Human Autosomal Dominant Polycystic Kidney Disease Research Articles

Somatic mutation in autosomal dominant polycystic kidney disease revealed by deep sequencing human kidney cysts

Autosomal Dominant Polycystic Kidney Disease (ADPKD) results in progressive cysts that lead to kidney failure, and is caused by heterozygous germline variants in PKD1 or PKD2. Cyst pathogenesis is not definitively understood. Somatic second-hit mutations have been implicated in cyst pathogenesis, though technical sequencing challenges have limited investigation. We used unique molecular identifiers, high-depth massively parallel sequencing and custom analysis techniques to identify somatic second-hit mutations in 24 whole cysts from disparate regions of six human ADPKD kidneys, utilising replicate samples and orthogonal confirmation. Average depth of coverage of 1166 error-corrected reads for PKD1 and 539 reads for PKD2 was obtained. 58% (14/24) of cysts had a detectable PKD1 somatic variant, with 5/6 participants having at least one cyst with a somatic variant. We demonstrate that low-frequency somatic mutations are detectable in a proportion of cysts from end-stage ADPKD human kidneys. Further studies are required to understand the drivers of this somatic mutation.

Integrin alpha1 beta1 promotes interstitial fibrosis in a mouse model of polycystic kidney disease.

Fibrosis is the cause of end-stage kidney failure in patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD). The molecular and cellular mechanisms involved in fibrosis are complex and anti-fibrotic therapies have so far failed to make an impact on patient welfare. Using unbiased proteomics analysis on the Pkd1 nl/nl mouse, we found that expression of the integrin α1 subunit is increased in this model of ADPKD. In human ADPKD tissue and two single cell RNA kidney disease datasets, ITGA1 was also upregulated. To investigate the functional role of this integrin subunit in ADPKD, we generated a Pkd1 nl/nl Itga1 -/- mouse. We observed a significant reduction in kidney volume and kidney dysfunction in mice lacking the integrin α1 subunit. Kidneys from Pkd1 nl/nl Itga1 -/- mice had smaller cysts and reduced interstitial expansion and tubular atrophy. Picrosirius red staining identified a restriction in collagen staining in the interstitium and the myofibroblast marker α smooth muscle actin was also downregulated. Myofibroblast cell proliferation was reduced in Pkd1 nl/nl Itga1 -/- mice and primary fibroblast cultures demonstrated an abrogated fibrogenic phenotype in integrin α1-depleted fibroblasts. These results highlight a previously unrecognised role for the integrin α1 subunit in kidney fibrosis.

Integrated mRNA-seq and miRNA-seq analysis reveals key transcription factors of HNF4α and KLF4 in ADPKD

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most prevalent genetic disorder affecting the kidneys. Understanding epigenetic regulatory mechanisms and the role of microRNAs (miRNAs) is crucial for developing therapeutic interventions. Two mRNA datasets (GSE7869 and GSE35831) and miRNA expression data (GSE133530) from ADPKD patients were used to find differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs), with a focus on genes regulated by hub transcription factors (TFs) and their target genes. The expression of hub TFs was validated in human kidneys and animal models through Western Blot (WB) and RT-PCR analysis. The location of the hub TF proteins in kidney cells was observed by a laser confocal microscope. A total of 2037 DEGs were identified. DEM analysis resulted in 59 up-regulated and 107 down-regulated miRNAs. Predicted target DEGs of DEMs indicated two top dysregulated TFs: hepatocyte nuclear factor 4 alpha (HNF4α) and Kruppel-like factor 4 (KLF4). RT-PCR, WB, and immunochemistry results showed that mRNA and protein levels of HNF4α were significantly decreased while KLF4 levels were significantly up-regulated in human ADPKD kidneys and Pkd1 conditional knockout mice compared with normal controls. Laser confocal microscopy revealed that KLF4 was mainly located in the cytoplasm while HNF4α was in the nucleus. Functional enrichment analysis indicated that genes regulated by HNF4α were mainly associated with metabolic pathways, while KLF4-regulated genes were linked to kidney development. Drug response prediction analysis revealed potential drug candidates for ADPKD treatment, including BI-2536, Sepantronium, and AZD5582. This integrated analysis provides new epigenetic insights into the complex miRNA-TF-mRNA network in ADPKD and identifies HNF4α and KLF4 as key TFs. These findings offer valuable resources for further research and potential drug development for ADPKD.

