Pharmaceutical composition for use in treatment of nephronophthisis

Abstract

One of the purposes of the present invention is to provide a pharmaceutical composition for use in the treatment of nephronophthisis, and in particular, one of the purposes of the present invention is to provide a pharmaceutical composition for use in the treatment of NPHP1-deficient nephronophthisis. As one of the solutions, the present invention provides a pharmaceutical composition for use in the treatment of nephronophthisis, the pharmaceutical composition containing a Hippo signaling inhibitor.

Description

Pharmaceutical composition for treating nephronophthisis

[0001] The present invention relates to a pharmaceutical composition for treating nephronophthisis.

[0002] Nephronophthisis (NPH) is an autosomal recessive renal disease accompanied by fibrosis and microcyst formation due to tubule dilation in the renal tubulointerstitium, and ultimately develops end-stage renal disease (ESKD; also referred to as ESRD) [Non-Patent Document 1]. There is no established treatment for NPH [Non-Patent Document 2]. It is a kidney disease in children and is the most common cause of ESKD in hereditary kidney diseases in children [Non-Patent Document 3].

[0003] As the causative gene, the frequency of NPHP1 is the highest, accounting for about 50% of all cases of NPH. Among them, homozygous complete gene deletion of NPHP1 is common [Non-Patent Document 4].

[0004] NPHP1-deficient NPH exhibits some characteristic phenotypes compared to NPH derived from other causative genes. The first is the onset time. NPH is clinically classified into an infantile type, a juvenile type, and a late-onset type according to the diagnosis time. Compared with other forms of NPH, patients with NPHP1-deficient NPH have a delayed onset of renal dysfunction and are observed at the highest frequency in the late-onset group [Non-Patent Documents 2, 5]. In a study of adult patients with ESKD, patients with NPHP1-deficient NPH accounted for 0.5% of the population. The median age at which patients developed ESKD was 30 years (range: 18 - 61 years) [Non-Patent Document 6]. Therefore, NPHP1-deficient NPH is important as a causative disease not only in pediatric patients with CKD but also in adult patients.

[0005] The second is histopathological observation. Characteristic histopathological findings of NPH include disruption of the tubular basement membrane with irregular thickness [Non-Patent Literature 7], interstitial fibrosis, tubular atrophy, and cysts at the corticomedullary junction [Non-Patent Literature 8]. NPHP1-deficient NPH is distinguished by the earlier onset of renal fibrosis compared to other forms of NPH [Non-Patent Literature 9, 10, 11]. In a histological study of renal biopsies from 45 pediatric patients with CKD, interstitial fibrosis was observed in 67% of NPHP1-deficient NPH patients, and cysts were observed in only 22%. The prevalence of cysts was not different compared to non-NPH CKD patients (P = 0.6) [Non-Patent Literature 12]. This indicates that, unlike NPH caused by genes other than NPHP1, fibrosis is the primary etiology of NPHP1-deficient NPH.

[0006] Numerous mouse models of NPHP1-deficient NPH exist [Non-Patent Literature 13, 14, 15, 16], but they cannot fully reproduce the pathology of the human kidney. This difference in renal phenotype between humans and mice poses a challenge in understanding the pathology of NPHP1-deficient NPH and developing treatments for it.

[0007] Recently, the present inventors have generated induced pluripotent stem cells (iPSCs) lacking NPHP1 using the CRISPR / Cas9 system [Non-Patent Literature 17].

[0008] For nephronophthria, no existing treatments (such as ACE inhibitors, ARBs, and SGLT2 inhibitors) have demonstrated a consistent therapeutic effect. In particular, there is a need for new drugs that fundamentally reduce the risk of end-stage renal failure.

[0009] Hildebrandt F, Benzing T, Katsanis N. Ciliopathies. N Engl J Med. 2011;364(16):1533-1543. doi:10.1056 / NEJMra1010172Petzold F, Billot K, Chen X, et al. The genetic landscape and clinical spectrum of nephronophthisis and related ciliopathies. Kidney Int. 2023;104(2):378-387. doi:10.1016 / j.kint.2023.05.007Gupta S, Ozimek-Kulik JE, Phillips JK. Nephronophthisis-Pathobiology and Molecular Pathogenesis of a Rare Kidney Genetic Disease. Genes (Basel). 2021;12(11). doi:10.3390 / genes12111762Hildebrandt F, Zhou W. Nephronophthisis-associated ciliopathies. J Am Soc Nephrol. 2007;18(6):1855-1871. doi:10.1681 / ASN.2006121344Li J, Su X, Zhang H, et al. Genotype and phenotype analysis and transplantation strategy in children with kidney failure caused by NPHP. Pediatric Nephrology. 2023;38(5):1609-1620. doi:10.1007 / s00467-022-05763-3Snoek R, Van Setten J, Keating BJ, et al. NPHP1 (Nephrocystin-1) gene deletions cause adult-onset ESRD. Journal of the American Society of Nephrology. 2018;29(6):1772-1779.doi:10.1681 / ASN.2017111200Fujimaru T, Kawanishi K, Mori T, et al. Genetic Background and Clinicopathologic Features of Adult-Onset Nephronophthisis. Kidney Int Rep. 2021;6(5):1346-1354. doi:10.1016 / j.ekir.2021.02.005Slaats GG, Lilien MR, Giles RH. Nephronophthisis: should we target cysts or fibrosis? Pediatr Nephrol. 2016;31(4):545-554. doi:10.1007 / s00467-015-3162-yKonig J, Kranz B, Konig S, et al. Phenotypic spectrum of children with nephronophthisis and related ciliopathies. Clinical Journal of the American Society of Nephrology. 2017;12(12):1974–1983. doi:10.2215 / CJN.01280217Stokman MF, van der Zwaag B, van de Kar NCAJ, et al. Clinical and genetic analyzes of a Dutch cohort of 40 patients with nephronophthisis-related ciliopathy. Pediatric Nephrology. 2018;33(10):1701-1712. doi:10.1007 / s00467-018-3958-7Leggatt G, Cheng G, Narain S, et al.A genotype-to-phenotype approach suggests under-reporting of single nucleotide variants in nephrocystin-1 (NPHP1) related disease (UK 100,000 Genomes Project). Sci Rep. 2023;13(1). doi:10.1038 / s41598-023-32169-4Larsen CP, Bonsib SM, Beggs ML, et al. Fluorescence in situ hybridization for the diagnosis of NPHP1 deletion-related nephronophthisis on renal biopsy. Hum Pathol. 2018;81:71-77. doi:10.1016 / j.humpath.2018.06.021Jiang ST, Chiou YY, Wang E, et al. Targeted disruption of Nphp1 causes male infertility due to defects in the later steps of sperm morphogenesis in mice. Hum Mol Genet. 2008;17(21):3368-3379. doi:10.1093 / hmg / ddn231Louie CM, Caridi G, Lopes VS, et al. AHI1 is required for photoreceptor outer segment development and is a modifier for retinal degeneration in nephronophthisis. Nat Genet. 2010;42(2):175-180. doi:10.1038 / ng.519Li D, Hu M, Chen H, et al. An Nphp1 knockout mouse model targeting exon 2-20 demonstrates characteristic phenotypes of human nephronophthisis.Hum Mol Genet. 2022;31(2):232-243. doi:10.1093 / hmg / ddab239Garcia H, Serafin AS, Silbermann F, et al. Agonists of prostaglandin E2 receptors as potential first in class treatment for nephronophthisis and related ciliopathies. Proc Natl Acad Sci US A. 2022;119(18):e2115960119. doi:10.1073 / pnas.2115960119Nakano Y, Susa K, Yanagi T, et al. Generation of NPHP1 knockout human pluripotent stem cells by a practical biallelic gene deletion strategy using CRISPR / Cas9 and ssODN. In Vitro Cell Dev Biol Anim. 2022;58(2):85-95. doi:10.1007 / s11626-022-00655-0.

[0010] Nephronophthisis (NPH) is a hereditary kidney disease characterized by progressive renal fibrosis. Originally, it was the primary underlying disease accounting for 5-10% of end-stage renal failure in children, but recently it has been revealed to account for 0.5% of end-stage renal failure in adults, increasing the importance of developing treatments. However, due to the difficulty in creating disease models that can reproduce the phenotype, such as the absence of renal phenotype in NPHP1-deficient gene-modified mice, not only treatments but also the molecular mechanisms of onset remained unclear, even for nephronophthisis caused by NPHP1 deficiency, which is the most common type. Therefore, the inventors of this study created 3D kidney organoids using human iPS cells lacking NPHP1 and investigated the disease mechanism and potential therapeutic agents.

