Cystogenesis In Autosomal Dominant Polycystic Kidney Disease Research Articles

Notch3 as a novel therapeutic target for the treatment of ADPKD by regulating cell proliferation and renal cyst development

Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic kidney disease. Emerging research indicates that the Notch signaling pathway plays an indispensable role in the pathogenesis of numerous kidney diseases, including ADPKD. Herein, we identified that Notch3 but not other Notch receptors was overexpressed in renal tissues from mice with ADPKD and ADPKD patients. Inhibiting Notch3 with γ-secretase inhibitors, which block a proteolytic cleavage required for Notch3 activation, or shRNA knockdown of Notch3 significantly delayed renal cyst growth in vitro and in vivo. Subsequent mechanistic study elucidated that the cleaved intracellular domain of Notch3 (N3ICD) and Hes1 could bind to the PTEN promoter, leading to transcriptional inhibition of PTEN. This further activated the downstream PI3K-AKT-mTOR pathway and promoted renal epithelial cell proliferation. Overall, Notch3 was identified as a novel contributor to renal epithelial cell proliferation and cystogenesis in ADPKD. We envision that Notch3 represents a promising target for ADPKD treatment.

Human iPSC-derived renal collecting duct organoid model cystogenesis in ADPKD

In autosomal dominant polycystic kidney disease (ADPKD), renal cyst lesions predominantly arise from collecting ducts (CDs). However, relevant CD cyst models using human cells are lacking. Although previous reports have generated invitro renal tubule cyst models from human induced pluripotent stem cells (hiPSCs), therapeutic drug candidates for ADPKD have not been identified. Here, by establishing expansion cultures of hiPSC-derived ureteric bud tip cells, an embryonic precursor that gives rise to CDs, we succeed in advancing the developmental stage of CD organoids and show that all CD organoids derived from PKD1-/- hiPSCs spontaneously develop multiple cysts, clarifying the initiation mechanisms of cystogenesis. Moreover, we identify retinoic acid receptor (RAR) agonists as candidate drugs that suppress invitro cystogenesis and confirm the therapeutic effects on an ADPKD mouse model invivo. Therefore, our invitro CD cyst model contributes to understanding disease mechanisms and drug discovery for ADPKD.

Nuclear Condensation of CDYL Links Histone Crotonylation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease.

Emerging evidence indicates that epigenetic modulation of gene expression plays a key role in the progression of autosomal dominant polycystic kidney disease (ADPKD). However, the molecular basis for how the altered epigenome modulates transcriptional responses, and thereby disease progression in ADPKD, remains largely unknown. Kidneys from control and ADPKD mice were examined for the expression of CDYL and histone acylations. CDYL expression and its correlation with disease severity were analyzed in a cohort of patients with ADPKD. Cdyl transgenic mice were crossed with Pkd1 knockout mice to explore CDYL's role in ADPKD progression. Integrated cistromic and transcriptomic analyses were performed to identify direct CDYL target genes. High-sensitivity mass spectrometry analyses were undertaken to characterize CDYL-regulated histone lysine crotonylations (Kcr). Biochemical analysis and zebrafish models were used for investigating CDYL phase separation. CDYL was downregulated in ADPKD kidneys, accompanied by an increase of histone Kcr. Genetic overexpression of Cdyl reduced histone Kcr and slowed cyst growth. We identified CDYL-regulated cyst-associated genes, whose downregulation depended on CDYL-mediated suppression of histone Kcr. CDYL assembled nuclear condensates through liquid-liquid phase separation in cultured kidney epithelial cells and in normal kidney tissues. The phase-separating capacity of CDYL was required for efficient suppression of locus-specific histone Kcr, of expression of its target genes, and of cyst growth. These results elucidate a mechanism by which CDYL nuclear condensation links histone Kcr to transcriptional responses and cystogenesis in ADPKD.

CAMP-Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease.