Circadian Clock Disruption and Growth of Kidney Cysts in Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 and PKD2 genes, and often progresses to kidney failure. ADPKD progression is not uniform among patients, suggesting that factors secondary to the PKD1/2 gene mutation could regulate the rate of disease progression. Here we tested the effect of circadian clock disruption on ADPKD progression. Circadian rhythms are regulated by cell-autonomous circadian clocks composed of clock proteins. BMAL1 is a core constituent of the circadian clock. To disrupt the circadian clock, we deleted Bmal1 gene in the renal collecting ducts of the Pkd1RC/RC (RC/RC) mouse model of ADPKD (RC/RC;Bmal1f/f;Pkhd1cre, called DKO mice), and in Pkd1 knockout mouse inner medullary collecting duct cells (Pkd1Bmal1KO mIMCD3 cells). Only male mice were used. Human nephrectomy ADPKD kidneys showed altered clock gene expression when compared to normal control human kidneys. When compared to RC/RC kidneys, DKO kidneys showed significantly altered clock gene expression, increased cyst growth, cell proliferation, apoptosis and fibrosis. DKO kidneys also showed increased lipogenesis and cholesterol synthesis-related gene expression, and increased tissue triglyceride levels compared to RC/RC kidneys. Similarly, in vitro, Pkd1Bmal1KO cells showed altered clock genes, increased lipogenesis and cholesterol synthesis-related genes, and reduced fatty-acid oxidation-related gene expression compared to Pkd1KO cells. The Pkd1Bmal1KO cells showed increased cell proliferation compared to Pkd1KO cells, which was rescued by pharmacological inhibition of lipogenesis. Renal collecting duct specific Bmal1 gene deletion disrupted the circadian clock and triggered accelerated ADPKD progression by altering lipid metabolism-related gene expression.

Circadian clock disruption and growth of kidney cysts in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 and PKD2 genes, and often progresses to kidney failure. ADPKD progression is not uniform among patients, suggesting that factors secondary to the PKD1/2 gene mutation could regulate the rate of disease progression. Here we tested the effect of circadian clock disruption on ADPKD progression. Circadian rhythms are regulated by cell-autonomous circadian clocks composed of clock proteins. BMAL1 is a core constituent of the circadian clock. To disrupt the circadian clock, we deleted Bmal1 gene in the renal collecting ducts of the Pkd1 RC/RC (RC/RC) mouse model of ADPKD (RC/RC; Bmal1 f/f ; Pkhd1 cre , called DKO mice), and in Pkd1 knockout mouse inner medullary collecting duct cells ( Pkd1Bmal1 KO mIMCD3 cells). Only male mice were used. Human nephrectomy ADPKD kidneys and Pkd1 KO mIMCD3 cells showed reduced Bmal1 gene expression compared to normal controls. When compared to RC/RC kidneys, DKO kidneys showed significantly altered clock gene expression, increased cyst growth, cell proliferation, apoptosis and fibrosis. DKO kidneys also showed increased lipogenesis and cholesterol synthesis-related gene expression, and increased tissue triglyceride levels compared to RC/RC kidneys. Similarly, in vitro, Pkd1Bmal1 KO cells showed altered clock genes, increased lipogenesis and cholesterol synthesis-related genes, and reduced fatty-acid oxidation-related gene expression compared to Pkd1KO cells. The Pkd1Bmal1 KO cells showed increased cell proliferation compared to Pkd1KO cells, which was rescued by pharmacological inhibition of lipogenesis. Renal collecting duct specific Bmal1 gene deletion disrupts the circadian clock and triggers accelerated ADPKD progression by altering lipid metabolism-related gene expression. Lack of BMAL1, a circadian clock protein in renal collecting ducts disrupted the clock and increased cyst growth and fibrosis in an ADPKD mouse model.BMAL1 gene deletion increased cell proliferation by increasing lipogenesis in kidney cells.Thus, circadian clock disruption could be a risk factor for accelerated disease progression in patients with ADPKD.