[0011] The present invention aims to provide a pharmaceutical composition for use in the treatment of nephronophthous phlebota, and more particularly, a pharmaceutical composition for use in the treatment of NPHP1-deficient nephronophthous phlebota.

[0012] In this invention, the inventors generated kidney organoids from NPHP1-deficient iPSCs. The inventors evaluated whether the organoid model could adequately reproduce fibrosis associated with NPHP1-deficient NPH. The inventors discovered a link between NPHP1 and the Hippo signaling pathway that leads to renal fibrosis. Finally, the inventors investigated whether Hippo signaling inhibitors act as therapeutic agents by suppressing fibrosis in NPHP1-deficient organoids.

[0013] Specifically, the inventors first conducted an evaluation using an experimental system in which IL-1β, an inflammatory cytokine known as a model for observing fibrosis in kidney organoids, is administered. They found that fibrosis was enhanced in NPHP1-deficient kidney organoids at a dose of IL-1β that did not enhance fibrosis in wild-type kidney organoids. This indicates that fibrosis is significantly promoted in NPHP1-deficient kidney organoids compared to wild-type organoids. This represents the world's first disease model successfully reproducing the phenotype of nephronophthiriasis using human cells. Next, the inventors searched for proteins that bind to NPHP1 through interactome analysis and co-immunoprecipitation experiments, and elucidated that LATS1 / 2, a major component of the Hippo signaling pathway, binds to and interacts with NPHP1.

[0014] The Hippo signaling pathway is a signaling system whose involvement in various biological phenomena, such as tumors and fibrosis, is being elucidated. Under normal conditions, LATS1 / 2 suppressively regulates the downstream YAP / TAZ pathway, thereby suppressing the expression of fibrosis-related genes. Therefore, the inventors hypothesized that "in nephronophthisis, NPHP1 is deficient, leading to a loss of interaction with LATS1 / 2, resulting in enhanced fibrosis." To verify this, they evaluated whether Hippo signaling inhibitors have therapeutic effects and conducted iPS cell-based drug discovery. Specifically, three types of Hippo signaling inhibitors—ivermectin, verteporfin, and bryostatin—were administered to NPHP1-deficient kidney organoids, and immunofluorescence staining was performed. The results showed that all three drugs had a significant inhibitory effect on both the upregulation of fibrosis-related genes and fibrosis markers in the Hippo signaling pathway induced by IL-1β. Furthermore, ivermectin and verteporfin are already drugs that have obtained domestic insurance approval and are marketed and clinically used for other diseases, and are therefore considered to have few toxicity issues.

[0015] These results represent the world's first discovery demonstrating that inhibiting the Hippo signaling pathway improves renal fibrosis in NPHP1-deficient nephronophthosis.

[0016] In other words, iPSC-based drug discovery identified the Hippo signaling pathway as a therapeutic target for fibrosis in NPHP1-deficient nephronophthiriasis. This is described in further detail below.

[0017] Nephronophthisis (NPH) is an autosomal recessive kidney disorder. NPHP1 is the most frequently affected gene, accounting for approximately 50% of all NPH cases. Tubulointerstitial fibrosis is the main phenotype of NPHP1-deficient NPH. The pathophysiology of NPHP1-deficient NPH is unclear due to a lack of models that accurately represent the disease's pathophysiology. Therefore, the inventors aimed to create a novel disease model of NPH using 3D kidney organoids derived from human induced pluripotent stem cells (iPSCs) and to explore potential treatments while elucidating the pathophysiology. NPHP1-deficient kidney organoids were generated from iPSCs. Induction of fibrosis by IL-1β significantly induced fibronectin expression and transcription of fibrosis-related genes such as FN1, COL1A, and COL3A in NPHP1-deficient organoids. Long-term culture of NPHP1-deficient organoids induced significantly greater fibril formation compared to wild-type organoids. Co-immunoprecipitation analysis revealed that NPHP1 binds to LATS1 / 2, a major component of the Hippo signaling pathway. Administration of IL-1β increased the expression of important Hippo signaling genes such as CTGF, CYR61, and ANKRD1 in NPHP1-deficient organoids. In contrast, Hippo signaling inhibitors improved IL-1β-induced fibrillation in NPHP1-deficient organoids. Since verteporfin, one of the inhibitors, is in clinical use, its practical application is expected from a drug repositioning perspective. The inventors created a pathophysiological model of NPHP1-deficient NPH for use in screening therapeutic drugs. The inventors' research results indicate that the Hippo signaling pathway is involved in fibrillatory changes associated with NPHP1-deficient NPH, and that Hippo signaling inhibitors can be therapeutic agents.

[0018] The above is summarized below. Patients with NPHP1-deficient nephronoplasia (NPH) almost always develop end-stage renal disease due to tubulointerstitial fibrosis. In this invention, the inventors generated NPHP1-deficient 3D kidney organoids derived from iPSCs. In fibrosis induction experiments using IL-1β, NPHP1-deficient organoids showed more advanced fibrosis than wild-type organoids. The Hippo signaling pathway is activated in fibrosis-induced NPHP1-deficient organoids. Treatment with Hippo signaling inhibitors, including drugs used in clinical practice, suppressed fibrosis. The inventors' research results indicate that the Hippo signaling pathway is involved in fibrosis in NPHP1-deficient NPH and that Hippo signaling inhibitors may be therapeutic agents.

[0019] In other words, the present invention provides the following: [Aspect 1] A pharmaceutical composition for use in the treatment of nephronophthosis, comprising a Hippo signaling inhibitor, wherein the Hippo signaling inhibitor is a substance that inhibits YAP / TAZ. [Aspect 2] The pharmaceutical composition according to Aspect 1, wherein the Hippo signaling inhibitor is a substance that inhibits the function of YAP / TAZ-TEAD. [Aspect 3] The pharmaceutical composition according to Aspect 1, wherein the Hippo signaling inhibitor is selected from the group consisting of peptide 17, ivermectin, verteporfin, bryostatin, and statin drugs. [Aspect 4] The pharmaceutical composition according to Aspect 3, wherein the statin drug is selected from the group consisting of atorvastatin and pitavastatin. [Aspect 5] The pharmaceutical composition according to Aspect 1, wherein the nephronophthosis is NPHP1-deficient nephronophthosis. [Aspect 6] A pharmaceutical composition for use in suppressing renal fibrosis of nephronophthosis, comprising a Hippo signaling inhibitor. [Aspect 7] A renal organoid lacking NPHP1. [Aspect 8] A kidney organoid according to Aspect 7, derived from stem cells lacking NPHP1. [Aspect 9] A kidney organoid according to Aspect 8, wherein the stem cells are iPS cells. [Aspect 10] A method for screening candidate substances for an active ingredient in a pharmaceutical composition for use in the treatment of nephronophthosis, comprising the step of identifying a candidate substance as the active ingredient that suppresses fibrosis in an NPHP1-deficient kidney organoid that has induced fibrosis upon administration to the said kidney organoid. [Aspect 11] The method according to Aspect 10, wherein the induction of fibrosis in the NPHP1-deficient kidney organoid is by IL-1β. [Aspect 12] A method for treating nephronophthosis, comprising the step of administering a therapeutically effective amount of a Hippo signaling inhibitor to a subject requiring treatment for nephronophthosis, wherein the Hippo signaling inhibitor is a substance that inhibits YAP / TAZ. [Aspect 13] The method according to Aspect 12, wherein the Hippo signaling inhibitor is a substance that inhibits the function of YAP / TAZ-TEAD. [Aspect 14] The method according to aspect 12, wherein the Hippo signaling inhibitor is selected from the group consisting of peptide 17, ivermectin, verteporfin, briostatin, and statin drugs.[Aspect 15] The method according to aspect 14, wherein the statin drug is selected from the group consisting of atorvastatin and pitavastatin. [Aspect 16] The method according to aspect 12, wherein the nephronophthria is NPHP1-deficient nephronophthria. [Aspect 17] A method for inhibiting renal fibrosis of nephronophthria, comprising the step of administering a therapeutically effective amount of a Hippo signaling inhibitor to a subject requiring inhibition of renal fibrosis of nephronophthria. [Aspect 18] Use of a Hippo signaling inhibitor in the manufacture of a pharmaceutical product for use in the treatment of nephronophthria, wherein the Hippo signaling inhibitor is a substance that inhibits YAP / TAZ. [Aspect 19] The use according to aspect 18, wherein the Hippo signaling inhibitor is a substance that inhibits the function of YAP / TAZ-TEAD. [Aspect 20] The use according to aspect 18, wherein the Hippo signaling inhibitor is selected from the group consisting of peptide 17, ivermectin, verteporfin, briostatin, and statin drugs. [Aspect 21] The use according to aspect 20, wherein the statin drug is selected from the group consisting of atorvastatin and pitavastatin. [Aspect 22] The use according to aspect 18, wherein the nephronophthae is NPHP1-deficient nephronophthae. [Aspect 23] The use of a Hippo signaling inhibitor in the manufacture of a pharmaceutical product for use in suppressing renal fibrosis of nephronophthae.