Formation of biomolecular condensates by phase separation has recently emerged as a new principle for regulating gene expression in response to extracellular signaling. However, the molecular mechanisms underlying the coupling of signal transduction and gene activation through condensate formation, and how dysregulation of these mechanisms contributes to disease progression, remain elusive. Here, the authors report that CREB‐regulated transcription coactivator 2 (CRTC2) translocates to the nucleus and forms phase‐separated condensates upon activation of cAMP signaling. They show that intranuclear CRTC2 interacts with positive transcription elongation factor b (P‐TEFb) and activates P‐TEFb by disrupting the inhibitory 7SK snRNP complex. Aberrantly elevated cAMP signaling plays central roles in the development of autosomal dominant polycystic kidney disease (ADPKD). They find that CRTC2 localizes to the nucleus and forms condensates in cystic epithelial cells of both mouse and human ADPKD kidneys. Genetic depletion of CRTC2 suppresses cyst growth in an orthologous ADPKD mouse model. Using integrative transcriptomic and cistromic analyses, they identify CRTC2‐regulated cystogenesis‐associated genes, whose activation depends on CRTC2 condensate‐facilitated P‐TEFb recruitment and the release of paused RNA polymerase II. Together, their findings elucidate a mechanism by which CRTC2 nuclear condensation conveys cAMP signaling to transcription elongation activation and thereby promotes cystogenesis in ADPKD.

Combination of curcumin and ginkgolide B inhibits cystogenesis by regulating multiple signaling pathways.

Autosomal dominant polycystic kidney disease (ADPKD), a common disease with a high incidence ratio of between 1/400 and 1/1,000 individuals, often results in kidney failure and even mortality. However, there are relatively few effective treatments available, and treatment is limited to lifelong hemodialysis or kidney transplant. Our previous studies have reported that curcumin (Cur) and ginkgolide B (GB) inhibited cystogenesis by regulating the Ras/ERK MAPK signaling pathway. In the present study, it was hypothesized that Cur and GB may have a synergistic effect on the inhibition of cystogenesis, and their synergistic effect may be the result of regulation of multiple signaling pathways. To assess this hypothesis, an in vitro Madin-Darby canine kidney (MDCK) cyst model and an in vivo kidney-specific polycystin 1 transient receptor potential channel interacting (Pkd1) knockout mouse model were established to observe the effects of the combination of Cur and GB. The cysts exposed to Cur, GB and Cur combined with GB became small thick-walled cysts, small thin-walled cysts and round shaped cell colonies, respectively. The combination of Cur and GB was more effective compared with either treatment alone in inhibiting cystogenesis. Additionally, to the best of our knowledge, the present study was the first to demonstrate the synergistic effect of Cur and GB on the inhibition of cystogenesis in Pkd1 knockout mice. Cur may have mediated its anti-cyst effects by blocking EGFR/ERK1/2, JNK and PI3K/mTOR signaling pathways, while GB may have inhibited cystogenesis via the downregulation of the EGFR/ERK1/2, JNK and p38 signaling pathways. These results provide a proof-of-concept for application of the combination of Cur and GB in inhibiting cystogenesis in ADPKD.

Suppression of Foxo3-Gatm by miR-132-3p Accelerates Cyst Formation by Up-Regulating ROS in Autosomal Dominant Polycystic Kidney Disease.

Accumulation of reactive oxygen species (ROS) is associated with the development of various diseases. However, the molecular mechanisms underlying oxidative stress that lead to such diseases like autosomal dominant polycystic kidney disease (ADPKD) remain unclear. Here, we observed that oxidative stress markers were increased in Pkd1f/f:HoxB7-Cre mice. Forkhead transcription factors of the O class (FOXOs) are known key regulators of the oxidative stress response, which have been observed with the expression of FoxO3a in an ADPKD mouse model in the present study. An integrated analysis of two datasets for differentially expressed miRNA, such as miRNA sequencing analysis of Pkd1 conditional knockout mice and microarray analysis of samples from ADPKD patients, showed that miR-132-3p was a key regulator of FOXO3a in ADPKD. miR-132-3p was significantly upregulated in ADPKD which directly targeted FOXO3 in both mouse and human cell lines. Interestingly, the mitochondrial gene Gatm was downregulated in ADPKD which led to a decreased inhibition of Foxo3. Overexpression of miR-132-3p coupled with knockdown of Foxo3 and Gatm increased ROS and accelerated cyst formation in 3D culture. This study reveals a novel mechanism involving miR-132-3p, Foxo3, and Gatm that is associated with the oxidative stress that occurs during cystogenesis in ADPKD.

TAZ/Wnt-β-catenin/c-MYC axis regulates cystogenesis in polycystic kidney disease

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common genetic renal disease, primarily caused by germline mutation of PKD1 or PKD2, leading to end-stage renal disease. The Hippo signaling pathway regulates organ growth and cell proliferation. Herein, we demonstrate the regulatory mechanism of cystogenesis in ADPKD by transcriptional coactivator with PDZ-binding motif (TAZ), a Hippo signaling effector. TAZ was highly expressed around the renal cyst-lining epithelial cells of Pkd1-deficient mice. Loss of Taz in Pkd1-deficient mice reduced cyst formation. In wild type, TAZ interacted with PKD1, which inactivated β-catenin. In contrast, in PKD1-deficient cells, TAZ interacted with AXIN1, thus increasing β-catenin activity. Interaction of TAZ with AXIN1 in PKD1-deficient cells resulted in nuclear accumulation of TAZ together with β-catenin, which up-regulated c-MYC expression. Our findings suggest that the PKD1-TAZ-Wnt-β-catenin-c-MYC signaling axis plays a critical role in cystogenesis and might be a potential therapeutic target against ADPKD.