Polycystin-1 loss of function increases susceptibility to atrial fibrillation through impaired DNA damage response.

The increasing prevalence of atrial fibrillation (AF) and chronic kidney diseases highlights the need for a deeper comprehension of the molecular mechanisms linking them. Mutations in PKD1, the gene encoding Polycystin-1 (PKD1 or PC1), account for 85% of autosomal dominant polycystic kidney disease (ADPKD) cases. This disease often includes cardiac complications such as AF. In cardiomyocytes, PC1 deletion reduces hypertrophic response to pressure overload but promotes baseline ventricular dysfunction, while deletion in fibroblasts ameliorates post-myocardial infarction fibrosis. Despite its known cardiac impact, the role of PC1 in atrial cardiomyocytes and arrhythmias is less understood. Here, we sought to investigate the role of PC1 in AF. We used intracardiac programmed stimulation and optical mapping to evaluate AF inducibility in two mouse models, Pkd1 R3277C, which recapitulates human ADPKD progression, and cardiomyocyte-specific Pkd1 deletion, and their respective controls. Isolated adult mouse atrial cardiomyocytes, human iPSC-derived atrial cardiomyocytes (hiPSC-aCM), and HL-1 cells served as in vitro cellular models. Molecular mechanisms were evaluated using optical mapping and molecular and biochemical approaches. Loss-of-function PC1 mutations significantly increased AF susceptibility in vivo and facilitated local reentry in ex vivo left atrial appendages. Comprehensive in vitro experiments supported a direct effect of PC1 in atrial cardiomyocytes. PC1-deficient monolayers exhibited increased arrhythmic events, escalating into reentrant spiral waves post-tachypacing. Transcriptomics analysis revealed PC1-dependent regulation of DNA repair, with PC1 deficiency leading to increased DNA damage under stress. PARP1 inhibitors or nicotinamide riboside, which counteract DNA damage-related metabolic consequences, reduced in vitro arrhythmias PC1-deficient monolayers. Overexpression of the C-terminus of PC1 had the opposite effects in DNA repair genes, suggesting its regulatory effects in atrial cardiomyocytes through retinoblastoma/E2F. Analyses of human atrial tissue from non-ADPKD patients showed reduced levels of mature PC1, suggesting a broader relevance of impaired PC1 in AF. Impaired PC1 increases in vivo AF inducibility under programmed electrical stimulation and promotes in vitro arrhythmias in hiPSC-aCM and HL-1 cells. Our findings indicate that PC1 protects against DNA damage to reduce AF susceptibility.

Overexpression of SMYD3 Promotes Autosomal Dominant Polycystic Kidney Disease by Mediating Cell Proliferation and Genome Instability.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disorder worldwide and progresses to end-stage renal disease (ESRD). However, its precise mechanism is not fully understood. In recent years, epigenetic reprogramming has drawn increasing attention regarding its effect on cyst growth. However, considering the complexity of epigenetic mechanisms and the broad range of alterations of epigenetic components in ADPKD, identifying more specific epigenetic factors and understanding how they are mechanistically linked to promote cyst growth is relevant for the development of treatment for ADPKD. Here, we find that the histone methyltransferase SMYD3, which activates gene transcription via histone H3 lysine 4 trimethylation (H3K4me3), is upregulated in PKD1 mutant mouse and human ADPKD kidneys. Genetic knockout of SMYD3 in a PKD1 knockout mouse model delayed cyst growth and improved kidney function compared with PKD1 single knockout mouse kidneys. Immunostaining and Western blot assays indicated that SMYD3 regulated PKD1-associated signaling pathways associated with proliferation, apoptosis, and cell cycle effectors in PKD1 mutant renal epithelial cells and tissues. In addition, we found that SMYD3 localized to the centrosome and regulated mitosis and cytokinesis via methylation of α-tubulin at lysine 40. In addition, SMYD3 regulated primary cilia assembly in PKD1 mutant mouse kidneys. In summary, our results demonstrate that overexpression of SMYD3 contributes to cyst progression and suggests targeting SMYD3 as a potential therapeutic strategy for ADPKD.