[0020] The pharmaceutical composition of the present invention can treat nephronoplasia. In a more specific embodiment, the pharmaceutical composition of the present invention can treat NPHP1-deficient nephronoplasia. The renal organoid of the present invention can be used as a disease model that reproduces the phenotype of nephronoplasia.

[0021] Generation of 3D kidney organoids using NPHP1-deficient iPSCs. (a) Bright-field imaging of wild-type and NPHP1-deficient kidney organoids at day 28. Black scale bar: 200 μm. (b) Representative images of the morphology of wild-type and NPHP1-deficient kidney organoids at day 28. Immunofluorescence (IF) staining of podocalyxin (PODXL, white), E-cadherin (CDH1, white), and lotus tetragonolobus lectin (LTL, white). White scale bar: 200 μm. (c) Representative IF images of fibrosis markers α-smooth muscle actin (α-SMA, white) or fibronectin (white) for comparison of fibrosis. White scale bar: 200 μm. The inset in the lower right shows DAPI staining. (d) Quantification of α-SMA-positive or fibronectin-positive regions in IF images. Values ​​are expressed as a ratio to the mean signal of wild-type organoids. Each dot represents one field (n = 9 images). Welch's t-test was used for comparison. Bar graphs show mean ± SEM. ns, not significant. (e) Expression of fibrosis-related genes FN1, ACTA2, COL1A, and COL3A was examined by quantitative PCR using cDNA from whole organoids. Transcription levels were normalized by GAPDH expression and expressed as a ratio to the mean level of wild-type organoids. Each dot represents one sample obtained from three organoids (n = 3 samples). Welch's t-test was used for comparison. Bar graphs show mean ± SEM. ns, not significant. Fibrosis in NPHP1-deficient organoids highlighted by IL-1β administration. (a) Representative IF images of fibronectin (white) expressed in wild-type or NPHP1-deficient organoids after low-dose IL-1β administration. White scale bar is 200 μm. The inset in the lower right shows DAPI. (b) Quantification of fibronectin-positive regions in IF images. Left: Representative image of the extracted region. White scale bar is 50 μm. Right: Bar graph comparing intensities. Values ​​are expressed as the ratio of the mean signal of wild-type organoids. Each dot represents one field (n = 15 images). Tukey's range test was used for comparison. Bar graphs are shown as mean ± SEM. ***P<0.001, ns, not significant.(c) Expression of fibrosis-related genes ACTA2, FN1, COL1A, and COL3A in wild-type or NPHP1-deficient organoids treated with low-dose IL-1β was examined by quantitative PCR. Transcriptional levels were normalized to GAPDH expression and expressed as a ratio to the mean level of wild-type organoids. Each dot represents one sample obtained from three organoids (n = 3 samples). Welch's t-test was used for comparison. Bar graphs show mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. Interaction of NPHP1 with components of the Hippo signaling pathway. (a) Binding of NPHP1 to LATS1 and LATS2. Myc-tagged LATS1 or LATS2 was co-expressed with Flag-tagged NPHP1 in HEK293T cells. Cell lysates were collected and the protein complexes were eluted by co-immunoprecipitation. Western blotting was performed using antibodies against Flag or Myc. (b) Hippo signaling pathway and NPHP1. YAP / TAZ binds to TEAD and induces transcription of downstream genes such as CTGF, CYR61, and ANKRD1. LATS1 / 2 inhibits YAP / TAZ activity. NPHP1 binds to LATS1 / 2. (c) Expression of CTGF, CYR61, and ANKRD1, Hippo pathway signature genes associated with fibrosis, was examined by quantitative PCR using cDNA from wild-type and NPHP1-deficient kidney organoids treated with low-dose IL-1β. Transcription levels were normalized to GAPDH expression and expressed as a ratio to the mean level of wild-type organoids. Each dot represents one sample obtained from three organoids (n = 3 samples). Welch's t-test was used for comparison. Bar graphs show mean ± SEM. **P<0.01, ***P<0.001, ns, not significant. Suppression of fibrillation in NPHP1-deficient organoids by Hippo pathway inhibitory peptide 17. (a) Representative IF image of fibronectin (white) showing the inhibitory effect of Hippo pathway inhibitory peptide 17 on IL-1β-mediated fibrosis in NPHP1-deficient kidney organoids. Organoids were collected and sectioned 7 days after drug exposure. White scale bar: 200 μm.The inset in the lower right shows DAPI staining. (b) Quantification of fibronectin-positive regions in IF images. Values ​​are expressed as a ratio to the mean signal of the control group. Each dot represents one field (n = 18 images). Tukey's range test was used for comparison. Bar graphs show mean ± SEM. ***P<0.001. (c) Expression of CTGF, CYR61, and ANKRD1 was examined using quantitative PCR of cDNA from whole organoids. Transcription levels were normalized to GAPDH expression and expressed as a ratio to the mean level of the control group. Each dot represents one sample obtained from three organoids (n = 3 samples). Tukey's range test was used for comparison. Bar graphs show mean ± SEM. ***P<0.001. (d) Expression of FN1, COL1A, and COL3A was examined using quantitative PCR of cDNA from whole organoids. Transcription levels are normalized to GAPDH expression and expressed as a ratio to the mean level of the control group. Each dot represents one sample obtained from three organoids (n = 3 samples). Tukey's range test was used for comparison. Bar graphs show mean ± SEM. *P<0.05, ***P<0.001. Inhibition of fibrillation in NPHP1-deficient organoids by the Hippo pathway inhibitor verteporfin. (a) Representative IF image of fibronectin (white) showing the inhibitory effect of the Hippo pathway inhibitor verteporfin on IL-1β-mediated fibrosis in NPHP1-deficient kidney organoids. Organoids were collected and sectioned 7 days after drug exposure. Representative IF image of fibronectin (white). White scale bar: 200 μm. The inset in the lower right shows DAPI staining. (b) Quantification of fibronectin-positive regions in IF images. Values ​​are expressed as a ratio to the mean signal of the control group. Each dot represents one field (n = 18 images). Tukey's range test was used for comparison. Bar graphs show mean ± SEM. ***P<0.001. (c) Expression of CTGF, CYR61, and ANKRD1 was examined using quantitative PCR of cDNA from whole organoids.Transcription levels are normalized to GAPDH expression and expressed as a ratio to the mean level of the control group. Each dot represents one sample obtained from three organoids (n = 3 samples). Tukey's range test was used for comparison. Bar graphs show mean ± SEM. ***P<0.001. (d) Expression of FN1, COL1A, and COL3A was examined using quantitative PCR of cDNA from the entire organoid. Transcription levels are normalized to GAPDH expression and expressed as a ratio to the mean level of the control group. Each dot represents one sample obtained from three organoids (n = 3 samples). Tukey's range test was used for comparison. Bar graphs show mean ± SEM. ***P<0.001. Figure 6 shows that ivermectin also showed an inhibitory effect on fibrosis in NPHP1-deficient organoids. Figure 7 shows that bryostatin also showed an inhibitory effect on fibrosis in NPHP1-deficient organoids. Figure 8 shows that atorvastatin and pitavastatin also demonstrated inhibitory effects on fibrosis in NPHP1-deficient organoids. Schematic diagram of research results. NPHP1 deficiency leads to LATS dysfunction and suppression of the Hippo pathway. Activation of nuclear YAP / TAZ increases the expression of fibrosis-inducing genes, leading to fibrosis. Hippo pathway inhibitors directly inhibit YAP / TEAD, suppressing fibrosis even in the absence of NPHP1 deficiency. Severe fibrosis of NPHP1-deficient kidney organoids induced by long-term culture. (a) Representative IF images of fibronectin (white) from wild-type and NPHP1-deficient kidney organoids cultured for 90 days after differentiation to compare fibrosis. White scale bar: 200 μm. The inset in the lower right shows DAPI staining. (b) Quantification of fibronectin-positive regions in IF images. Values ​​are expressed as the ratio of the mean signal of wild-type organoids. Each dot represents one field (n = 7 images). Welch's t-test was used for comparison. Bar graphs show mean ± SEM. ***P<0.001. (c) Expression of FN1, COL1A, and COL3A was examined using quantitative PCR of cDNA from whole organoids.Transcription levels were normalized to GAPDH expression and expressed as a ratio to the mean level of wild-type organoids. Each dot represents one sample obtained from three organoids (n = 3 samples). Welch's t-test was used for comparison. Bar graphs show mean ± SEM. *P<0.05, ***P<0.001, ns, not significant. (d) Expression of CTGF, CYR61, and ANKRD1 was examined using quantitative PCR of cDNA from whole organoids. Transcription levels were normalized to GAPDH expression and expressed as a ratio to the mean level of wild-type organoids. Each dot represents one sample obtained from three organoids (n = 3 samples). Welch's t-test was used for comparison. Bar graphs show mean ± SEM. ***P<0.001. Fibrosis of renal organoids by activation of the Hippo signaling pathway via etaclidin. (a) Representative IF images of fibronectin (white) showing fibrillation in wild-type and NPHP1-deficient kidney organoids mediated by Hippo pathway activator etaclidin. Organoids were collected and sectioned 7 days after drug treatment. White scale bar: 200 μm. The inset in the lower right shows DAPI staining. (b) Quantification of fibronectin-positive areas in IF images. Values ​​are expressed as a ratio to the mean signal of wild-type organoids. Each dot represents one field (n = 3 images). Tukey's range test was used for comparison. Bar graphs show mean ± SEM. **P<0.01, ***P<0.001.

