor is selected from senicapoc, clotrimazole, TRAM-34, NS6180, nifedipine, 4-Phenyl-4H-pyran or bicyclic hexadiene lactone, and a combination thereof.

35. The KCa3.1 inhibitor for use of claim 33, wherein the KCa3.1 inhibitor is senicapoc.

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