The Han:SPRD Rat: A Preclinical Model of Polycystic Kidney Disease.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) stands as the most prevalent hereditary renal disorder in humans, ultimately culminating in end-stage kidney disease. Animal models carrying mutations associated with polycystic kidney disease have played an important role in the advancement of ADPKD research. The Han:SPRD rat model, carrying an R823W mutation in the Anks6 gene, is characterized by cyst formation and kidney enlargement. The mutated protein, named Samcystin, is localized in cilia of tubular epithelial cells and seems to be involved in cystogenesis. The homozygous Anks6 mutation leads to end-stage renal disease and death, making it a critical factor in kidney development and function. This review explores the utility of the Han:SPRD rat model, highlighting its phenotypic similarity to human ADPKD. Specifically, we discuss its role in preclinical trials and its importance for investigating the pathogenesis of the disease and developing new therapeutic approaches.

Prostaglandin E2, Osmoregulation, and Disease Progression in Autosomal Dominant Polycystic Kidney Disease.

Prostaglandin E2 (PGE2) plays a physiological role in osmoregulation, a process that is affected early in autosomal dominant polycystic kidney disease (ADPKD). PGE2 has also been implicated in the pathogenesis of ADPKD in preclinical models, but human data are limited. Here, we hypothesized that urinary PGE2 excretion is associated with impaired osmoregulation, disease severity, and disease progression in human ADPKD. Urinary excretions of PGE2 and its metabolite (PGEM) were measured in a prospective cohort of patients with ADPKD. The associations between urinary PGE2 and PGEM excretions, markers of osmoregulation, eGFR and height-adjusted total kidney volume were assessed using linear regression models. Cox regression and linear mixed models were used for the longitudinal analysis of the associations between urinary PGE2 and PGEM excretions and disease progression defined as 40% eGFR loss or kidney failure, and change in eGFR over time. In two intervention studies, we quantified the effect of starting tolvaptan and adding hydrochlorothiazide to tolvaptan on urinary PGE2 and PGEM excretions. In 562 patients with ADPKD (61% female, eGFR 63±28 ml/min per 1.73 m 2 ), higher urinary PGE2 or PGEM excretions were independently associated with higher plasma copeptin, lower urine osmolality, lower eGFR, and greater total kidney volume. Participants with higher baseline urinary PGE2 and PGEM excretions had a higher risk of 40% eGFR loss or kidney failure (hazard ratio, 1.28; 95% confidence interval [CI], 1.13 to 1.46 and hazard ratio, 1.50; 95% CI, 1.26 to 1.80 per two-fold higher urinary PGE2 or PGEM excretions) and a faster change in eGFR over time (-0.39 [95% CI, -0.59 to -0.20] and -0.53 [95% CI, -0.75 to -0.31] ml/min per 1.73 m 2 per year). In the intervention studies, urinary PGEM excretion was higher after starting tolvaptan, while urinary PGE2 excretion was higher after adding hydrochlorothiazide to tolvaptan. Higher urinary PGE2 and PGEM excretions in patients with ADPKD are associated with impaired osmoregulation, disease severity, and progression.

Probenecid slows disease progression in a murine model of autosomal dominant polycystic kidney disease.

Development of autosomal dominant polycystic kidney disease (ADPKD) involves renal epithelial cell abnormalities. Cystic fluid contains a high level of ATP that, among other effects, leads to a reduced reabsorption of electrolytes in cyst-lining cells, and thus results in cystic fluid accumulation. Earlier, we demonstrated that Pkd1RC/RC mice, a hypomorphic model of ADPKD, exhibit increased expression of pannexin-1, a membrane channel capable of ATP release. In the current study, we found that human ADPKD cystic epithelia have higher pannexin-1 abundance than normal collecting ducts. We hypothesized that inhibition of pannexin-1 function with probenecid can be used to attenuate ADPKD development. Renal function in male and female Pkd1RC/RC and control mice was monitored between 9 and 20 months of age. To test the therapeutic effects of probenecid (a uricosuric agent and a pannexin-1 blocker), osmotic minipumps were implanted in male and female Pkd1RC/RC mice, and probenecid or vehicle was administered for 42 days until 1 year of age. Probenecid treatment improved glomerular filtration rates and slowed renal cyst formation in male mice (as shown in histopathology). The mechanistic effects of probenecid on sodium reabsorption and fluid transport were tested on polarized mpkCCDcl4 cells subjected to short-circuit current measurements, and in 3D cysts grown in Matrigel. In the mpkCCDcl4 epithelial cell line, probenecid elicited higher ENaC currents and attenuated in vitro cyst formation, indicating lower sodium and less fluid retention in the cysts. Our studies open new avenues of research into targeting pannexin-1 in ADPKD pathology.

