Methods of treating and / or preventing polycystic kidney disease

Abstract

Methods for treating and / or preventing polycystic kidney disease (PKD) are disclosed. The methods can include administering one or more sigma- 1 receptor agonists to a subject in need thereof.

Description

[0001] METHODS OF TREATING AND / OR PREVENTING POLYCYSTIC KIDNEY DISEASE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Serial No. 63 / 710,866 filed October 23, 2024, and is incorporated herein by reference for all purposes.

[0003] BACKGROUND OF THE INVENTION

[0004] Autosomal-dominant polycystic kidney disease (ADPKD) is a common genetic disease that counts for 5-10% of kidney failure patients. The affected individuals who carry the genetic mutations develop fluid-filled cysts in the kidney that enlarge over time to destroy the kidneys. Mutations in PKD1 and PKD2 genes coding for polycystin-1 (PCI) and polycystin-2 (PC2), respectively, are responsible for the majority of ADPKD cases, featured by mitochondrial dysfunction, metabolic reprogramming, cell proliferation, and fluid-filled cysts in kidneys. The function of these genes and why mutations cause the disease remain elusive. There is a need to understand the function of PKD1 and PKD2 in ADPKD and develop effective treatments.

[0005] SUMMARY OF THE INVENTION

[0006] In one aspect, a method for treating and / or preventing polycystic kidney disease (PKD) is provided. The method can include administering one or more sigma- 1 receptor agonists to a subject in need thereof.

[0007] In another aspect, a method for reducing a number of cysts in a kidney of a subject in need thereof is provided. The method can include administering one or more sigma-1 receptor agonists to a subject in need thereof.

[0008] BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figures 1A-1ES. Kidney histology and mitochondrial morphology changes in Pkd2 mice at cystic and pre-cystic stages. (A) Hematoxylin and eosin-stained images of kidney sections from Pkd2-cKO mice at pre-cystic and cystic stages in comparison to control mice. Scale bars, 5 mm. (B) (C) (D) Parameters of mitochondrial morphology: area, perimeter, roundness, cristae density and cristae number in control mice as compared to that in Pkd2-cKO mice at pre-cystic and cystic stages. Mitochondrial roundness is represented by 0-1 scale, where 0 refers to a straight line and 1 as a perfect circle. Data include >150 mitochondria from 3 mice. At least 50 mitochondria (from multiple images) per mouse were analyzed. Images were from PTs. Data are shown as mean ± SEM. Unpaired student t-test with p< 0.05 is considered significant. (E) Representative TEM images of proximal tubules from kidneys of control and Pkd2-cKO mice at pre-cystic and cystic stages. Upper panel, at lower magnification; lower panel, at higher magnification. Arrows denotes normal mitochondria; arrowheads, abnormal mitochondria with indistinct cristae; asterisk, cystic cavity. Scale bars, low magnification- 10pm and high magnification- 2pm.

[0010] Figures 2A-2B. TricB expression rescues mitochondrial morphological changes in Pkd2-cKO mice. (A) and (B) represent morphological parameters of mitochondria that are rescued by Tg-TricB in Pkd2-cKO mice at both cystic and pre-cystic stages, respectively. Data include >150 mitochondria from 3 mice. At least 50 mitochondria (from multiple images) per mouse were analyzed. Images were from PTs. Data are shown as mean ± SEM. Unpaired student t-test with p< 0.05 is considered significant.

[0011] Figure 3A-3C. 3D quantitative analysis of mitochondrial volume and density by serial block face scanning electron microscopy reveals altered mitochondrial structure in Pkd2-cKO mice kidneys. (A) Histograms showing distribution frequency vs mitochondrial volume (pm3) for wildtype (WT) control, pre-cystic, cystic mice with or without TricB, respectively. (B) Mean mitochondrial volume in the kidneys of corresponding mice groups. Each data point in the bar graph represents a single mitochondrion. Volume analysis of mitochondria was carried out from 20 serial block SEM images of each mouse. (C) Mean mitochondrial density in the mice groups are presented as number of mitochondria per pm2. Mitochondrial density was carried out from five randomly selected areas within a SEM image and total three images for each mouse. Data are shown as mean ± SD. Significance for volume analysis was calculated through two-tailed Mann-Whitney test and for density analysis through one-way ANOVA with post-hoc Tukey test. p< 0.05 is considered significant.

[0012] Figures 4A-4E. Mitochondria-associated ER membrane (MAM) structural alterations in Pkd2-cKO mice is mitigated by TricB expression. (A) (B) (C) TEM images illustrating altered MAMs structure in Pkd2-cKO mice in comparison to control mice. Tg-TricB expression reverses structural changes in Pkd2-cKO at both pre-cystic and cystic stages. Scale bars, 500 nm. (D) mean MAMs distance, length and coverage in control, pre-cystic, and cystic Pkd2-cKO mice. (E, F) Effect of TricB expression on Pkd2-cK0 cystic (E) and pre-cystic kidneys (F). Only those contact sites with a maximum distance of 30nm between ER and mito were used for the comparison. Data include 75-100 mitochondria from 3-4 mice. At least 25 mitochondria (from multiple images) per mouse were analyzed. Images were from PTs. Data are shown as mean ± SEM. Unpaired student t-test with p< 0.05 is considered significant.

[0013] Figures 5A-5B. Reversal of MAMs alteration by TricB in Pkd2-cKO mice analyzed by proximity ligation assays (PLA). Representative in situ PLA images illustrating IP3R-VDAC interactions corresponding to ER-mito contacts as red dots in the kidney tissue sections. Intensity and numbers of PLA signals (red dots) is proportional to IP3R-VDAC interactions. Dapi staining for nuclei. In comparison to control mice, number of red dots reflecting ER-Mito contact sites is reduced in Pkd2-cKO at both cystic (A) and pre-cystic stages (B) and is rescued by TricB expression. Bar graphs: PLA signals as relative mean intensity from at least 3 images per mice from 3 mice in each group. Data are shown as mean ± SEM. Unpaired student t-test with p< 0.05 is considered significant. Scale bars, 20 pm.

[0014] Figures 6A-6C. Alterations of mitochondrial membrane potential, calcium concentration and content in proximal tubules isolated from Pkd2-cKO mice. (A) Phase contrast (DIC) and TMRM fluorescence (Cy3 channel) images of live renal tubules incubated in HBSS buffer containing lOnM TMRM are presented. MitoG is a mitochondrial marker. (B) Phase contrast (DIC) and Rhod2 fluorescence images of live renal tubules. Non-cystic proximal tubules were isolated from dissected kidneys of control or Pkd2-cKO mice with or without TricB at cystic stages. Scale bars, 200 pm. Bar graphs are mean ± sem of 3 mice. Data from each mouse are averaged from at least 10 separate measurements. (C) Mean ± sem of calcium contents of mitochondria isolated from control, Pkd2-cKO pre-cystic, and cystic kidneys with or without TricB expression.

[0015] Figures 7A-7B. Oxygen consumption rate (OCR) in proximal tubules (PTs) freshly isolated from control, Pkd2-cKO with or without TricB expression. (A) Non-cystic tubules from cystic kidneys. (B) Tubules from pre-cystic kidneys. Left panels are representative line tracings from one experiment. Data points in each of 4 groups represent mean ± SD of 5 wells with each well containing 9-10 freshly dissected PTs. OCR values are normalized to 18S rRNA measured by rt-PCR. Bar graphs on the right panels represent mean ± SEM of >10 single experiments. Describe about O, FCCP and R / A, and how basal and maximal respiration are defined. Unpaired student t- test with p< 0.05 is considered significant.

[0016] Figures 8A-8B. Expression levels of genes modulating mitochondrial biogenesis, dynamics and ER-mitochondria connection in Pkd2-cKO mice. (A, B) Quantitative real-time PCR analysis of mitochondria DNA copy number per cell, genes related to mitochondrial and MAMs function, miRNA 17-5p clusters in kidney tissues dissected from control and Pkd2-cKO ± TricB cystic (A) and pre-cystic stages (B). Data shown are mean ± SEM. Unpaired student t-test with p< 0.05 is considered significant.

[0017] Figures 9A-9F. TricB expression rescues cystic phenotypes in Pkdl-cKO mice kidneys. (A) H&E-stained kidney sections of control, Pkdlkf; Pax8-LC 1 , and PkdP7f; Pax8-LCl; Tg-TricB mice. Scale bars, 5mm. (B) Kidney to body weight ratio. (C) Cyst index. (D) Blood BUN levels. (E, F) Fold changes of genes normalized to the control for cystic kidney (D) and pre-cystic kidneys (F). Data shown are mean ± SEM. Unpaired student t-test with p< 0.05 is considered significant.