[0022] Pharmaceutical composition: The present invention provides a pharmaceutical composition for use in the treatment of nephronophthria, comprising a Hippo signaling inhibitor. More specifically, the present invention provides a pharmaceutical composition for use in the treatment of nephronophthria, comprising a Hippo signaling inhibitor as an active ingredient.

[0023] Examples of Hippo signaling inhibitors include substances that inhibit YAP / TAZ, particularly substances that inhibit the function of YAP / TAZ-TEAD. However, Hippo signaling inhibitors may also be substances that specifically inhibit YAP / TAZ, particularly substances that specifically inhibit the function of YAP / TAZ-TEAD. In this disclosure, "specifically inhibiting" means that the substance has significantly higher affinity or selective inhibitory activity towards the Hippo signaling pathway, i.e., YAP / TAZ or the YAP / TAZ-TEAD complex, compared to other signaling pathways or other protein complexes; or, even if the substance has multiple mechanisms of action, at the effective dose, the suppression of the Hippo signaling pathway (particularly inhibition of nuclear translocation of YAP / TAZ or inhibition of binding to TEAD) is the dominant effect; or, the inhibitory activity (IC50 value, etc.) towards Hippo signaling-related factors is significantly lower than the inhibitory activity towards other target proteins. However, the Hippo signaling inhibitors in this disclosure may also act on targets other than YAP / TAZ, and therefore do not necessarily have to be "specific."

[0024] YAP / TAZ inhibition can also be described as suppression of YAP / TAZ activation. As mentioned above, YAP / TAZ binds to TEAD and induces transcription of downstream genes such as CTGF, CYR61, and ANKRD1. Therefore, inhibition of YAP / TAZ-TEAD function includes inhibition of the binding of YAP / TAZ to TEAD. Furthermore, inhibition of YAP / TAZ-TEAD function includes not only direct inhibition of complex formation, but also reducing the concentration of YAP / TAZ that can interact with TEAD in the nucleus by suppressing the translocation of YAP / TAZ from the cytoplasm to the nucleus or promoting its export from the nucleus to the cytoplasm (suppression of nuclear translocation); promoting the phosphorylation of YAP / TAZ by activating or preventing the inactivation of upstream factors of the Hippo pathway (such as LATS1 / 2), thereby keeping them in the cytoplasm or inducing degradation by the proteasome (promotion of inactivation via upstream kinases); and inhibiting the function of YAP / TAZ as a coupled transcription activator even when it is bound to TEAD, thereby reducing the transcription efficiency of downstream fibrillation-related genes (such as CTGF, CYR61, and ANKRD1) (suppression of transcriptional activation ability).

[0025] Examples of Hippo signaling inhibitors include statins. Examples of statins include atorvastatin, pitavastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, and lovastatin, which are used to treat dyslipidemia. In some embodiments, the pharmaceutical composition according to this disclosure may be a pharmaceutical composition for use in the treatment of nephronophthiriasis, comprising a statin. Furthermore, in some embodiments, the pharmaceutical composition according to this disclosure may be a pharmaceutical composition for use in the treatment of nephronophthiriasis, comprising a drug selected from the group consisting of atorvastatin, pitavastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, and lovastatin. More specifically, the pharmaceutical composition according to this disclosure may be a pharmaceutical composition for use in the treatment of nephronophthiriasis, comprising atorvastatin, or a pharmaceutical composition for use in the treatment of nephronophthiriasis, comprising pitavastatin.

[0026] Drugs that activate AMP-activated protein kinase (AMPK) also have a Hippo signaling inhibitory effect, including metformin, teneligliptin, and miglitol used in the treatment of diabetes, as well as bempedoic acid used in the treatment of dyslipidemia.

[0027] Examples of Hippo signaling inhibitors include peptide-17, ivermectin, verteporfin, and briostatin. Peptide-17, ivermectin, verteporfin, and briostatin may also be derivatives.

[0028] Peptide 17 is YAP-TEAD Inhibitor 1, an inhibitor of the YAP-TEAD protein-protein interaction. Peptide 17 is a 17-residue cyclic peptide obtained by structure-based design, and because it has a higher binding affinity to TEAD1 than YAP (50-171), it inhibits the YAP-TEAD protein-protein interaction (PPI) as a high-affinity ligand for the YAP binding site.

[0029] Ivermectin is an antiparasitic drug classified as a macrolide. Ivermectin is a chemical derivative of avermectin, which is produced by actinomycetes. Ivermectin specifically and with high affinity binds to glutamatergic chloride ion channels (GluCl) present in the nerve and muscle cells of invertebrates, and binds to chloride ions (Cl - It increases the permeability of the cell membrane to )

[0030] Verteporfin is a benzoporphyrin derivative. Verteporfin can be used as a photosensitizer in photodynamic therapy to remove abnormal blood vessels in the eye associated with conditions such as wet macular degeneration.

[0031] Briostatin is a macrolactone compound (macrolide). Briostatin 1 is a potent activator of protein kinase C, similar to 12-O-tetradecanoylphorbol 13-acetate (TPA), a carcinogenic promoter, but it also acts as an antagonist to TPA (e.g., anti-cancer promotion).

[0032] The pharmaceutical composition of the present invention can treat the target nephronophthys. Treatment of nephronophthys includes not only the treatment of nephronophthys but also its prevention. Treatment of nephronophthys includes not only complete cure of nephronophthys but also improvement of the symptoms of nephronophthys and alleviation of the symptoms of nephronophthys. Examples of nephronophthys include NPHP1-deficient nephronophthys.

[0033] The present invention provides a pharmaceutical composition for use in suppressing renal fibrosis, particularly nephronophthiriasis fibrosis, comprising a Hippo signaling inhibitor. The pharmaceutical composition of the present invention can suppress renal fibrosis, particularly nephronophthiriasis fibrosis, in a target population. In some embodiments, the pharmaceutical composition according to the present disclosure may be a pharmaceutical composition for use in suppressing nephronophthiriasis fibrosis, comprising a statin. Furthermore, in some embodiments, the pharmaceutical composition according to the present disclosure may be a pharmaceutical composition for use in suppressing nephronophthiriasis fibrosis, comprising an agent selected from the group consisting of atorvastatin, pitavastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, and lovastatin. More specifically, the pharmaceutical composition according to the present disclosure may be a pharmaceutical composition for use in suppressing nephronophthiriasis fibrosis, comprising atorvastatin, or a pharmaceutical composition for use in suppressing nephronophthiriasis fibrosis, comprising pitavastatin.