Metabolism-based approaches for autosomal dominant polycystic kidney disease.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) leads to end stage kidney disease (ESKD) through the development and expansion of multiple cysts throughout the kidney parenchyma. An increase in cyclic adenosine monophosphate (cAMP) plays an important role in generating and maintaining fluid-filled cysts because cAMP activates protein kinase A (PKA) and stimulates epithelial chloride secretion through the cystic fibrosis transmembrane conductance regulator (CFTR). A vasopressin V2 receptor antagonist, Tolvaptan, was recently approved for the treatment of ADPKD patients at high risk of progression. However additional treatments are urgently needed due to the poor tolerability, the unfavorable safety profile, and the high cost of Tolvaptan. In ADPKD kidneys, alterations of multiple metabolic pathways termed metabolic reprogramming has been consistently reported to support the growth of rapidly proliferating cystic cells. Published data suggest that upregulated mTOR and c-Myc repress oxidative metabolism while enhancing glycolytic flux and lactic acid production. mTOR and c-Myc are activated by PKA/MEK/ERK signaling so it is possible that cAMPK/PKA signaling will be upstream regulators of metabolic reprogramming. Novel therapeutics opportunities targeting metabolic reprogramming may avoid or minimize the side effects that are dose limiting in the clinic and improve on the efficacy observed in human ADPKD with Tolvaptan.

Defining cellular complexity in human autosomal dominant polycystic kidney disease by multimodal single cell analysis

Autosomal dominant polycystic kidney disease (ADPKD) is the leading genetic cause of end stage renal disease characterized by progressive expansion of kidney cysts. To better understand the cell types and states driving ADPKD progression, we analyze eight ADPKD and five healthy human kidney samples, generating single cell multiomic atlas consisting of ~100,000 single nucleus transcriptomes and ~50,000 single nucleus epigenomes. Activation of proinflammatory, profibrotic signaling pathways are driven by proximal tubular cells with a failed repair transcriptomic signature, proinflammatory fibroblasts and collecting duct cells. We identify GPRC5A as a marker for cyst-lining collecting duct cells that exhibits increased transcription factor binding motif availability for NF-κB, TEAD, CREB and retinoic acid receptors. We identify and validate a distal enhancer regulating GPRC5A expression containing these motifs. This single cell multiomic analysis of human ADPKD reveals previously unrecognized cellular heterogeneity and provides a foundation to develop better diagnostic and therapeutic approaches.

Adult human kidney organoids originate from CD24+ cells and represent an advanced model for adult polycystic kidney disease.

Adult kidney organoids have been described as strictly tubular epithelia and termed tubuloids. While the cellular origin of tubuloids has remained elusive, here we report that they originate from a distinct CD24+ epithelial subpopulation. Long-term-cultured CD24+ cell-derived tubuloids represent a functional human kidney tubule. We show that kidney tubuloids can be used to model the most common inherited kidney disease, namely autosomal dominant polycystic kidney disease (ADPKD), reconstituting the phenotypic hallmark of this disease with cyst formation. Single-cell RNA sequencing of CRISPR-Cas9 gene-edited PKD1- and PKD2-knockout tubuloids and human ADPKD and control tissue shows similarities in upregulation of disease-driving genes. Furthermore, in a proof of concept, we demonstrate that tolvaptan, the only approved drug for ADPKD, has a significant effect on cyst size in tubuloids but no effect on a pluripotent stem cell-derived model. Thus, tubuloids are derived from a tubular epithelial subpopulation and represent an advanced system for ADPKD disease modeling.