[0018] Figures 10. Mitochondrial structural changes in Pkdl-cKO and effect of TricB expression. (A) representative TEM images. (B) Mitochondrial parameters in control, pre-cystic and cystic Pkdl-cKO kidneys. (C.D) Effect of TricB expression on pre-cystic (C) and cystic kidneys (D). Data include >150 mitochondria from 3 mice. At least 50 mitochondria (from multiple images) per mouse were analyzed. Images were from PTs. Data shown are mean ± SEM. Unpaired student t-test with p< 0.05 is considered significant.

[0019] Figures 11A-11E. Effect of TricB expression on ER-mitochondria connection in Pkdl-cKO mice. (A) MAMs distance, length, and coverage in Pkdl-cKO at pre-cystic and cystic stages in comparison to control. (B, C) Effect of TricB expression on MAMs structure in pre-cystic (B) and cystic kidneys (C). MAMs with a contact distance of < 30nm were analyzed. Data include 75-100 mitochondria from 3-4 mice. At least 25 mitochondria (from multiple images) per mouse were analyzed. Images were from PTs. Data are shown as mean ± SEM. Unpaired student t-test with p< 0.05 is considered significant. (D, E) Representative in situ PLA images illustrating IP3R-VDAC interactions corresponding to ER-mitochondria contacts as red dots in the kidney tissue sections. Intensity and numbers of PLA signals (red dots) is proportional to IP3R-VDAC interactions. Dapi staining for nuclei. In comparison to control mice, number of red dots reflecting ER-mitochondria contact sites is reduced in Pkdl-cKO at both pre-cystic (D) and cystic stages (E) and is rescued by TricB expression. Bar graphs: PLA signals as relative mean intensity from at least 3 images per mice from 3 mice in each group. Data are shown as mean ± SEM. Unpaired student t-test with p< 0.05 is considered significant. Scale Bar, 20 pm.

[0020] Figures 12A-12B. Oxygen consumption rate (OCR) in proximal tubules (PTs) freshly isolated from control, Pkdl-cKO with or without TricB expression. (A) Non-cystic tubules from cystic kidneys. (B) Tubules from pre-cystic kidneys. Left panels are representative line tracings from one experiment. Data points in each of 4 groups represent mean ± SD of 5 wells with each well containing 9-10 freshly dissected PTs. OCR values are normalized to 18S rRNA measured by rt-PCR. Bar graphs on the right panels represent mean ± SEM of >10 single experiments. Unpaired student t-test with p< 0.05 is considered significant.

[0021] Figures 13A-13B. Acute effect of pre-084 on OCR of proximal tubules isolated from WT and Pkdl-cKO kidneys. (A) Representative western blot of sigma-1 receptor (SIR) protein in MAMs fraction isolated from control (Ctrl), Pkdl-cKO cystic kidney ± TricB. Voltage-dependent anion channel (VDAC), a known protein localized to MAMs, is used as protein loading control. (B) OCR measurement in control and Pkdl-cKO proximal tubules (PTs) with or without Pre084. Freshly isolated PTs were incubated with or without pre-084 for 2 hrs before OCR measurement. Left panel shows representative line plot from one experiment. Data points in each of 4 groups represent mean ± SD of 5 wells with each well containing 9-10 freshly dissected PTs. OCR values are normalized to 18S rRNA measured by rt-PCR. Bar graphs on the right represent mean ± SEM of >3 single experiments. Unpaired student t-test with p< 0.05 is considered significant. Please note that the modest effect of pre-084 on Pkdl-cKO tubules (-20% increases) reflects short-term incubation in vitro.

[0022] Figures 14A-14B. Pre-084 reverses cystogenesis in Pkdl-cKO mice. (A) H&E-stained kidney sections of control (Ctrl), and Pkdl-cKO treated with saline (vehicle) or pre-084. Pkdl-cKO mice received daily intraperitoneal injection of pre-084 (1 mg / kg body weight) or saline at the same time of doxycycline induction. Kidneys were harvested at 12 weeks after induction. (B) cyst index. Paired student t-test with p< 0.05 is considered significant.

[0023] Figure 15. Epigenetic landscape in Pkdl-cKO and Pkd2-cKO and effect of TricB and MAMs activator. H3K27 acetylated histone was measured by ELISA.

[0024] Figure 16. Working model for ER-mitochondria connection disruption in mitochondrial dysfunction and pathogenesis of ADPKD. Polycystin-1 (PCI) and -2 (PC2) are present in multiple subcellular sites including ER, primary cilia, basolateral membranes, but not in mitochondria. ER-resident PCs are important for ER Ca2+homeostasis. Loss of PCs-regulated ER Ca2+release decreases mitochondrial Ca2+concentration ([Ca2+]), leading to reduced TCA enzyme activities (“functional” - red). Decreases in localized cytosol [Ca2+], through decreases in calmodulindependent kinase-II (CaMKII) and calcineurin (CaN) activity, downregulates the mitochondrial biogenesis master regulator PGCla, which leads to downregulation of MFN2 and PPARa. Reduced activity of master regulators PGCla and PPARa leads downregulation of gene expression of TCA enzymes (“transcriptional” - blue). Functional and transcriptional decreases of mitochondrial function, plus other mitochondrial dynamics changes, lead to mitochondria stress and metabolic disturbances, alters mitochondrial metabolites, which communicates to nucleus through epigenetic rewiring (i.e., mitochondrial retrograde signaling). Epigenetic changes cause widespread transcriptional dysregulation and rewiring contributing to cystogenesis, metabolic reprogramming, and further downregulation mitochondrial function. Downregulation of MFN2 contributes to MAM structure disruption. Decreases in local [Ca21] may directly impact MAMs structure. Enhancing ER-mitochondria connection and MAMs structure and function by TricB expression or MAMs activator improves mitochondrial function and ameliorates cystogenesis in ADPKD. Functions of PCs in cilia and basolateral membranes also contribute to anti-cystogenesis.

[0025] Figures 17A-17B. Evaluation of mitochondrial morphology using additional parameters such as circularity, ferret, aspect ratio (AR) and solidity in control, Pkd2-cKO mice at pre-cystic and cystic stages. At least, 50 mitochondria per mice from multiple images of PTs and 3 mice per groups were analyzed. Data are shown as mean ± SEM. Unpaired student t-test with p< 0.05 is considered significant.

[0026] Figures 18A-18C. Restoration of mitochondrial morphology by TricB expression in Pkd2-cKO mice. (A) TEM images showing expression of TricB ameliorates morphological changes seen in PTs’ mitochondria of Pkd2-cKO mice in both cystic and pre-cystic stages. Arrows denotes normal mitochondria; arrowheads denote abnormal mitochondria with indistinct cristae and asterisk symbolizes cystic cavity. Scale bars, low magnification- 10pm and high magnification- 2pm. (B) Assessment of auxiliary parameters related to mitochondrial morphology is shown in TricB expressing Pkd2-cKO mice at cystic (upper panel) and pre-cystic (lower panel) stages. Data are shown as mean ± SEM. Unpaired student t-test with p< 0.05 is considered significant. Figures 19A-19C. Serial block face SEM analysis of mitochondria in Pkd2-cKO mice at pre-cystic stage. (A) Representative image of 3D SEM sections of Pkd2-cKO pre-cystic kidney show numerous mitochondria. (B-C) Electron micrograph with a subset of traced mitochondria in the pre-cystic kidney of Pkd2-cKO mice. B represent the traced / selected mitochondria while C shows the post traced / selected. (D-F) Lower panel represents the reconstruction of mitochondria in magenta (D), grey (E), magenta with black background (F) from pre-cystic kidney of Pkd2-cKO mice.

[0027] Figures 20A-20B. Additional structural analysis exhibits reversal of mitochondrial integrity by TricB in Pkdl-cKO mice. (A) TEM images of PTs’ at low and high magnification in TricB expressing Pkdl-cKO mice at pre-cystic and cystic stages. Arrows denotes normal mitochondria; arrowheads denote abnormal mitochondria with indistinct cristae. Scale bars, low magnification-10pm and high magnification- 2pm. (B) Upper panel presents subsidiary morphological parameters of mitochondria in control, Pkdl-cKO pre-cystic mice compared to that at cystic stage. Middle and lower panel corresponds to assessment in Pkdl-cKO mice at cystic and pre-cystic stages, respectively to that of mice expressing TricB. Data are shown as mean ± SEM. Unpaired student t-test with p< 0.05 is considered significant.