[0034] Examples of subjects include vertebrates. Examples of vertebrates include mammals such as mice, rats, rabbits, pigs, cattle, monkeys, and humans. Mammals are preferably humans. Subjects may be of any age, including infants, young people, adolescents, adults, and the elderly.

[0035] The pharmaceutical composition of the present invention is intended to be administered by means of, for example, intrathecal administration, oral administration, intravenous administration, subcutaneous administration, transdermal administration, intramuscular administration, intra-articular administration, nasal administration, intraperitoneal administration, direct injection into target tissue, inhalation administration, enteral administration, enema administration, enteral nutrition, etc.

[0036] The pharmaceutical composition of the present invention may be formulated in an orally administered dosage form such as tablets, capsules, elixirs, or microcapsules, or it may be formulated in a parenterally administered dosage form such as injections, ointments, or patches.

[0037] The pharmaceutical composition of the present invention may consist of a Hippo signaling inhibitor, or it may be formulated as a pharmaceutical composition mixed with a pharmaceutically acceptable carrier.

[0038] Examples of pharmaceutically acceptable carriers include solvents such as sterile water and physiological saline; binders such as gelatin, corn starch, tragacanth gum, and gum arabic; excipients such as crystalline cellulose; and leavening agents such as corn starch, gelatin, and alginic acid.

[0039] Examples of pharmaceutically acceptable carriers include additives. These additives include lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, and saccharin; flavoring agents such as peppermint and red ginger oil; stabilizers such as benzyl alcohol and phenol; buffering agents such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives; surfactants; and emulsifiers.

[0040] The pharmaceutical composition can be formulated, for example, by appropriately combining the above-mentioned carriers and mixing them in a unit dose form generally accepted for pharmaceutical production.

[0041] When the pharmaceutical composition is an injectable preparation, suitable solvents for injection include, for example, physiological saline, glucose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other adjuvants in an isotonic solution. The solvent for injection may also contain alcohols such as ethanol; polyalcohols such as propylene glycol and polyethylene glycol; and nonionic surfactants such as polysorbate 80 (trademark) and HCO-50.

[0042] The pharmaceutical composition of the present invention may be accompanied by instructions. The instructions may include, for example, descriptions of the active ingredients contained therein, the dosage per administration of the Hippo signal inhibitor as the active ingredient, the number of administrations per day, the administration method, the total number of administrations, the dosage per administration, and the like. The attachment of the instructions does not necessarily require the physical presence of a written document and may be carried out via a telecommunication line such as the Internet.

[0043] The present invention provides a method for treating nephron sclerosis in a subject, which comprises administering a Hippo signal inhibitor to a subject requiring treatment. In some embodiments, the method for treating nephron sclerosis according to the present disclosure may be a method for treating nephron sclerosis that includes administering a statin drug. Further, in some embodiments, the method for treating nephron sclerosis according to the present disclosure may be a method for treating nephron sclerosis that includes administering a drug selected from the group consisting of atorvastatin, pitavastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, and lovastatin. More specifically, the method for treating nephron sclerosis according to the present disclosure may be a method for treating nephron sclerosis that includes administering atorvastatin, or a method for treating nephron sclerosis that includes administering pitavastatin.

[0044] The present invention provides a method for suppressing renal fibrosis in a subject, which comprises administering a Hippo signal inhibitor to a subject requiring treatment. In some embodiments, the method for suppressing renal fibrosis according to the present disclosure may be a method for suppressing renal fibrosis that includes administering a statin drug. Further, in some embodiments, the method for suppressing renal fibrosis according to the present disclosure may be a method for suppressing renal fibrosis that includes administering a drug selected from the group consisting of atorvastatin, pitavastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, and lovastatin. More specifically, the method for suppressing renal fibrosis according to the present disclosure may be a method for suppressing renal fibrosis that includes administering atorvastatin, or a method for suppressing renal fibrosis that includes administering pitavastatin.

[0045] The present invention provides a treatment for nephronophthosis comprising a Hippo signaling inhibitor as an active ingredient. In some embodiments, the treatment according to the present disclosure may be a treatment for nephronophthosis comprising a statin. Furthermore, in some embodiments, the treatment according to the present disclosure may be a treatment for nephronophthosis comprising a drug selected from the group consisting of atorvastatin, pitavastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, and lovastatin. More specifically, the treatment according to the present disclosure may be a treatment for nephronophthosis comprising atorvastatin, or a treatment for nephronophthosis comprising pitavastatin.

[0046] The present invention provides an inhibitor of renal fibrosis comprising a Hippo signaling inhibitor as an active ingredient. In some embodiments, the inhibitor of renal fibrosis according to the present disclosure may be an inhibitor of renal fibrosis comprising a statin. Furthermore, in some embodiments, the inhibitor of renal fibrosis according to the present disclosure may be an inhibitor of renal fibrosis comprising a drug selected from the group consisting of atorvastatin, pitavastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, and lovastatin. More specifically, the inhibitor of renal fibrosis according to the present disclosure may be an inhibitor of renal fibrosis comprising atorvastatin, or an inhibitor of renal fibrosis comprising pitavastatin.

[0047] The present invention provides the use of Hippo signaling inhibitors in the manufacture of pharmaceutical compositions or pharmaceuticals for the treatment of nephronophthosis. In some embodiments, the use relating to the present disclosure may be the use of statin agents in the manufacture of pharmaceutical compositions or pharmaceuticals for the treatment of nephronophthosis. Furthermore, in some embodiments, the use relating to the present disclosure may be the use of agents selected from the group consisting of atorvastatin, pitavastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, and lovastatin in the manufacture of pharmaceutical compositions or pharmaceuticals for the treatment of nephronophthosis. More specifically, the use relating to the present disclosure may be the use of atorvastatin or pitavastatin in the manufacture of pharmaceutical compositions or pharmaceuticals for the treatment of nephronophthosis.

[0048] The present invention provides the use of a Hippo signaling inhibitor in the manufacture of a pharmaceutical composition or a medicament for use in suppressing renal fibrosis. In some embodiments, the use according to the present disclosure may be the use of a statin drug in the manufacture of a pharmaceutical composition or a medicament for use in suppressing renal fibrosis. Further, in some embodiments, the use according to the present disclosure may be the use of a drug selected from the group consisting of atorvastatin, pitavastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, and lovastatin in the manufacture of a pharmaceutical composition or a medicament for use in suppressing renal fibrosis. More specifically, the use according to the present disclosure may be the use of atorvastatin or the use of pitavastatin in the manufacture of a pharmaceutical composition or a medicament for use in suppressing renal fibrosis.

[0049] Kidney organoid: The present invention provides a kidney organoid lacking NPHP1. The kidney organoid lacking NPHP1 of the present invention may be derived from stem cells lacking NPHP1. Here, examples of the stem cells include iPS cells and the like. The kidney organoid of the present invention can be used as a disease model that reproduces the phenotype of nephronophthisis.

[0050] Screening method: The present invention provides a method for screening an active ingredient in a pharmaceutical composition for use in treating nephronophthisis from a candidate substance, the method including the step of identifying, as the active ingredient, a candidate substance that suppresses fibrosis in a kidney organoid lacking NPHP1 induced to undergo fibrosis by administering the candidate substance to the kidney organoid lacking NPHP1. Here, the induction of fibrosis in the kidney organoid lacking NPHP1 may be, for example, by IL-1β. The induction of fibrosis in the kidney organoid lacking NPHP1 may be, for example, by long-term culture.