Myofibroblast depletion reduces kidney cyst growth and fibrosis in autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) involves the development and persistent growth of fluid filled kidney cysts. In a recent study, we showed that ADPKD kidney cyst epithelial cells can stimulate the proliferation and differentiation of peri-cystic myofibroblasts. Although dense myofibroblast populations are often found surrounding kidney cysts, their role in cyst enlargement or fibrosis in ADPKD is unclear. To clarify this, we examined the effect of myofibroblast depletion in the Pkd1RC/RC (RC/RC) mouse model of ADPKD. RC/RC;αSMAtk mice that use the ganciclovir-thymidine kinase system to selectively deplete α-smooth muscle actin expressing myofibroblasts were generated. Ganciclovir treatment for four weeks depleted myofibroblasts, reduced kidney fibrosis and preserved kidney function in these mice. Importantly, myofibroblast depletion significantly reduced cyst growth and cyst epithelial cell proliferation in RC/RC;αSMAtk mouse kidneys. Similar ganciclovir treatment did not alter cyst growth or fibrosis in wild-type or RC/RC littermates. Invitro, co-culture with myofibroblasts from the kidneys of patients with ADPKD increased 3D microcyst growth of human ADPKD cyst epithelial cells. Treatment with conditioned culture media from ADPKD kidney myofibroblasts increased microcyst growth and cell proliferation of ADPKD cyst epithelial cells. Further examination of ADPKD myofibroblast conditioned media showed high levels of protease inhibitors including PAI1, TIMP1 and 2, NGAL and TFPI-2, and treatment with recombinant PAI1 and TIMP1 increased ADPKD cyst epithelial cell proliferation invitro. Thus, our findings show that myofibroblasts directly promote cyst epithelial cell proliferation, cyst growth and fibrosis in ADPKD kidneys, and their targeting could be a novel therapeutic strategy to treat PKD.

Kidney Cyst Lining Epithelial Cells Are Resistant to Low-Dose Cisplatin-Induced DNA Damage in a Preclinical Model of Autosomal Dominant Polycystic Kidney Disease.

Increased DNA damage response (DDR) signaling in kidney cyst-lining epithelial cells (CECs) may provide an opportunity for cell-specific therapeutic targeting in autosomal dominant polycystic kidney disease (ADPKD). We hypothesized that inhibiting ataxia telangiectasia mutated (ATM; a proximal DDR kinase) together with low-dose cisplatin overwhelms the DDR response and leads to selective apoptosis of cyst-lining epithelial cells (CECs). Pkd1RC/RC/Atm+/− mice were treated with either vehicle or a single low-dose cisplatin, and the acute effects on CECs (DNA damage and apoptosis) after 72 h and chronic effects on progression (cyst size, inflammation, fibrosis) after 3 weeks were investigated. At 72 h, cisplatin caused a dose-dependent increase in γH2AX-positive nuclei in both CECs and non-cystic tubules but did not cause selective apoptosis in Pkd1RC/RC/Atm+/− mice. Moreover, the increase in γH2AX-positive nuclei was 1.7-fold lower in CECs compared to non-cystic epithelial cells (p < 0.05). Low-dose cisplatin also did not alter long-term disease progression in Pkd1RC/RC/Atm+/− mice. In vitro, human ADPKD cyst-derived cell lines were also resistant to cisplatin (WT9-12: 61.7 ± 4.6%; WT9-7: 64.8 ± 2.7% cell viability) compared to HK-2 (25.1 ± 4.2%), and 3D cyst growth in MDCK cells was not altered. Finally, combined low-dose cisplatin with AZD0156 (an ATM inhibitor) non-selectively reduced γH2AX in both cystic and non-cystic tubular cells and exacerbated cystic kidney disease. In conclusion, these data suggest that CECs are resistant to DNA damage, and that the combination of cisplatin with ATM inhibitors is not an effective strategy for selectively eliminating kidney cysts in ADPKD.