[0028] Figure 21. Western blot showing TricB, IP3R, Calnexin, and PC2 levels in various samples.

[0029] DETAILED DESCRIPTION

[0030] Aspects of the present disclosure relate, in part, to methods for treating and / or preventing a polycystic kidney disease (PKD) in a subject in need thereof. In all aspects, the PKD may be autosomal dominant PKD (ADPKD).

[0031] In one or more aspect, the methods can include administering one or more sigma- 1 receptor agonists to a subject. The one or more sigma- 1 receptor agonists can include any sigma- 1 receptor agonists, for example a selective sigma-1 receptor agonist. In certain aspects, the one or more sigma-1 receptor agonists are selected from one or more of pre-084 and pridopidine. In one aspect, pre-084 is a selective sigma-1 receptor agonist and has the following structure:

[0032]

[0033] In one aspect, pridopidine has the following structure:

[0034]

[0035] Aspects disclosed include one or more sigma- 1 receptor agonist for treating and / or preventing a polycystic kidney disease (PKD). Further aspects include use of one or more sigma-1 receptor agonist for treating and / or preventing PKD. The one or more sigma- 1 receptor agonists used for treating and / or preventing a PKD, for example ADPKD, can include any sigma- 1 receptor agonists, such as a selective sigma-1 receptor agonist. In the aspects, the one or more sigma-1 receptor agonists are selected from pre-084 and pridopidine.

[0036] In the aspects, the one or more sigma-1 receptor agonist can be administered to the subject according to any regimen suitable for treating PKD. In certain aspects, the one or more sigma- 1 receptor agonists can be administered daily, weekly, monthly, or yearly.

[0037] In one or more aspects, the one or more sigma- 1 receptor agonist can be administered orally, subcutaneously, intravenously, via transdermal patches, or any other suitable route of administration.

[0038] In certain aspects, the subject in need thereof can be one who has, or is at risk of having, PKD. In one or more aspects, the subject in need thereof has one or more cysts present in a kidney. In the same or alternative aspects, the subject in need thereof exhibits chronic kidney disease and / or is at risk of kidney failure. In various aspects, the subject in need thereof, is diagnosed with autosomal dominant PKD (ADPKD). In the same or alternative aspects, the subject in need thereof has one or more mutations, deletions, or insertions to the PKD1 and / or PKD2 genes.

[0039] In various aspects, the subject in need thereof can be at least 30 years old, at least 35 years old, or at least 40 years old. In the same or alternative aspects, the subject has one or more cysts present in a kidney. In certain aspects, the subject in need thereof can be: i) at least 30 years old, at least 35 years old, or at least 40 years old; ii) has one or more cysts present in a kidney; and iii) a family history of ADPKD.

[0040] In certain aspects, the subject in need thereof can be any mammal. In various aspects, the subject is a human.

[0041] In certain aspects, the method can include performing an imaging examination of a kidney of the subject in need thereof and / or reviewing imaging data or results of a kidney of the subject in need thereof. In various aspects, this imaging may occur prior to, during, or after the administration of the one or more sigma- 1 receptor agonists. In certain aspects, the imaging can include one or more of an ultrasound, CT scan, or MRI. In various aspects, the imaging can be utilized to identify one or more cysts in the kidney of the subject for diagnostic purposes and / or to assess treatment with the one or more sigma-1 receptor agonists.

[0042] In various aspects, administering the one or more sigma-1 receptor agonists can do one or more of the following: improve kidney function, e g., as evidenced by kidney function tests; reduce kidney size; or reduce a number and / or size of cysts in a kidney of the subject. In the same or alternative aspects, administering the one or more sigma- 1 receptor agonists can prevent formation of cysts in a kidney of the subject and / or the formation of additional cysts in a kidney of the subject.

[0043] The definitions and terminology used herein are for the purpose of describing particular aspects only and are not intended to be limiting.

[0044] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise. As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0045] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0046] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0047] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.” All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, 5, or 6 members, and so forth.

[0048] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.” Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes including describing and disclosing the chemicals, instruments, statistical analyses and methodologies which are reported in the publications which might be used in connection with the invention. All references cited in this specification are to be taken as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0049] Aspects of the present disclosure that are described with respect to methods can be utilized in the context of the compositions of matter or kits discussed in this disclosure. Similarly, aspects of the present disclosure that are described with respect to compositions of matter can be utilized in the context of the methods and kits, and aspects of the present disclosure that are described with respect to kits can be utilized in the context of the methods and compositions of matter.

[0050] The above description, attached figures, and claims listed below are intended to be illustrative and not limiting of this invention. In light of the invention described herein, many themes and variations to this invention will be suggested to one skilled in the art. All such themes and variations are within the contemplation hereof. For instance, while this invention has been described in conjunction with the various exemplary embodiments outlined above and in the below claims, various alternatives, modifications, variations, improvements, and / or substantial equivalents, whether known or that are rare or may be presently unforeseen, may become apparent to those having at least ordinary skill in the art. Various changes may be made without departing from the spirit and scope of the invention. Therefore, the invention is intended to embrace all known or later-developed alternatives, modifications, variations, improvements, and / or substantial equivalents of these exemplary embodiments.

[0051] It is to be understood that the invention is not limited to the particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The scope of the present invention will be limited only by the claims.

[0052] It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context.

[0053] The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.

[0054] ILLUSTRATIVE EMBODIMENTS

[0055] 1. A method for treating and / or preventing polycystic kidney disease (PKD), comprising: administering one or more sigma- 1 receptor agonists to a subject in need thereof.

[0056] 2. The method of embodiment 1, wherein the subject in need thereof is diagnosed with autosomal dominant PKD.

[0057] 3. The method of embodiment 1 or 2, wherein the subject in need thereof has one or more mutations, deletions, or insertions to the PKD1 and / or PKD2 genes.

[0058] 4. The method of any one of embodiments 1-3, wherein the subject in need thereof exhibits chronic kidney disease and / or kidney failure.

[0059] 5. The method of any one of embodiments 1-4, wherein the one or more sigma- 1 receptor agonists are selected from pre-084 and pridopidine.

[0060] 6. The method of any one of embodiments 1-5, wherein the one or more sigma- 1 receptor agonists comprises a selective sigma-1 receptor agonist. 7. The method of any one of embodiments 1 -6, wherein the administering one or more sigma- 1 receptor agonists to a subject in need thereof comprises administration of the one or more sigma-1 receptor agonists daily, weekly, monthly, or yearly.

[0061] 8. A method for reducing a number of cysts in a kidney of a subject in need thereof, comprising administering one or more sigma- 1 receptor agonists to a subject in need thereof.

[0062] 9. The method of embodiment 8, wherein the subject in need thereof is diagnosed with autosomal dominant PKD.

[0063] 10. The method of embodiments 8 or 9, wherein the subject in need thereof has one or more mutations, deletions, or insertions to the PKD1 or PKD2 genes.

[0064] 11. The method of any one of embodiments 8-10, wherein the subject in need thereof exhibits chronic kidney disease and / or kidney failure.

[0065] 12. The method of any one of embodiments 8-11, wherein the one or more sigma-1 receptor agonists are selected from pre-084 and pridopidine.

[0066] 13. The method of any one of embodiments 8-12, wherein the one or more sigma- 1 receptor agonists comprises a selective sigma- 1 receptor agonist.

[0067] 14. The method of any one of embodiments 8-13, wherein the administering one or more sigma-1 receptor agonists to a subject in need thereof comprises administration of the one or more sigma- 1 receptor agonists daily, weekly, monthly, or yearly.

[0068] 15. The method of any one or embodiments 1-14, further comprising performing an imaging examination of a kidney of the subject in need thereof, before, after, or during treatment with the one or more sigma-1 receptor agonists.

[0069] 16. The method of embodiment 15, wherein the imaging examination comprises one or more of an ultrasound, CT scan, or MRI.

[0070] The invention will be more fully understood upon consideration of the following nonlimiting examples.