[0051] [Method]:

[0052] Antibodies: The primary antibodies used in the immunofluorescence (IF) experiments consisted of goat anti-PODXL (R&D, AF1658), mouse anti-CDH1 (Invitrogen, 13-1700), biotinylated anti-LTL (Vector Lab, B-1325), rabbit anti-α-SMA (Abcam, ab5694), rabbit anti-fibronectin (Abcam, ab2413), and rabbit anti-SIX2 (Proteintech, 11562-1-AP). The secondary antibodies used in the IF experiment consisted of Alexa Fluor 488 donkey anti-mouse IgG (Thermo Fisher Scientific, A21202), Alexa Fluor 488 donkey anti-rabbit IgG (Thermo Fisher Scientific, A21206), Alexa Fluor 555 donkey anti-goat IgG (A11056), Alexa Fluor 555 donkey anti-mouse IgG (A31570), Alexa Fluor 555 donkey anti-rabbit IgG (A31572), streptavidin, and Alexa Fluor 633 (Thermo Fisher Scientific, S21375). The primary antibodies used for Western blotting were mouse anti-Flag M2 (Sigma, F3165) and mouse anti-Myc-Tag (Cell Signaling Technology, Myc-tag). The secondary antibody used for Western blotting consisted of anti-mouse IgG (H+L) and AP conjugate (Promega, S372B).

[0053] Cell Culture: Human iPSC strain 1231A3 was provided by the RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National Bioresource Project and generated NPHP1-deficient iPSCs as reported [In Vitro Cell Dev Biol Anim. 2022;58(2):85-95.]. All iPSCs were maintained in StemFit AK02N (Ajinomoto, RCAK02N) supplemented with 10 μM Y-27632 (Nacalai Tesque, 18188-04) and 100 ng / ml FGF2 (PeproTech, 100-18B). Cell culture plates were coated with hESC-certified Geltrex (Thermo Fisher Scientific, A1413302). HEK 293T cells were maintained in DMEM (Nacalai Tesque, 0845935) supplemented with 10% fetal bovine serum.

[0054] Differentiation of 3D kidney organoids: We followed Morizane's method for inducing differentiation of iPSCs into 3D kidney organoids [Nat Biotechnol. 2015;33(11):1193-1200.;Nat Protoc. 2017;12(1):195-207.]. During differentiation, iPSCs were maintained in Advanced RPMI 1690 (Thermo Fisher, 12633020) supplemented with GlutaMax (Gibco, 35050-061). Initially, iPSCs were treated with 4.5 μM CHIR 99021 (R&D, 252917-06-9) from day 0 to day 4. Next, 10 ng / ml Activin A (R&D, 338-AC) was added from day 4 to day 6, followed by 10 ng / ml FGF-9 (R&D, 273-F9) on day 7, which differentiated the cells into metanephromesenchymal cells, including nephron progenitor cells, with an efficiency of 80%–90%. On day 8, the resulting metanephromesenchymal cells were transferred to a 96-well ultra-low adhesion plate (Sumitomo Bakelite, MS-9096S). The cells were treated with 2.5 μM CHIR 99021 and 10 ng / ml FGF-9 from day 8 to day 10, and with 10 ng / ml FGF-9 from day 10 to day 13. After 28 days of culture, organoids were used in experiments. On day 8, the expression of SIX-2 as a marker for nephron progenitor cells was evaluated by immunofluorescence staining for quality control. On day 21, organoids were immunofluorescently stained using antibodies against CDH1, PODXL, and LTL to identify nephron-like structures. In all experiments, the phenotype of organoids derived from NPHP1-deficient iPSCs was compared to the phenotype of wild-type organoids derived from the parent iPSC 1231A3 strain.

[0055] Immunofluorescence staining of kidney organoids: Organoids were rinsed once with PBS (Nacalai Tesque, 27575-31), fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed three times with PBS, and immersed in 30% sucrose / PBS until submerged. Next, the organoids were immersed in cryomold (Sakura Finetek, 4583) containing OCT compound (optimal cutting temperature compound) and frozen using liquid nitrogen. The frozen blocks were cut into 8 μm slices using Tissue-Tek Polar Cryostat (Sakura Finetek, POLAR-D). Sections were blocked with Blocking One Histo (Nacalai Tesque, 06349-64) at room temperature for 1 hour, washed three times with PBS, and incubated overnight at 4°C with primary antibody. Primary antibody was diluted with 5% Blocking One Histo in PBS. For staining biotinylated LTL, the Avidin / Biotin Blocking Kit (Vector Labs, SP-2001) was used. The following day, slides were washed three times with PBS and incubated with the corresponding secondary antibody combination. Finally, samples were mounted using ProLong Glass Antifade Mountant with NucBlue Stain (Invitrogen, P36981). All slides were examined with a fluorescence microscope (Keyence, BZ-X800) or a confocal microscope (Leica, SP-8, and Nikon, AX80). Sections were scanned with 10x, 20x, or 60x lenses. Images used for comparison were acquired with the same settings. Stained areas were quantified using ImageJ Fiji (https: / / imagej.net / software / fiji / downloads).

[0056] Quantitative PCR: Organoids were lysed using Sepazol-RNA I Super G (Nacalai Tesque, 09379). Total RNA was extracted using Direct-Zol RNA MicroPrep Kit (Zymo Research R2060) according to the manufacturer's instructions. RNA concentration and purity were measured using NanoDrop OneC (Thermo Fisher, ND-ONE-W). RNA was reverse transcribed using ReverTra Ace qPCR RT Master Mix (TOYOBO, FSQ-201). Quantitative PCR was performed using PCR Thermal Cycler Dice (TaKaRa Bio). Transcription levels were normalized to GAPDH expression.

[0057] The following primers were used. Human genes were detected with all primers.

[0058] GAPDH: 5'-GTCTCCTCTGACTTCAACAGCG-3' (SEQ ID NO: 1), and 5'-ACCACCCTGTTGCTGTAGCCAA-3' (SEQ ID NO: 2). ACTA2: 5'-CTATGCCTCTGGACGCACAACT-3' (SEQ ID NO: 3), and 5'-CAGATCCAGACGCATGATGGCA-3' (SEQ ID NO: 4). FN1: 5'-ACAACACCGAGGTGACTGAGAC-3' (SEQ ID NO: 5), and 5'-GGACACAACGATGCTTCCTGAG-3' (SEQ ID NO: 6). COL1A: 5'-GATTCCCTGGACCTAAAGGTGC-3' (SEQ ID NO: 7), and 5'-AGCCTCTCCATCTTTGCCAGCA-3' (SEQ ID NO: 8). COL3A: 5'-TGGTCTGCAAGGAATGCCTGGA-3' (SEQ ID NO: 9), and 5'-TCTTTCCCTGGGACACCATCAG-3' (SEQ ID NO: 10). CTGF: 5'-CTTGCGAAGCTGACCTGGAAGA-3' (SEQ ID NO: 11), and 5'-CCGTCGGTACATACTCCACAGA-3' (SEQ ID NO: 12). CYR61: 5'-GGAAAAGGCAGCTCACTGAAGC-3' (SEQ ID NO: 13), and 5'-GGAGATACCAGTTCCACAGGTC-3' (SEQ ID NO: 14). ANKRD1: 5'-GGAGATACCCCGTTGCATGAT-3' (SEQ ID NO: 15), and 5'-GTAGCACCAGATCCATCGGC-3' (SEQ ID NO: 16).

[0059] Drug induction: NPHP1-deficient organoids were administered 5 μM etaclizine (Selleck S4945), 5 μM peptide-17 (Selleck S8164), and 1 μM verteporfin (Selleck S1786). The concentrations of the reagents used were as reported [J Biochem. 2015;158(5):413-423.; Cancer Res. 2021;81(22):5642-5665.; Nature. 2022;601(7894):600-605.].

[0060] Cell fractionation: Plasmid transfection of HEK 293T cells was performed using Lipofectamine 2000 Reagent (Invitrogen, 11668500) and Opti-MEM (Gibco, 31985-070). After culturing for 3 days, cells were fractionated by centrifugation and total protein was extracted. For immunoprecipitation, the protein fraction was treated with anti-Flag M2 affinity gel (Sigma, A2220) or anti-c-Myc magnetic beads (Thermo Scientific, 88842) according to the manufacturer's instructions. Treated samples were retained using Tris SDS β-ME sample buffer (Cosmo-Bio, DCB-423437).

[0061] Western blotting: Protein samples were loaded onto sodium dodecyl sulfate (SDS) polyacrylamide gels and subjected to electrophoresis. After SDS polyacrylamide gel electrophoresis, the samples were transferred to a nitrocellulose membrane. The blots were blocked with 5% skim milk for 1 hour, incubated overnight at 4°C with primary antibodies against Flag (1:1000) and Myc (1:1000), washed, and incubated at room temperature for 1 hour with secondary antibodies.