Identifying cystogenic paracrine signaling molecules in cyst fluid of patients with polycystic kidney disease.

In autosomal dominant polycystic kidney disease (ADPKD) paracrine signaling molecules in cyst fluid can induce proliferation and cystogenesis of neighboring renal epithelial cells. However, the identity of this cyst-inducing factor is still unknown. The aim of this study was to identify paracrine signaling proteins in cyst fluid using a 3D in vitro cystogenesis assay. We collected cyst fluid from 15 ADPKD patients who underwent kidney or liver resection (55 cysts from 13 nephrectomies, 5 cysts from 2 liver resections). For each sample, the ability to induce proliferation and cyst formation was tested using the cystogenesis assay (RPTEC/TERT1 cells in Matrigel with cyst fluid added for 14 days). Kidney cyst fluid induced proliferation and cyst growth of renal epithelial cells in a dose-dependent fashion. Liver cyst fluid also induced cystogenesis. Using size exclusion chromatography, 56 cyst fluid fractions were obtained of which only the fractions between 30 and 100 kDa showed cystogenic potential. Mass spectrometry analysis of samples that tested positive or negative in the assay identified 43 candidate cystogenic proteins. Gene ontology analysis showed an enrichment for proteins classified as enzymes, immunity proteins, receptors, and signaling proteins. A number of these proteins have previously been implicated in ADPKD, including secreted frizzled-related protein 4, S100A8, osteopontin, and cysteine rich with EGF-like domains 1. In conclusion, both kidney and liver cyst fluids contain paracrine signaling molecules that drive cyst formation. Using size exclusion chromatography and mass spectrometry, we procured a candidate list for future studies. Ultimately, cystogenic paracrine signaling molecules may be targeted to abrogate cystogenesis in ADPKD.

Ouabain Regulates CFTR-Mediated Anion Secretion and Na,K-ATPase Transport in ADPKD Cells.

Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) requires the transepithelial secretion of fluid into the cyst lumen. We previously showed that physiological amounts of ouabain enhance cAMP-dependent fluid secretion and cyst growth of human ADPKD cyst epithelial cells in culture and formation of cyst-like dilations in metanephric kidneys from Pkd1 mutant mice. Here, we investigated the mechanisms by which ouabain promotes cAMP-dependent fluid secretion and cystogenesis. Ouabain (3 nM) enhanced cAMP-induced cyst-like dilations in embryonic kidneys from Pkd1 (m1Bei) mice, but had no effect on metanephroi from Pkd1 (m1Bei) mice that lack expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, ouabain stimulation of cAMP-induced fluid secretion and in vitro cyst growth of ADPKD cells were abrogated by CFTR inhibition, showing that CFTR is required for ouabain effects on ADPKD fluid secretion. Moreover, ouabain directly enhanced the cAMP-dependent Cl(-) efflux mediated by CFTR in ADPKD monolayers. Ouabain increased the trafficking of CFTR to the plasma membrane and up-regulated the expression of the CFTR activator PDZK1. Finally, ouabain decreased plasma membrane expression and activity of the Na,K-ATPase in ADPKD cells. Altogether, these results show that ouabain enhances net fluid secretion and cyst formation by activating apical anion secretion via CFTR and decreasing basolateral Na(+) transport via Na,K-ATPase. These results provide new information on the mechanisms by which ouabain affects ADPKD cells and further highlight the importance of ouabain as a non-genomic stimulator of cystogenesis in ADPKD.

Inactivation of integrin-β1 prevents the development of polycystic kidney disease after the loss of polycystin-1.