[0071] EXAMPLES Autosomal-dominant polycystic kidney disease (ADPKD) is the common genetic disease that counts for 5-10% of kidney failure patients. The affected individuals carry the genetic mutations develop fluid-filled cysts that enlarge over time to destroy the kidneys. The mutation genes have been discovered since early 1990’s. The function of these genes and why mutations cause the disease remains elusive. Endoplasmic reticulum (ER) is the intracellular organelle where proteins are made. The proteins coding for ADPKD genes are very abundantly present in the ER. Mitochondrion is the powerhouse of the cells. The inventors discovered that defects in ER-mitochondrion connection plays an important role in causing ADPKD. Sigma- 1 receptor (SIR) is a protein present in ER-mitochondrion connection and improves the function of the connection. In mouse models of ADPKD, the inventors found that chemicals that enhance ER-mitochondrion connection, e.g., SIR receptor agonists, ameliorate the cystic disease and kidney function of ADPKD. Defects in ER-mitochondrion connection has also been implicated in many neurodegenerative diseases including Alzheimer’s disease. These classes of chemicals are safe in human use and are currently in phase 3 clinical trials for treating neurodegenerative diseases. Materials and Methods

[0072] Western blotting

[0073] Total kidney lysates were prepared by homogenization in RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail (Roche). Total protein concentration was measured by Bradfords (Bio-Rad, 5000006) or Pierce™ BCA Protein Assay Kits (Thermo Fisher Scientific 23225). Protein lysates were boiled in 1 x NuPAGE LDS Sample Buffer with lOmM DTT, loaded on to NuPAGE 4% to 12% Bis-Tris Gels (Thermo Fisher Scientific) and run at 130 V. Proteins are then transferred to 0.45 pm PVDV membrane (Thermo Fisher Scientific) at constant 300 mA for 80 minutes at 4°C. The membrane was blocked in 5% skim milk in TBS- Tween 20 (0.01%) for 60 min at RT and incubated in following primary antibodies overnight at 4°C; ATF4 (Biolegend, 693902), SIR (Santa Cruz, sc-137075), Phospho-Gcn2 (Cell signaling, 94668), IP3R1 (Thermofisher PAI -901), VDAC (Thermofisher, 600-101-HB2) and 0-actin. Membranes are then washed three times in lx TBST each for 10 min and incubated in HRP-conjugated secondary antibodies for Ihr at RT. Afterwards, membranes are washed three times in TBST for 10 min each and developed in Clarity Western ECL Substrate (BioRad, 1705061) or SuperSignal™ West Femto (ThermoFisher Scientific, 34096X4). Signal is then detected in ChemiDoc XRS+ System with Image Lab Software (Bio-Rad).

[0074] Real-time PCR

[0075] Kidney tissues were dissected and saved in RNAlater. Total RNA from kidney tissues or micro-dissected proximal tubules were isolated by homogenization in TRIzol (ThermoFisher Scientific, 15596026) following manufacturer’s instructions. Subsequently, cDNA was synthesized from I pg of RNA by using iScript cDNA Synthesis Kit (Bio-Rad, 1708891). Primers were designed through Primer-BLAST and procured from IDT. 18s rRNA or GAPDH gene expression was used as an endogenous control for normalization of targeted gene expression. Quantitative real-time PCR was then performed using iTaq Universal SYBR Green Supermix (Bio-Rad, 1725124) in the Cl 000 Touch Thermal Cycler (Bio-Rad) following the manufacturer’s instructions. AACt method was used to express the fold difference of targeted gene expression.

[0076] Immunohistochemistry

[0077] Dissected kidney tissues were fixed in 4% paraformaldehyde overnight at 4°C. Following fixation, tissues were embedded in OCT and sectioned at 6 pm using a cryostat. Tissue sections on slides were then rinsed in water and washed twice 5 min each in TBS with 0.025% Triton X-100. Slides were then blocked in 10% normal horse serum with 1% BSA in TBS for 2hr at RT. Following blocking, tissue sections were incubated in primary antibody in 1% BSA in TBS at 4°C overnight. Afterwards, it is rinsed twice 5min each in TBS with 0.025% Triton X-100 and incubated in fluorophore-conjugated secondary antibody in 1% BSA in TBS for 1 hr in dark at RT. Then slides were washed three times 5min each in TBS and mounted with DAPI mounting medium. The following primary and secondary antibodies are used for the studies: polycystin-1, polycystin-2 (sc-28331, 1:500; Santa Cruz Biotechnology), acetyl-alpha tubulin (ThermoFisher, PA5-105102), anti-mouse, anti-. Leica SP8 confocal microscope (Leica) was used on the LAS X 3.0.14 software platform (Leica) to image the specimens and processed in ImageJ 1.54f.

[0078] Mitochondrial membrane potential assay

[0079] Mitochondrial membrane potential is measured by using cell permeant fluorescent dye, T etram ethylrhodamine, methyl ester (TMRM) (ThermoFisher, T668) following manufacturer’s protocol and McKezie etal. (PMID: 28190045). Briefly, PTs were isolated as described elsewhere from dissected kidneys and seeded on glass cover slips coated with Cell-Tak (Corning™) and cultured in complete media (Lifeline, RenaLife ™ Epithelial Medium Complete LL-0025) for 72 hr at 37°C in the CO2 incubator. On the day of experiment, seeded cells were incubated at 37°C for 30 min with TMRM (100 nM) in the cell growth media along with MitoTracker Green (200 nM) (ThermoFisher, M7514), a mitochondrial-selective dye that is used to track mitochondria.

[0080] Mitochondrial calcium measurement (both Rhod2 AM and Ca2+ content)

[0081] Measurement of Ca2+concentration in the mitochondria of cultured primary proximal tubule cells were performed following Maxwell et al. (PMID: 29757281). Accordingly, Rhod-2 AM (ThermoFisher, R1244) is used as a fluorescent labeled calcium indicator rearrange Rhod-2 AM (20 pm) and MitoTracker green (200 nM) in Tyrode’s solution (140 mM NaCl, 5 mM KC1, 1.2 mM CaC12, 1 mM MgC12, 0.33 mM NaH2PO4, 5.5 mM glucose, 10 mM HEPES at pH 7.4) for 30 min at room temperature in dark. Afterwards, de-esterification of Rhod-2 AM is done by replacing the dye solution with fresh Tyrode’s solution and incubated for another 30 min. Cover slips were then transferred to imaging chambers and plasma membrane of cells were permeabilized by 0.005% saponin solution for 1 min to offload cytosol-localized Rhod-2 AM while retaining mitochondria localized Rhod-2 AM. Buffer in the microscope chamber is then immediately replaced by Ca2+-free Tyrode’s solution with 2 mM EGTA. Subsequently, fluorescent images showing colocalization of Rhod-2 AM (Ex / Em 552 / 581 nm) and MitoTracker Green (Ex / Em 490 / 516 nm) were taken under the microscope (Nikon Eclipse E600) and processed in Imaged (1.54f).

[0082] Absolute Ca2+content was measured in isolated mitochondrial fractions using colorimetric Calcium assay kit from Abeam (abl02505) and following Kwong et al. (PMID: 26119742). Briefly, mitochondrial pellets were resuspended in calcium assay buffer supplied in the kit and solubilized using a sonicator. Afterwards, the lysates were centrifuged at 10,000 rpm for 3 min at 4°C to clear insoluble material. Protein quantification was done through BCA assay and lysates were loaded onto wells followed by chromogenic reagent and calcium assay buffer provided in the kit. After mixing and incubating in the dark at room temperature for 5 min, the absorbance is measured at 570 nm using a microplate reader. Observed OD is then used to calculate the absolute Ca2+content by using a series of Calcium standards supplied in the kit.

[0083] Measurement of total H3K27ac

[0084] Total H3K27ac (acetylation of lysine 27 on histone H3 protein subunit; marker of epigenetic modification to the histone 3 DNA packaging protein) was measured in mice kidney tissues by using EpiQuik Global Acetyl Histone H3K27 Quantification kit (Epigentek, P-4059) and following Lakhia et al (PMID: 36283570). Histone extracts were prepared through acidextraction method following manufacturers protocol. Kidney tissues were minced and disaggregated in TEB (PBS containing 0.5% Triton X 100, 2 mM PMSF and 0.02% NaN ) buffer using a Dounce homogenizer. Tissue homogenate is then centrifuged at 10,000 rpm for 1 min at 4°C and the collected tissue pellet after resuspending in 3 volumes of extraction buffer (0.5N HC1 + 10% glycerol) incubated on ice for 30 min. After centrifugation at 12,000 rpm for 5 min at 4°C, the supernatant is collected, and 8 volumes of acetone is added and incubated for overnight at -20°C. Afterwards, it is centrifuged again at 12,000 rpm for 5 min, air-dried the pellet and dissolved in nuclease free water. Protein extracts were then quantitated through BCA protein assay. Following manufacturer’s protocol, 200 ng of histone extracts were added per well for H3K27ac quantification. Briefly, acetylated histone H3 at lysine 27 from the extracts were captured onto the strip wells coated with anti-acetyl H3K27 antibody. Captured H3K27ac were then detected through a colorimetric assay with the help of a labeled detection antibody specific to H3K27ac and the absorbance is measured in a microplate reader at 450 nm. Using a reference standard control supplied in the kit the absolute amount of H3K27ac is calculated.