[0062] Statistical Analysis: R (version 4.3.2) was used for data analysis. Unless otherwise specified, Welch's t-test was used to compare two means. Tukey's multiple comparison test was used to compare three or more means. Results are presented as mean ± SEM, and statistical significance was set as *P<0.05, **P<0.01, ***P<0.001, and no significant difference for ns.

[0063] [result]:

[0064] NPHP1 deficiency in human iPSCs 3D kidney organoid generation:

[0065] The inventors generated kidney organoids using NPHP1-deficient iPSCs according to the method of Morizane [In Vitro Cell Dev Biol Anim. 2022;58(2):85-95.; Nat Biotechnol. 2015;33(11):1193-1200.; Nat Protoc. 2017;12(1):195-207.]. SIX-2 staining was used during the 2D culture stage to evaluate the optimal conditions for organoid induction. Finally, 3D kidney organoids were generated from NPHP1-deficient iPSCs (Figure 1a).

[0066] To evaluate the developmental state and morphology of nephron-like structures, organoids were cultured for 28 days and analyzed by immunofluorescence (IF). Co-staining with podocalyxin (PODXL), lotus tetragonolobus lectin (LTL), and E-cadherin (CDH1) revealed nephron-like structures within the organoids. No specific changes were detected between wild-type and NPHP1-deficient organoids, particularly in the structure of the proximal tubule (LTL) (Figure 1b).

[0067] The inventors compared the potential fibrotic state of these early-stage organoids. Immunofluorescence staining was used to evaluate the expression of α-smooth muscle actin (α-SMA) and fibronectin (FN) (Figure 1c). Fluorescence intensity within the region of interest in each section was measured and quantified. No statistically significant differences were observed between the two groups (α-SMA: P = 0.303, FN: P = 0.468) (Figure 1d). Finally, mRNA was extracted from the organoids and quantitative PCR was performed. No significant changes were observed in the expression of ACTA2 (P = 0.392), FN1 (P = 0.107), COL1A (P = 0.453), and COL3A (P = 0.497) (Figure 1e).

[0068] In other words, the inventors generated 3D kidney organoids from iPSCs lacking NPHP1. These organoids did not exhibit a characteristic phenotype. Generally, kidney organoids mimic the fetal kidney, and patients with NPHP1-deficient NPH do not show renal dysfunction during the fetal period. Consistent with this finding, the inventors' model did not exhibit a specific phenotype. However, disease models are necessary to elucidate the pathogenesis and to aid in the development of therapeutic drugs.

[0069] The application of fibrosis-stimulating agents to 3D renal organoids revealed that NPHP1 deficiency promotes fibrotic changes:

[0070] After differentiation was induced, NPHP1-deficient organoids resembled wild-type organoids. This indicates that NPHP1-deficient organoids did not acquire morphological or fibrous phenotypes in the early stages. Based on the clinical finding that most NPHP1-deficient NPH patients develop ESKD in adulthood, we hypothesized that additional stimulation is required for NPHP1-deficient organoids to develop fibrosis. Administration of the inflammatory cytokine IL-1β induced fibrosis in renal organoids [Journal of the American Society of Nephrology. 2018;29(6):1690-1705.]. Therefore, we used IL-1β to induce fibrosis.

[0071] Wild-type and NPHP1-deficient organoids were treated with low doses of IL-1β. The control group received the same dose of D-PBS. After 7 days, organoids were harvested and fibrosis was evaluated (Figure 2a-c).

[0072] Immunofluorescence staining of tissue sections showed that IL-1β administration did not induce fibrosis in wild-type organoids. However, in NPHP1-deficient organoids, FN expression increased even at this low concentration of IL-1β (wild-type IL-1β vs. NPHP1-deficient IL-1β P<0.001) (Figure 2a,b). Quantitative PCR showed elevated expression of FN1, COL1A, and COL3A (P = 0.003, 0.036, and 0.024, respectively). However, no elevation in ACTA2 expression was observed (P = 0.306) (Figure 2c).

[0073] Interestingly, similar to IL-1β administration, fibrosis increased in NPHP1-deficient organoids with extended culture periods (Figure 10a-c).

[0074] In this way, we generated the first organoid model representing the fibrous phenotype of NPHP1-deficient NPH. This model can be used to investigate the pathophysiology of NPHP1-deficient NPH and develop therapeutic strategies.

[0075] NPHP1 binds to LATS1 / 2, a component of the Hippo signaling pathway:

[0076] To elucidate the pathogenesis of NPHP1-deficient NPH, we investigated molecules that interact with NPHP1.

[0077] First, we investigated proteins that interact with NPHP1. Flag-NPHP1 and Flag-empty were overexpressed in HEK 293T cells. The proteins were isolated by immunoprecipitation and analyzed using mass spectrometry. Our results reaffirm the strong binding of NPHP1 to NPHP4 and are consistent with other reports [Cell. 2011;145(4):513-528.].

[0078] Among the molecules that interact with NPHP4, we focused on LATS1 and LATS2, which are components of the Hippo signaling pathway [Cells. 2019;8(5):468.]. The Hippo signaling pathway is involved in renal fibrosis. We hypothesized that NPHP1 binds to LATS. To confirm protein binding, Myc-LATS1 or Myc-LATS2 was expressed together with Flag-NPHP1 in HEK 293T cells. Co-immunoprecipitation was performed, and the binding of NPHP1 to the LATS protein was confirmed by immunoblotting results (Figure 3a).

[0079] Next, the role of Hippo signaling was evaluated in NPHP1-deficient organoids of a fibrosis model. Transcription of CTGF, CYR61, and ANKRD1, representative fibrosis-inducing genes located downstream of Hippo signaling, was evaluated by qPCR (Figure 3c). The variation in response to IL-1β administration was compared between wild-type organoids and NPHP1-deficient organoids. Changes in gene expression of CTGF and ANKRD were observed only in NPHP1-deficient organoids (P = 0.002 and <0.001, respectively) (Figure 3c). CYR61 was transcriptionally upregulated in wild-type organoids, but the fold change was larger in NPHP1-deficient organoids (fold change = 1.849 and 3.072, respectively) (Figure 3c). Similarly, in NPHP1-deficient organoids, the expression of CTGF, CYR61, and ANKRD1 increased with longer culture periods (Figure 10d).

[0080] For further investigation, we examined whether fibrosis could be induced in NPHP1-deficient organoids by stimulating Hippo signaling. Ethacridine, a Hippo signaling pathway enhancer, was administered to wild-type and NPHP1-deficient organoids. The control group received PBS. Ethacridine induced fibrosis in both types of renal organoids. Furthermore, NPHP1-deficient organoids showed increased sensitivity to etacridine (Figure 11a, b).

[0081] These findings indicate that the Hippo signaling pathway plays a role in the progression of fibrosis in NPHP1-deficient NPH.

[0082] Hippo pathway inhibitory peptide 17 improved fibrosis in organoid models:

[0083] Next, using our organoid model, we investigated the inhibitory effect of Hippo pathway inhibitors on fibrosis and evaluated whether these drugs have potential as therapeutic agents for NPH.

[0084] Based on the fact that NPHP1 binds to LATS, we hypothesized that insufficient inhibition of YAP-TAZ by the LATS complex promotes fibrosis through enhanced Hippo signaling. Therefore, we first evaluated peptide 17, which specifically inhibits the binding of YAP / TAZ-TEAD, located downstream of LATS1 / 2 (and NPHP1) (Figure 4).

[0085] Immunofluorescence staining demonstrated that peptide 17 significantly suppressed IL-1β-induced fibrillation in NPHP1-deficient organoids (IL-1β vs. IL-1β with peptide 17, P<0.001) (Figure 4a,b). Quantitative PCR revealed transcriptional repression of CTGF, CYR61, and ANKRD1 downstream of the YAP / TAZ-TEAD complex (Figure 4c). Transcription of the fibrillation markers FN1, COL1A, and COL3A was significantly suppressed (IL-1β vs. IL-1β with peptide 17, P<0.001, <0.001, and 0.017, respectively) (Figure 4d).

[0086] These results demonstrate that peptide 17 is a potential therapeutic candidate for the treatment of NPHP1-deficient NPH.