Dysregulation of polycystin-1 (PC1) leads to autosomal dominant polycystic kidney disease (ADPKD), a disorder characterized by the formation of multiple bilateral renal cysts, the progressive accumulation of extracellular matrix (ECM), and the development of tubulointerstitial fibrosis. Correspondingly, cystic epithelia express higher levels of integrins (ECM receptors that control various cellular responses, such as cell proliferation, migration, and survival) that are characteristically altered in cystic cells. To determine whether the altered expression of ECM and integrins could establish a pathologic autostimulatory loop, we tested the role of integrin-β1 in vitro and on the cystic development of ADPKD in vivo. Compared with wild-type cells, PC1-depleted immortalized renal collecting duct cells had higher levels of integrin-β1 and fibronectin and displayed increased integrin-mediated signaling in the presence of Mn(2+). In mice, conditional inactivation of integrin-β1 in collecting ducts resulted in a dramatic inhibition of Pkd1-dependent cystogenesis with a concomitant suppression of fibrosis and preservation of normal renal function. Our data provide genetic evidence that a functional integrin-β1 is required for the early events leading to renal cystogenesis in ADPKD and suggest that the integrin signaling pathway may be an effective therapeutic target for slowing disease progression.

Osmoregulation, vasopressin, and cAMP signaling in autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent inherited nephropathy. This review will focus on the vasopressin and 3'-5'-cyclic adenosine monophosphate (cAMP) signaling pathways in ADPKD and will discuss how these insights offer new possibilities for the follow-up and treatment of the disease. Defective osmoregulation is an early manifestation of ADPKD and originates from both peripheral (renal effect of vasopressin) and central (release of vasopressin) components. Copeptin, which is released from the vasopressin precursor, may identify ADPKD patients at risk for rapid disease progression. Increased levels of cAMP in tubular cells, reflecting modifications in intracellular calcium homeostasis and abnormal stimulation of the vasopressin V2 receptor (V2R), play a central role in cystogenesis. Blocking the V2R lowers cAMP in cystic tissues, slows renal cystic progression and improves renal function in preclinical models. A phase III clinical trial investigating the effect of the V2R antagonist tolvaptan in ADPKD patients has shown that this treatment blunts kidney growth, reduces associated symptoms and slows kidney function decline when given over 3 years. These advances open perspectives for the understanding of cystogenesis in ADPKD, the mechanisms of osmoregulation, the role of polycystins in the brain, and the pleiotropic action of vasopressin.

What Is the Role of Somatic Mutation in Autosomal Dominant Polycystic Kidney Disease?

A 50% dosage reduction of gene product, known as haploinsufficiency, as found in heterozygotes with an inactivating mutation, is not usually associated with a detectable physiologic effect. Consequently, either loci associated with dominant disease are those rare ones that are sensitive to haploinsufficiency, or an additional mechanism plays a role to enhance the effect of the single mutant allele. In the case of autosomal dominant polycystic kidney disease (ADPKD), adult-onset disease is characterized by focal cyst development and expansion as a result of heterozygous mutation in PKD1 or PKD2 . In mice, homozygous inactivating mutations to either Pkd1 or Pkd2 results in embryonic lethality with the development of renal and pancreatic cysts from embryonic day 13.5.1,2 Humans with two fully penetrant mutations are also thought to have a nonviable phenotype. The wide range of human mutations causing ADPKD, approximately 70% of which are clearly inactivating, and the lack of clear genotype–phenotype correlations suggest that a dominant negative mechanism, whereby the mutant product adversely affects the function of the normal ADPKD allele product, is unlikely to be important in the human disease.3 Rather, a widely accepted view is that ADPKD is recessive at the cellular level and that cysts develop clonally from a tubular cell only once the cell has acquired a second, somatic mutation to inactivate the remaining normal allele. There are considerable data to support this two-hit hypothesis for cystogenesis in ADPKD. This includes the demonstration of deletion (loss of heterozygosity) or inactivating point mutations to the remaining normal PKD1 or PKD2 allele in epithelial cyst linings isolated from single cysts, in either the kidney or the liver.4–8 In addition, there is evidence for the clonality of cysts.7 Further data come from a Pkd2 mouse model ( Pkd2 WS25) that is prone …

Polycystin-2 Expression Is Regulated by a PC2-binding Domain in the Intracellular Portion of Fibrocystin