[0085] Mitochondria and MAMs isolation

[0086] Mitochondria and mitochondria-associated ER membranes (MAMs) were isolated from kidney following Wieckowski et al. (PMID: 19816421). Briefly, both kidneys were homogenized in the homogenization buffer (225 mM mannitol, 75 mM sucrose, 0.5% BSA, 0.5 mM EGTA and 30 mM Tris-HCl at pH 7.4) using homogenizer and then centrifuged at 740g for 5 min to remove unbroken cell debris. Collected supernatant again centrifuged at 9,000g for 10 min at 4°C and the pellet containing mitochondria sequentially washed as per recommended protocol. Finally, crude mitochondrial pellet is resuspended in MRB (mitochondria resuspending buffer: 250 mM mannitol, 5 mM HEPES and 0.5-mM EGTA at pH 7.4) and layered on top of percoll medium (225 mM mannitol, 25 mM HEPES, 1 mM EGTA and 30% Percoll at pH 7.4) followed by MRB solution and centrifuged at 95,000g for 30 min at 4°C in an ultracentrifuge. Following centrifugation, floating diffused white band representing MAM and dense band at the bottom for mitochondria were collected and rewashed accordingly to obtain pure mitochondria and MAM in MRB solution. The purity of mitochondria and MAM preparation was checked by western blot analysis of targeted markers.

[0087] Proximity ligation assays

[0088] Proximity ligation assays were performed using the kit Duolink® In Situ Red Starter Kit Goat / Rabbit (Sigma DUO92105). Steps were followed according to the manufacturer’s protocol. Briefly, mice kidney tissues were fixed in 4% formaldehyde, embedded in OCT and 6 um sections were cut through a cryostat. Tissue sections on slide were rinsed twice in water, washed in TBS plus 0.025% Triton X-100 twice for 5 min each and then blocked using Duolink® Blocking Solution for 60 minutes at 37 °C in a humidity chamber. After incubation, blocking solution is drained and Duolink® Antibody Diluent containing primary antibodies specific to IP3R1 (Thermofisher PAI -901, 1:250 dilution) and VDAC (Thermofisher, 600-101-HB2, 1:250 dilution) were added and incubated overnight at 4°C. Slides were then washed in lx wash buffer A twice for 5 min each at RT and incubated in PLA probe solution in a humidity chamber for 1 hour at 37°C. Subsequently, slides were washed again with lx Wash Buffer A twice for 5min each at RT and incubated with ligation solution for 30 minutes at 37 °C. Following washing with lx wash buffer A twice, slides were incubated with amplification solution in a pre-heated humidity chamber for 100 minutes at 37 °C. Afterwards, slides were washed twice with lx wash buffer B for 10 min each followed by O.Olx wash buffer B for 1 min. Sections were then mounted with Duolink® In Situ Mounting Medium with DAPI and visualized on Leica SP8 confocal microscope (Leica) through LAS X 3.0.14 software platform (Leica) with a 1 OOx objective. At least three images from each mouse were processed and mean fluorescence intensity were analyzed in Imaged 1 54f Microdissection of proximal renal tubules

[0089] Proximal tubules were dissected and isolated following Glaudemans et al. (PMID: 23887378) and Cheng et al (PMID: 22791335). Briefly, mice kidneys were dissected, sliced, and incubated in prewarmed Hank’s balanced salt solution (137mMNaCl, 5mMKCl, 0.8mM MgSO4, 0.33mM Na2HPO4, 0.44mM KH2PO4, ImM MgC12, lOmM Trishydroxtmethyl aminomethane hydrochloride, 0.25mM CaC12, 2mM L-glutamine, 2mM L-lactate, 295 osmolality at pH 7.4) containing collagenase type I (1.5 mg / ml) and bovine serum albumin (Img / ml) and shaken vigorously on a titer plate shaker at 37°C for 30 min. Following incubation, the digested segments were passed through 200 pM fdter and collagenase digested tissue segments were collected. Microdissection of individual proximal renal tubules were then performed under a stereo microscope based on their morphological characteristics following Glaudemans et al. (PMID: 23887378).

[0090] Oxygen consumption rate assay

[0091] The measurement of oxygen consumption rate (OCR) is carried out according to manufacturer’s protocol (Agilent Seahorse XF Cell Mito Stress Test Kit). Individually isolated 8-10 proximal renal tubules were seeded on a coated (Coming™ Cell-Tak) Seahorse XFp Cell Culture Miniplates (Seahorse XFp FluxPak, Agilent) with Seahorse XF DMEM media supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose and incubated in a non-CO2 incubator for Ihr at 37°C. For PRE-084 treatment, media is also supplemented with 10 pM of PRE-084 (Bio-techne, 0589). Following incubation, media is replaced with fresh media and OCR is measured in the seeded tubules using Seahorse XF Cell Mito Stress Test Kit (Agilent) in a Seahorse XF HS Mini (Agilent) as per manufacturer’s instructions. Preoptimized concentrations of modulators provided in the kit were used, oligomycin (3 pM), FCCP (1 pM) and rotenone and antimycin A (0.5 pM). Each OCR assay is performed in 2-3 technical replicates per group. After OCR measurement, total RNA from the seeded tubules was extracted by Trizol and gene expression of 18s rRNA were quantitated using qRT-PCR and cycle threshold (Ct) values were used for normalization across wells. Data were analyzed by Seahorse XF Mito Stress Test Report Generator.

[0092] Transmission and scanning electron microscopy

[0093] Structural analysis of mitochondria and MAMs in kidney tissue samples were analyzed as per Lam et al. 2021 (PMID: 34571826) and Giacomello et al. 2016 (PMID: 27341186) using ImageJ vl.53t. Accordingly, 5-10 TEM random images were taken from multiple embedded sections derived from each sample at 10k magnification. Subsequently, each image was divided into quadrants by using ImageJ plugin quadrant. After splitting into four quadrants, two quadrants were randomly selected for mito and MAMs structural analysis. Randomly, 50 mitochondria from multiple images were included for analysis per sample. Similarly, for MAMs characterization at least 20 mitochondria were selected randomly from multiple images per mice. Within the mitochondria, only those MAMs were analyzed where the ER-mito contacts distance was less than 30 nm. (PMID: 27341186)

[0094] Statistical analysis Quantitative data are presented as mean ± SEM. Statistical significance was analyzed by two-tailed, unpaired Student’s t test or by one-way ANOVA followed by Tukey’s multiple comparison tests in GraphPad Prism (Version 10.2.0). All experiments were repeated at least three times, p < 0.05 was considered as statistically significant.

[0095] Results

[0096] Mitochondrial structural alterations in Pkd2-cKO kidneys

[0097] Mitochondrial dysfunction is evident in ADPKD mice models (PMID: 29426897, 32239723, 28993480). Since cyst growth in ADPKD is progressive, it was speculated that a gradual deterioration of mitochondrial function in ADPKD mice models at early and late stages. To examine this, inducible adult-onset kidney specific Pkd2-defici ent mice models were generated and analyzed at 4 weeks (pre-cystic) and 12-16 weeks (cystic) after doxycycline treatment to induce Pkd2 deletion. Compared to the control wildtype kidneys, Pkd2-cKO mice kidneys were mostly normal in size at pre-cystic stage while markedly enlarged at cystic stage (Fig 1A,B). Occasionally a mildly enlarged pre-cystic kidney could be observed. In H&E staining histology, the pre-cystic kidneys exhibited variable degrees of dilation in some renal tubules and occasionally few small cysts in comparison to cystic kidneys (not shown). To assess the relationship between mitochondria structure (and function) and cystogenesis overall mitochondria structure in Pkd2-cKO mice was examined by transmission electron microscopy (TEM) at pre-cystic and cystic stages (Fig. ID, IE and 17A). TEM images of proximal renal tubules (PTs) in both pre-cystic and cystic mice revealed abnormal mitochondria characterized by reduced mitochondria area, perimeter and increased roundness accompanied by altered auxiliary parameters (Fig. ID, IE and 17B). The mitochondrial cristae density and numbers were reduced at pre-cystic as well as cystic stages of Pkd2-cKO mice in comparison to control (Fig. ID, IE). Notably, all above changes in mitochondrial structure already occurred at pre-cystic stage and no further progression from pre-cystic to cystic stages.