[0087] Drug repositioning:

[0088] Several clinically used drugs are known to inhibit Hippo signaling. These drugs were used in drug repositioning to confirm their protective effects against NPHP1-deficient organoids. Drug repositioning is of great significance in establishing treatments because no effective animal models exist. Furthermore, results obtained with a single drug are insufficient to confirm the efficacy of Hippo pathway inhibitors. Therefore, the inventors conducted this experiment.

[0089] Verteporfin was administered to NPHP1-deficient organoids. Verteporfin is used as a photosensitizer in photodynamic therapy for the treatment of macular degeneration. Immunofluorescence staining demonstrated that verteporfin significantly inhibited the proliferation of IL-1β-induced fibrillation (IL-1β vs. verteporfin, P<0.001) (Figure 5a,b). qPCR analysis confirmed transcriptional inhibition of genes downstream of the Hippo pathway and fibrosis marker genes (Figure 5c,d). Similarly, ivermectin, a marketed anthelmintic, bryostatin, which is undergoing clinical trials for malignant tumors and Alzheimer's disease, and the dyslipidemia treatments atorvastatin and pravastatin also showed inhibitory effects on fibrosis in NPHP1-deficient organoids. (Figures 6-8)

[0090] In conclusion, we confirmed that several Hippo pathway inhibitors suppress fibrous changes in NPHP1-deficient organoids (Figure 9), and identified verteporfin, ivermectin, briostatin, atorvastatin, and pitavastatin for drug repositioning.

[0091] [Consideration]:

[0092] Since NPH was first reported in 1945, several causative genes have been identified. NPHP1 deficiency is the most common and is a key target for intervention. Generally, fibrosis and microcysts associated with tubular dilation are observed in NPH. However, unlike other forms of NPH, NPHP1-deficient NPH is characterized by early-stage tubulointerstitial fibrosis, and the incidence of cystic formation is low in NPHP1-deficient NPH patients. Therefore, a model that mimics fibrosis is needed to elucidate the pathophysiology of NPHP1-deficient NPH.

[0093] Although there are several reports on mouse models, it has been difficult to reproduce the phenotype of the human kidney. Therefore, it has been difficult to elucidate the pathophysiology and develop therapeutic strategies.

[0094] Recent advances in biotechnology, including in vitro direct differentiation protocols, have enabled the study of disease pathogenesis and the discovery of iPSC-based drugs using kidney organoids generated from iPSCs. Due to a lack of suitable animal models, 3D kidney organoids were generated from NPHP1-deficient iPSCs. The inventors generated the first model that reproduces the fibrosis of NPHP1-deficient NPH using human cells. Furthermore, progress has been made in elucidating the pathogenesis of NPHP1-deficient NPH and identifying therapeutic agents.

[0095] Several studies have documented interactions between NPHP1-differentiated NPH-inducing proteins and the Hippo signaling pathway. However, there are no reports investigating these interactions in the context of fibrillation.

[0096] The inventors revealed that the Hippo signaling pathway is involved in the pathogenesis of NPHP1-deficient NPH. They confirmed the binding of LATS1 / 2, a key component of the Hippo pathway, to NPHP1. Furthermore, they demonstrated that administration of IL-1β induces excessive activation of the Hippo pathway, particularly in NPHP1-deficient organoids, leading to fibrosis. This fibrosis was mitigated by administration of a Hippo signaling inhibitor.

[0097] The Hippo signaling pathway has a wide range of functions, including cell growth, proliferation, tissue or organ regeneration, and wound healing [Signal Transduct Target Ther. 2022;7(1):376.]. Furthermore, Hippo signaling plays a crucial role in fibrosis [Cells. 2019;8(5):468.]. Dysregulation of Hippo signaling can lead to diseases in various organs, including fibrosis of the lungs, liver, and kidneys. The contribution of Hippo signaling dysregulation to fibrosis during the transition from AKI to CKD in the kidney has recently been reported [Cell Death Dis. 2021;12(8).]. Loss of LATS or overexpression of YAP / TAZ has been widely reported to lead to activation of Hippo signaling, causing renal fibrosis in mice [Journal of Clinical Investigation. 2021;131(11).; Sci Rep. 2016;6.; JCI Insight. 2022;7(4).; Fibrogenesis Tissue Repair. 2012;5(Suppl 1):S24.; PLoS One. 2013;8(2).].

[0098] Peptide 17, a Hippo signaling inhibitor, significantly reduced fibrillation in NPHP1-deficient organoids. Peptide 17 specifically and potently inhibited the binding of YAP / TAZ to TEAD, suggesting that the primary pathogenesis of enhanced fibrosis in NPHP1-deficient organoids lies upstream of the YAP / TAZ-TEAD interaction within the Hippo pathway. Considering that NPHP1 binds to LATS, deletion of NPHP1 inhibits LATS-mediated YAP / TAZ inhibition, thereby inducing fibrosis.

[0099] The inventors demonstrated that verteporfin, another Hippo signaling inhibitor, has an inhibitory effect on fibrosis of NPHP1-deficient organoids. Several drugs on the market have been reported to inhibit Hippo signaling, and research into their potential applications is ongoing. For diseases where in vivo models are not available, such as NPHP1-deficient NPH, a drug repositioning approach is beneficial in terms of human safety. Verteporfin showed good tolerability in humans. The inventors' results suggest that verteporfin administration is effective in patients with NPHP1-deficient NPH.

[0100] NPHP1-deficient organoids did not show a phenotype compared to wild-type organoids in the early stages of maturation. Among NPHs, NPHP1-deficient NPH does not show a phenotype at birth, and renal function is gradually lost as the person grows. Our group previously reported that even twins with the same genetic background show a large difference in the age at which ESKD develops. We hypothesized that an external factor, which could be called a "third hit," is involved in the progression of the disease in patients with NPHP1-deficient NPH.

[0101] Extending the culture period revealed a significant difference in FN expression between wild-type organoids and NPHP1-deficient organoids. The time-dependent progression of fibrosis suggests a mechanism common to age-related disease progression in NPHP1-deficient NPH.

[0102] In experiments using low-dose IL-1β, fibrosis was induced only in NPHP1-deficient organoids, while no changes were observed in wild-type organoids. This finding suggests the importance of a third hit in promoting fibrosis in NPHP1-deficient NPH.

[0103] Since most of the causative genes for NPH are highly expressed in primary cilia, research aimed at elucidating the pathogenesis of NPH has been centered on the "cilia / centrosome hypothesis." However, because NPHP1 is expressed not only inside but also outside the cilia, further investigation is needed to confirm whether cilia are involved in Hippo dysfunction caused by NPHP1 deficiency. In summary, NPHP1 deficiency induced fibrosis via hyperactivation of the Hippo signaling pathway. Furthermore, Hippo pathway inhibitors may be usable as novel therapeutic agents for treating NPHP1-deficient NPH.

Claims

1. A pharmaceutical composition for use in the treatment of nephronophthria, comprising a Hippo signaling inhibitor, wherein the Hippo signaling inhibitor is a substance that inhibits YAP / TAZ.

2. The pharmaceutical composition according to claim 1, wherein the Hippo signaling inhibitor is a substance that inhibits the function of YAP / TAZ-TEAD.

3. The pharmaceutical composition according to claim 1, wherein the Hippo signaling inhibitor is selected from the group consisting of peptide 17, ivermectin, verteporfin, briostatin, and statin drugs.

4. The pharmaceutical composition according to claim 3, wherein the statin drug is selected from the group consisting of atorvastatin and pitavastatin.

5. The pharmaceutical composition according to claim 1, wherein the nephrona is an NPHP1-deficient nephrona.

6. A pharmaceutical composition containing a Hippo signaling inhibitor for use in suppressing renal fibrosis of nephronophthiriasis.

7. Renal organoid lacking NPHP1.

8. The kidney organoid according to claim 7, derived from stem cells lacking NPHP1.

9. The kidney organoid according to claim 8, wherein the stem cells are iPS cells.

10. A method for screening candidate substances for an active ingredient in a pharmaceutical composition for use in the treatment of nephronitis, comprising the step of identifying a candidate substance as the active ingredient that suppresses fibrosis in a renal organoid lacking NPHP1, which has been administered to the renal organoid that has induced fibrosis.

11. The method according to claim 10, wherein fibrosis in renal organoids lacking NPHP1 is induced by IL-1β.

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