Autosomal dominant (ADPKD) and autosomal recessive (ARPKD) polycystic kidney disease are caused by mutations in Pkd1/Pkd2 and Pkhd1, which encode polycystins (PCs) and fibrocystin/polyductin (FPC). Our recent study reported that a deficiency in FPC increases the severity of cystic disease in Pkd2 mutants and down-regulates PC2 in vivo, but the precise molecular mechanism of these effects is unknown (Kim, I., Fu, Y., Hui, K., Moeckel, G., Mai, W., Li, C., Liang, D., Zhao, P., Ma, J., Chen, X.-Z., George, A. L., Jr., Coffey, R. J., Feng, Z. P., and Wu, G. (2008) J. Am. Soc. Nephrol. 19, 455-468). In this study, through the use of deletion and mutagenesis strategies, we identified a PC2-binding domain in the intracellular C terminus of FPC and an FPC-binding domain in the intracellular N terminus of PC2. These binding domains provide a molecular basis for the physical interaction between PC2 and FPC. In addition, we also found that physical interaction between the binding domains of PC2 and FPC is able to prevent down-regulation of PC2 induced by loss of FPC. In vivo, we generated a mouse model of ADPKD with hypomorphic Pkd2 alleles (Pkd2nf3/nf3) and show that PC2 down-regulation is accompanied by a phenotype similar to that of Pkhd1(-/-) mice. These findings demonstrate a common mechanism underlying cystogenesis in ADPKD and ARPKD and provide insight into the molecular relationship between PC2 and FPC.

Mitotic activation of Akt signalling pathway in Han:SPRD rats with polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by an imbalance between tubular epithelial cell proliferation and apoptosis. We have previously shown that the mammalian target of rapamycin (mTOR) signalling pathway is aberrantly activated in the cystic kidneys of Han:SPRD rats with ADPKD. Because the Akt kinase is an upstream regulator of mTOR, we hypothesized that the activity of Akt could be enhanced in the kidneys of Han:SPRD rats. Reverse transcription-polymerase chain reaction, western blot, enzyme-linked immunosorbent assay and immunohistochemistry were used to analyse Akt expression in rat polycystic kidneys. Wild-type (+/+) and heterozygous (Cy/+) Han:SPRD rats showed constitutive expression of Akt-1, -2 and -3 mRNA by reverse transcription-polymerase chain reaction analysis with no significant difference between Cy/+ and +/+ kidneys. Western blotting and enzyme-linked immunosorbent assay showed a significant increase in phosphorylated Akt in Cy/+ compared with +/+ kidneys. The pattern of immunoreactivity for phosphorylated Akt in kidney sections was the same in +/+ and in Cy/+ rats, with very low levels in interphase cells, but extremely bright signals in mitotic cells, beginning with the onset of the prophase. The in vivo incorporation of bromo-deoxyuridine revealed approximately a ninefold higher rate of proliferation in Cy/+ cyst epithelia compared with normal tubule epithelia in +/+ rats, while the expression of the cell cycle marker Ki67 revealed approximately a sixfold higher rate of proliferation. In summary, enhanced phosphorylation of Akt can be demonstrated in Cy/+ kidneys which correlates with a markedly elevated proliferation rate of epithelial cells in cysts. Mitotic but not resting cells display strong phosphorylation of Akt. Because Akt is a proximal target of mTOR, its inhibition with specific antagonists could be useful to prevent or halt cystogenesis in ADPKD.

Polycystic kidney disease: genes, proteins, animal models, disease mechanisms and therapeutic opportunities

An increased understanding of the genetic, molecular and cellular mechanisms responsible for the development of polycystic kidney disease has laid out the foundation for the development of rational therapies. Many animal models where these therapies can be tested are currently available. This review summarizes the rationale for these treatments, the results of preclinical trials and the prospects for clinical trials, some already in early phases of implementation.

Somatic PKD2 mutations in individual kidney and liver cysts support a "two-hit" model of cystogenesis in type 2 autosomal dominant polycystic kidney disease.

An intriguing feature of autosomal dominant polycystic kidney disease (ADPKD) is the focal and sporadic formation of renal and extrarenal cysts. Recent documentation of somatic PKD1 mutations in cystic epithelia of patients with germ-line PKD1 mutations suggests a "two-hit" model for cystogenesis in type 1 ADPKD. This study tests whether the same mechanism for cystogenesis might also occur in type 2 ADPKD. Genomic DNA was obtained from 54 kidney and liver cysts from three patients with known germ-line PKD2 mutations, using procedures that minimize contamination of cells from noncystic tissue. Using intragenic and microsatellite markers, these cyst samples were screened for loss of heterozygosity. The same samples were also screened for somatic mutations in five of the 15 exons in PKD2 by single-stranded conformational polymorphism analysis. Loss of heterozygosity was found in five cysts, and unique intragenic mutations were found in seven other cysts. In 11 of these 12 cysts, it was also determined that the somatic mutation occurred nonrandomly in the copy of PKD2 inherited from the unaffected parent. These findings support the "two-hit" model as a unified mechanism for cystogenesis in ADPKD. In this model, the requirement of a somatic mutation as the rate-limiting step for individual cyst formation has potential therapeutic implications.