[0098] Reversal of Pkd2-cKO mitochondrial structure alterations by TricB transgene expression It is reported that Pkd2-deleted cells have impaired agonist- stimulated Ca2+release from ER and expression of an ER-restricted K+-permeable channel TricB restores ER Ca2+release in cultured cells and ameliorated cystogenesis in Pkd2-deleted mice (PMID: 35835458). TricB does so by facilitating K+-Ca2+exchange in ER to promote Ca2+release in Pkd2-cKO mice. ER and mitochondrion are two abundant intracellular organelles in close contact and form functional coupling. TricB transgene expression was employed as a tool to investigate the role of ER calcium homeostasis on mitochondrial alterations seen in Pkd2-cKO mice. TEM images taken from Pkd2-cKO mice expressing transgenic TricB exhibit small cystic lumen and reduced mitochondrial aberration (Fig. 2B; compare Fig. 18A vs Fig. IE). For pre-cystic Pkd2-cKO kidneys, TricB expression reversed key mitochondrial morphological parameters while other parameters showed trends of improvement (Figure 2A). The decreases in the cristae density and number in Pkd2-cKO mice at both pre-cystic and cystic stages were reversed by expressing TricB (Figure 2A, 2B). Morphometric analysis of mitochondria from these TEM images reveals a significant improvement in all measured mitochondrial parameters in TricB expressing Pkd2-cKO mice compared to Pkd2-cKO mice alone at cystic stage (Fig. 2B, and Fig. 18B). At pre-cystic stages TricB expression restored most but not all mitochondrial morphological parameter (Fig. 2A and Fig. 18C). Importantly, the key parameters such as cristae density and cristae number that reflecting functional mitochondrial ETC capacity were restored to the control values at pre-cystic and cystic stages by expressing TricB in Pkd2-cKO mice (Fig. 2A, 2B).

[0099] Mitochondria volume in Pkd2-cKO mice analyzed by scanning EM and 3D reconstruction To gain insights into individual mitochondrion volume and the density per cell, Pkd2-cKO kidneys were analyzed using scanning EM (SEM) and 3D volume rendering. Images deck taken through serial block face SEM were subjected through manual contour tracing in Amira software for 3D volume rendering (Fig. 19A). Figure 3 shows histogram of frequency distribution over range of induvial mitochondrial volume. At both pre-cystic and cystic stages, the frequency of smaller mitochondria is relatively higher as compared to wild type. Cumulative mean mitochondrial volume as well as mitochondrial density is significantly reduced in cystic Pkd2-cKO mice compared to control mice but not in pre-cystic mice (Fig. 3B-C). Expression of TricB transgene partially restores the frequency of mitochondria with larger volume, mean mitochondrial volume and density in comparison to cystic Pkd2-cKO mice (Fig. 3A-C). Thus, Pkd2 deletion drives mitochondria toward decreased fusion and / or increased fission / fragmentation that involves defects in ER Ca2+homeostasis.

[0100] Deletion of Pkd2 alters mitochondria-associated ER membranes Mitochondria-associated ER membranes (MAMs) are close contact sites between endoplasmic reticulum (ER) and mitochondria and play vital roles in regulating mitochondrial dynamics, protein transfer and notably, transport of lipid and Ca2+into the mitochondrial matrix necessary for its function (PMID: 32138899). The distance between ER and mitochondria at MAMs depends on type of ER. The large ribosome constrains the distance between rough ER and mitochondria to 50-80 nm. The contact between smooth ER and mitochondria is indispensable for direct ER-mitochondria Ca2+transfer and the distance is a critical determinant of the amount of Ca2+transferred (ref). Normally, it ranges 10-30 nm with an optimal distance ~15 nm. Shorter distance prevents proper opposition between the pore of the inositol trisphosphate receptor (IPBR) at the ER membrane and the voltage-dependent anion channel (VDAC) at the outer mitochondria membrane (OMM). Longer separation limits the diffusion. Our findings that TricB reverses ER Ca2+release defect and ameliorates cystogenesis in Pkd2-cKO raise an interesting possibility Pkd2 deletion affects MAMs structure. Next, MAMs structure in TEM images were analyzed focusing on the structure of MAMs up to 30 nm in distance. In addition to the distance, MAMs length that measures the contact length between ER and mitochondria as well as MAMs coverage defining the percentage of mitochondrial surface covered by MAMs were also analyzed (Fig. 4A-C). As shown, representative images showing MAMs structure is altered with increased distance between ER and mitochondria in both pre-cystic and cystic stages of Pkd2-cKO mice as opposed to control mice (Fig. 4A-B).

[0101] Detailed quantitative analysis revealed that mean MAMs distance in the control kidneys at 17 nm and was significantly increased to 18- and 19 nm in pre-cystic and cystic stage kidneys, respectively (Fig. 4D). The average MAMs length was significantly reduced from 1150 nm in length in control kidneys to 650 and 550 nm in length in pre-cystic and cystic kidneys. The percentage of mitochondrial surface covered by MAMs was also decreased in Pkd2-cKO vs control kidneys. Notably, these changes in MAMs structure occurred predominantly between control and pre-cystic kidneys with no significant difference between pre-cystic and cystic kidneys. As in other mitochondria parameters shown in Figs. 1-3, TricB expression reversed MAMs structural changes caused by Pkd2 deletion (Fig. 4B, 4C, 4E). TricB is restricted to ER, not present in mitochondria (Fig. 21).

[0102] To validate the findings that Pkd2 deletion increases MAMs distance analyzed using TEM and to provide additional functional implication of widening MAMs structure, independent proximity ligation assay that probes the distance between IP3R and VDAC was employed. In the assay, proximity between IP3R and VDAC below a distance threshold triggers chemical reaction generating red fluorescent signals. As shown, Pkd2 deletion markedly reduced fluorescent signal intensity in pre-cystic as well cystic kidneys (Fig. 5A, 5B). TricB expression partially reversed the decrease in Pkd2-deleted kidneys. The results corroborate with findings by TEM and supports functional implication that Ca2+transfer between IP3R and VADC at MAMs are impaired.

[0103] Pkd2-cKO alters mitochondrial membrane potential and Ca2+levels

[0104] The mitochondrial membrane potential (ATm) is generated by ETC complexes and an essential component in the process of energy storage during oxidative phosphorylation. Together with the proton gradient (ApH), A 'Pm forms the transmembrane potential of hydrogen ions which is harnessed to make ATP. Although there are some fluctuations reflecting normal physiological activity, the levels of A 'Pm are kept relatively stable. A long-lasting drop or rise of A 'Pm vs reflects pathologies, playing a key role in mitochondrial homeostasis by serving as a signal for selective elimination of dysfunctional mitochondria. Mitochondria are an important regulator of cellular Ca2+concentration ([Ca2+]) by sequestering and releasing Ca2+(ref). Mitochondrial [Ca2+] also has other important functions, such as regulation of mitochondrial metabolism, ATP production and cell death.

[0105] Mitochondrial membrane potential was measured in proximal tubules (PTs) freshly isolated from Pkd2-cKO and control kidneys. Note that while cystic stage Pkd-cKO kidneys were studied here, only non-cystic tubules could be manually dissected for isolation. The T etram ethylrhodamine, methyl ester (TMRM) is a cell-permeant potential-sensitive red fluorescent dye that selectively accumulates in active mitochondria (Fig. 6A-6C). Selective partition to mitochondrial is verified by colocalization with mitochondrial marker MitoG. A 'Pm in proximal tubules of Pkd2-cKO kidneys was much higher (hyperpolarization) than in those of control kidneys (Fig. 6A). TricB expression in Pkd2-cKO normalized the ATm. Mitochondrial [Ca2+] was measured using a calcium-sensitive fluorescent dye rhodamine-2-acetoxymethy ester (Rhod2AM). The dye enters cytosol and cellular organelles in acetoxymethyl ester form and retained after esterase cleavage. Selective permeabilization of cell and ER membrane by digitoxin allows preferential retention of Rhod2 in mitochondria (ref). As shown, mitochondrial [Ca2+] was reduced in Pkd2-cKO proximal tubules relative to the control (Fig. 6B). TricB expression partially reversed the decreases in Pkd2-cK0. To support that the above [Ca2+] measurement by Rhod2 reflects predominantly, if not solely, changes in mitochondria, studies were performed to measure Ca2+contents in isolated mitochondria. Mitochondrial Ca2+content was significantly reduced in pre-cystic Pkd2-cKO kidneys compared to the control (Fig. 6C). There was no difference between pre-cystic and cystic kidneys. TricB expression partially restored mitochondrial Ca2+content in Pkd2-cKO.

[0106] Decreased mitochondrial respiration in proximal tubules of Pkd2-cKO mice and reversal by TricB expression

[0107] Mitochondrial respiratory function in Pkd2-cKO was studied by real-time oxygen consumption rate (OCR) in PTs isolated from dissected kidneys (Fig. 7). OCR assay revealed a decrease in oxidative phosphorylation activity (OXPHOS) in PTs isolated from Pkd2-cKO cystic kidneys compared to the control (Fig. 7A). Both normalized basal respiration and maximal respiration in a mitochondrial stress state is significantly reduced in Pkd2-cKO compared to control. Further, transgenic overexpression of TricB partially restores the mitochondrial respiratory capacity in the PTs of Pkd2-cKO mice by elevating both basal and maximal respiration. For pre-cystic kidneys, OXPHOS activity at both basal and stressed state revealed a declining trend but did not reach statistical significance (Fig. 7B).

[0108] Aberrant expression of mitochondrial biogenesis and dynamic regulator genes in Pkd2-cKO kidneys.

[0109] The expression of genes involved in regulating mitochondrial function, biogenesis, and dynamics was examined. The number of mitochondria per cell analyzed by the mitochondrial / nuclear DNA ratio (ND1 / HK2, labeled “Ndl”) was significantly reduced in Pkd2-cKO cystic kidneys (Figure 8A, 8B). The master regulators of mitochondrial biogenesis, peroxisome proliferator-activated receptor-a (Ppara) and PPAR-y coactivator 1-a (Pgcla), were reduced (Figure C). cMyc is an early gene that promotes cyst formation; microRNA cluster miR-17-5p is reported to suppress Ppara. Both were increased in Pkd2-cKO kidneys (Figure 8A). The pro-fusion genes mitofusin-1 (Mfnl), mitofusin-2 (Mfn2), and optic atrophy 1 (Opal) were downregulated in Pkd2-cKO cystic kidneys. Conversely, the expression of pro-fission gene dynamin-related protein 1 (Drpl) was upregulated. Transgenic overexpression of TricB reversed these changes in gene expression in Pkd2-cKO cystic kidneys, restoring the levels to the control (Figure 8A). At the pre-cystic stage, Pgcla, Ppara and Mfn2 were significantly downregulated in Pkd2-cK0 vs controls (Figure 8B).

[0110] Pkd2-cKO kidneys show aberrant expression of genes involved in mitochondrial biogenesis and dynamics

[0111] Cumulatively, the aforesaid results implicate a significant decline of mitochondrial health in Pkd2-cKO mice. To explore the role of genes involved in regulating mitochondrial number and function, their expression was checked in dissected kidney tissues. As shown through qRT-PCR assays, mitochondrial DNA copy number (mt-DCN) that signifies mitochondrial density (number of mitochondria per cell) and function were significantly reduced in Pkd2-cKO mice at cystic but not pre-cystic stage (Fig. 9A, 9B). Transgenic overexpression of TricB restored the mt-DCN in cystic Pkd2-cKO mice to the control level. Next, PGCla and PPARa genes known to be master regulators of mitochondrial biogenesis and function were examined, and results show that they were downregulated in both pre-cystic and cystic stages of Pkd2-cKO mice than that of control kidney. This is in line with previous studies implying PGCla and PPARa playing a key role in modulating mitochondrial function underlying pathogenesis of ADPKD (PMID: 28205547). cMyc is believed an early gene that promotes cyst formation, microRNA clusters miR-17-5p is reported to suppress PGCla and PPARa (PMID: 1646908, 28205547). Both were upregulated in Pkd2-cKO cystic kidneys (Fig. 9A). Genes promoting mitochondrial fusion and MAMs integrity such as Mfnl and OPA1 were reduced while DRP1, a pro-fission gene, was upregulated in Pkd2-cKO cystic kidney. MFN2, a pro-fusion gene also implicated important for formation and maintenance of MAMs structure (ref) was significantly downregulated in pre-cystic and cystic Pkd2-cKO (Fig.

[0112] 9). TricB expression reversed these gene alterations in Pkd2-cKO cystic kidneys. At the pre-cystic stage, TricB expression showed a trend toward reversing Pkd-cKO-induced changes. Overall, these gene signature supports the mitochondrial structural and functional changes observed in Pkd2-cKO and the effect of TricB.

[0113] Cystic phenotypes in Pkdl-cKO mice are suppressed by TricB overexpression

[0114] PCI and PC2 are two structurally very diverse proteins. The precise molecular mechanisms underlying the phenocopy remain elusive. One hypothesis is that PCI and PC2 work as a receptorchannel complex. However, the subcellular distribution of PCI and PC2 are not completely congruent. Another possibility complementary to the receptor-channel complex hypothesis that PCI and PC2 function converge on a common downstream target. To test the hypothesis that ER-mitochondria connection may be a common target that modify the pathogenesis of ADPKD, the effect of TricB expression on cystogenesis of Pkdl-cKO was examined.

[0115] Mice carrying doxycycline inducible kidney-specific / / -floxed allele (Pkdl^,Pax8-LC1) with or without allele for inducible kidney-specific expression of TricB transgene (cTg-TricB) were generated. As shown, adult-onset Pkdl-cKO kidneys 12 weeks after induction showed markedly enlarged cystic kidneys while coexpressing TricB transgene in Pkdl-cKO ameliorated cyst formation with decreased kidney size (Fig. 9A, 9B). Cyst index and BUN levels were elevated in Pkdl-cKO and partially reversed by TricB expression (Fig. IOC, 10D). Thus, TricB expression suppresses cystogenesis in Pkdl-cKO as is for Pkd2-cKO mice (PMTD: 35835458).

[0116] Next, the expression level of genes that are involved in the process of mitochondrial biogenesis as well as its function was measured. As is for Pkd2-cKO, the number of mitochondria per cell as reflected by mitochondrial DNA / nuclear DNA ratio, master regulators for mitochondria biogenesis and function and genes regulating mitochondrial dynamics were altered in Pkdl-cKO cystic kidneys and reversed by TricB expression. Many of these alterations occurred early at Pkdl-cKO pre-cystic stage, some showed a trend but not reaching statistical significance at the pre-cystic stage.

[0117] Mitochondrial structural and functional changes in Pkdl-cKO and reversal by TricB expression

[0118] Previous studies reported an impaired mitochondrial structure in the absence of PCI including human kidney biopsy samples (PMID: 32239723, 29426897). To compare the structural changes of mitochondria in Pkdl-cKO with Pkd2-cKO and the effect of TricB expression, the mitochondrial morphology in the dissected kidney tissues was analyzed by TEM. Mitochondria in Pkdl-cKO mice are smaller in size and rounded in both pre-cystic and cystic stages compared to control mice (Fig. 10A). These findings were supported by quantitate analysis of morphometric parameters including mitochondrial area, perimeter, and roundness (Fig. 10B). Other morphological parameters were also significantly altered (Fig. 20B). The cristae density and cristae numbers per mitochondria were significantly reduced in Pkdl-cKO (Fig. 10B). As in Pkd2-cKO, the mitochondrial changes in Pkdl-cKO occurred early beginning in the pre-cystic kidneys. Unlike in Pkd2-cKO, the changes in the cystic stage are significantly different from in the pre- cystic stage. The implication of progressive worsening of mitochondrial structures from pre-cystic to cystic stage in Pkdl-cKO is unclear but may reflect underlying differences in PCI and PC2 function. Nonetheless, TricB expression in Pkdl-cKO reversed all measured mitochondrial parameter in pre-cystic as well cystic kidneys (Fig. IOC, 10D; Fig. 20A, 20B).

[0119] The importance of MAMs contact sites in ER-mitochondria communication, particularly Ca2+transfer, prompted us to examine MAMs structure in Pkdl-cKO. MAMs distance, ER-Mito contact lengths and MAMs coverage were analyzed from TEM images (Suppl Fig 7). As shown, MAMs distance in Pkdl pre-cystic and cystic were significantly increased compared to control kidneys (Fig. 11 A). MAMs length and mitochondrial surface covered by ER were both decreased in pre-cystic and cystic Pkdl-cKO kidneys compared to control mice. TricB expression in Pkdl-cKO mice reversed all Pkdl -cKO-induced MAMs parameters in pre-cystic as well cystic kidneys except for MAMs distance in the pre-cystic kidneys (Fig. 11B,C). The changes in MAMs contact in Pkdl-cKO were further examined by the proximity ligation assays. Supporting the findings by TEM, MAMs contacts analyzed by probing the interaction between IP3R and VDAC revealed reduced contacts in Pkdl-cKO pre-cystic and cystic kidneys and reversed by TricB expression (Fig. 11D,E).

[0120] Mitochondrial respiration in Pkdl-cKO and the effect of TricB expression was examined by real-time oxygen consumption rate (OCR) in freshy isolated PTs. Real-time OCR was significantly reduced in non-cystic PTs isolated from cystic stage ofPkdl-cKO kidneys (Fig. 12 A). TricB expression restored OCR to the level of control kidneys. In pre-cystic PTs, OCR showed a trend of decrease but was not insignificantly different from the control (Fig. 12B).

[0121] Enhancing MAMs function reverses mitochondrial dysfunction and cystogenesis in Pkdl-cKO

[0122] Impaired mitochondrial structure and function in Pkdl- and Pkd2-cKO is shown. Cystogenesis and mitochondrial changes can be reversed by TricB, an ER-restricted channel that has been shown to correct PC2-deficient ER Ca2+release defect (ref). Importantly, ER-mitochondria contact sites as MAMs are disrupted early in the pre-cystic stages of Pkdl- and Pkd2-cKO. Here the hypothesis that enhancing MAMs function can improve mitochondrial structure and function and reverse cystogenesis in Pkd-cKO was examined. SIR is markedly upregulated in Pkdl-cKO kidneys (Fig. 13A). TricB expression reversed Pkdl -cKO-induced upregulation (Fig. 13A). Under normal conditions, SIR forms complex with BiP (GRP78), another chaperon commonly upregulated during ER stress. SIR agonist bind to and dissociate SIR from BiP allowing activation of IP3R to promote Ca2+release at MAMs (PMID: 20869780, 17981125 and 32668330). Thus, simple upregulation of SIR protein may not confer increased activity, activation by agonist is required. Consistent with this notion, treatment of isolated PTs from Pkdl-cKO with SIR agonist pre-084 augmented the basal and maximal mitochondrial respiration (Fig. 13B).

[0123] Next, the effect of pre-084 on Pkdl-cKO mice was examined. Pkdl-cKO mice received pre-084 or saline daily via intraperitoneal injection for 3 months beginning 2 weeks after doxycycline induction. As shown, Pkdl-cKO mice received pre-084 had smaller kidneys with few cysts compared to saline injected mice (Fig. 14A). Blood BUN levels and cyst index are significantly lower in pre-084-treated than saline-treated mice (Fig. 14B, 14C). TEM analysis revealed improved mitochondrial structures in pre-84-treated kidneys vs saline-treated kidneys (Fig. 14D, 14E).

[0124] Altered epigenetic landscape in Pkdl- and Pkdl-cKO and reversal by TricB expression and by MAMs agonist

[0125] Widespread transcriptional dysregulation is a prominent feature of ADPKD. Recent evidence indicates that epigenetic rewiring may be behind the transcriptomic dysregulation. How mutation of PKD genes lead to epigenetic rewiring is unknown. Thus, whether mitochondrial dysfunction in Pkd-cKO may alter epigenetic landscape and the effect of enhancing MAMs function was examined. A recent study by Lakhia et al reported that H3K27acetylated histone (H3K27ac) is increased in Pkdl-mutant cells (Kidney Int. 2023 ; 103(1): 87-99). Here, lysates from Pkdl- and Pkd2- kidneys had increased levels of H3K27ac (Fig. 15). The increases were evident in pre-cystic as well as cystic kidneys, and were reversed in Pkd-cKO by TricB expression or treated with pre-084.

[0126] Summary

[0127] Mutations on PKD1 and PKD2 genes coding for PCI and PC2 respectively cause ADPKD. Metabolic reprogramming / mitochondrial dysfunction is an important modifier of ADPKD cystogenesis. The cause of mitochondrial dysfunction in ADPKD remains elusive. PC2 is a cation channel that conducts monovalent cation K+ and Na+ more than Ca2+. The inventors have recently shown that ER-localized PC2 functions as a K+-permeable channel to mediate K+-Ca2+ exchange to facilitate ER Ca2+ release. TricB is an ER-restricted K+ channel. The inventors showed that TricB reverses ER Ca2+ release defect in a PC2-deficient cell line and cystogenesis in Pkd2-cKO mice. ER and mitochondrion each occupies a large share of cell volume and form close interorganelle contact known as mitochondria-associated ER membranes (MAMs). The role of ER-mitochondria connection in ADPKD pathogenesis was examined.

[0128] Mitochondria morphology in pre-cystic (4 weeks) and cystic (16 weeks) kidneys after doxy induction in Pax8-LCl driven Pkd2-cKO mice were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Mitochondrial respiration in isolated proximal tubules were studied by Seahorse oxygen consumption rate (OCR) assays. Mitochondrial DNA mass and regulator gene expression were examined by real-time PCR.

[0129] TEM showed that Pkd2-cK0 kidneys vs control have altered mitochondria morphology including decreased area and cristae density, and increased roundness. About 60% mitochondria surface was in contact with ER forming MAMs. The distance between ER-mitochondria contact was increased and the length of contact was decreased in Pkd2-cKO kidney. These changes occurred in pre-cystic kidneys, and no further progression from precystic to cystic kidneys occurred. SEM with 3D rendering showed decreased individual mitochondria size. Proximal tubules isolated from cKO kidneys showed decreased OCR. Mitochondria master regulators PGCla and PPARa, DNA mass, and mitofusin-2 (a mitochondria profusion factor and MAM regulator) were decreased in cKO kidneys. All above changes in cKO were reversed by transgenic expression of TricB or by SIR agonist pre-084.

[0130] Conclusion

[0131] Mitochondria defects occur in early precystic stages. ER and mitochondria are in close contact with 60% mitochondria surface in contact with ER. TricB transgene reverses cystogenesis and mitochondria defects in Pkd2-cKO mice. The results support that ER Ca2+ homeostasis defect is important in mitochondrial dysfunction and the pathogenesis of ADPKD. Moreover, enhancing ER-mitochondria connection by an SIR agonist may be an effective treatment for ADPKD.

Claims

CLAIMSWe claim:

1. A method for treating and / or preventing polycystic kidney disease (PKD), comprising administering one or more sigma- 1 receptor agonists to a subject in need thereof.

2. The method of claim 1, wherein the subject in need thereof is diagnosed with autosomal dominant PKD.

3. The method of claim 1, wherein the subject in need thereof has one or more mutations, deletions, or insertions to the PKD1 and / or PKD2 genes.

4. The method of claim 1, wherein the subject in need thereof exhibits chronic kidney disease and / or kidney failure.

5. The method of claim 1, wherein the one or more sigma- 1 receptor agonists are selected from pre-084 and pridopidine.

6. The method of claim 1, wherein the one or more sigma- 1 receptor agonists comprises a selective sigma- 1 receptor agonist.

7. The method of claim 1, wherein the administering one or more sigma- 1 receptor agonists to a subject in need thereof comprises administration of the one or more sigma-1 receptor agonists daily, weekly, monthly, or yearly.

8. A method for reducing a number of cysts in a kidney of a subject in need thereof, comprising administering one or more sigma- 1 receptor agonists to a subject in need thereof.

9. The method of claim 8, wherein the subject in need thereof is diagnosed with autosomal dominant PKD.

10. The method of claim 8, wherein the subject in need thereof has one or more mutations, deletions, or insertions to the PKD1 or PKD2 genes.

11. The method of claim 8, wherein the subject in need thereof exhibits chronic kidney disease and / or kidney failure.

12. The method of claim 8, wherein the one or more sigma-1 receptor agonists are selected from pre-084 and pridopidine.

13. The method of claim 8, wherein the one or more sigma- 1 receptor agonists comprises a selective sigma- 1 receptor agonist.

14. The method of claim 8, wherein the administering one or more sigma-1 receptor agonists to a subject in need thereof comprises administration of the one or more sigma-1 receptor agonists daily, weekly, monthly, or yearly.

15. The method of claim 1 or claim 8, further comprising performing an imaging examination of a kidney of the subject in need thereof, before, after, or during treatment with the one or more sigma-1 receptor agonists.

16. The method of claim 15, wherein the imaging examination comprises one or more of an ultrasound, CT scan, or MRI.

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