The lonidamine derivative H2-gamendazole reduces cyst formation in polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a debilitating renal neoplastic disorder with limited treatment options. It is characterized by the formation of large fluid-filled cysts that develop from kidney tubules through abnormal cell proliferation and cyst-filling fluid secretion driven by cAMP-dependent Cl- secretion. We tested the effectiveness of the indazole carboxylic acid H2-gamendazole (H2-GMZ), a derivative of lonidamine, to inhibit these processes using in vitro and in vivo models of ADPKD. H2-GMZ was effective in rapidly blocking forskolin-induced, Cl--mediated short-circuit currents in human ADPKD cells, and it significantly inhibited both cAMP- and epidermal growth factor-induced proliferation of ADPKD cells. Western blot analysis of H2-GMZ-treated ADPKD cells showed decreased phosphorylated ERK and decreased hyperphosphorylated retinoblastoma levels. H2-GMZ treatment also decreased ErbB2, Akt, and cyclin-dependent kinase 4, consistent with inhibition of heat shock protein 90, and it decreased levels of the cystic fibrosis transmembrane conductance regulator Cl- channel protein. H2-GMZ-treated ADPKD cultures contained a higher proportion of smaller cells with fewer and smaller lamellipodia and decreased cytoplasmic actin staining, and they were unable to accomplish wound closure even at low H2-GMZ concentrations, consistent with an alteration in the actin cytoskeleton and decreased cell motility. Experiments using mouse metanephric organ cultures showed that H2-GMZ inhibited cAMP-stimulated cyst growth and enlargement. In vivo, H2-GMZ was effective in slowing postnatal cyst formation and kidney enlargement in the Pkd1flox/flox: Pkhd1-Cre mouse model. Thus, H2-GMZ treatment decreases Cl- secretion, cell proliferation, cell motility, and cyst growth. These properties, along with its reported low toxicity, suggest that H2-GMZ might be an attractive candidate for treatment of ADPKD.NEW & NOTEWORTHY Autosomal dominant polycystic kidney disease (ADPKD) is a renal neoplastic disorder characterized by the formation of large fluid-filled cysts that develop from kidney tubules through abnormal cell proliferation and cyst-filling fluid secretion driven by cAMP-dependent Cl- secretion. This study shows that the lonidamine derivative H2-GMZ inhibits Cl- secretion, cell proliferation, and cyst growth, suggesting that it might have therapeutic value for the treatment of ADPKD.

Disruption of polycystin-1 cleavage leads to cardiac metabolic rewiring in mice

Cardiovascular manifestations account for marked morbi-mortality in autosomal dominant polycystic kidney disease (ADPKD). Pkd1- and Pkd2-deficient mice develop cardiac dysfunction, however the underlying mechanisms remain largely unclear. It is unknown whether impairment of polycystin-1 cleavage at the G-protein-coupled receptor proteolysis site, a significant ADPKD mutational mechanism, is involved in this process. We analyzed the impact of polycystin-1 cleavage on heart metabolism using Pkd1V/V mice, a model unable to cleave this protein and with early cardiac dysfunction. Pkd1V/V hearts showed lower levels of glucose and amino acids and higher lipid levels than wild-types, as well as downregulation of p-AMPK, p-ACCβ, CPT1B-Cpt1b, Ppara, Nppa and Acta1. These findings suggested decreased fatty acid β-oxidation, which was confirmed by lower oxygen consumption by Pkd1V/V isolated mitochondria using palmitoyl-CoA. Pkd1V/V hearts also presented increased oxygen consumption in response to glucose, suggesting that alternative substrates may be used to generate energy. Pkd1V/V hearts displayed a higher density of decreased-size mitochondria, a finding associated with lower MFN1, Parkin and BNIP3 expression. These derangements were correlated with increased apoptosis and inflammation but not hypertrophy. Notably, Pkd1V/V neonate cardiomyocytes also displayed shifts in oxygen consumption and p-AMPK downregulation, suggesting that, at least partially, the metabolic alterations are not induced by kidney dysfunction. Our findings reveal that disruption of polycystin-1 cleavage leads to cardiac metabolic rewiring in mice, expanding the understanding of heart dysfunction associated with Pkd1 deficiency and likely with human 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.

Feline Polycystic Kidney Disease: An Update.

Polycystic kidney disease (PKD) is a disease that affects felines and other mammals, such as humans. The common name is autosomal dominant polycystic kidney disease (ADPKD) and causes a progressive development of fluid-filled cysts in the kidney and sometimes in other organs as the liver and pancreas. The formation and growth of cysts progress slowly, causing deterioration of kidney tissue and a gradual decrease in kidney function, leading to irreversible kidney failure. Feline PKD or ADPKD in humans are hereditary pathologies of autosomal dominant transmission. ADPKD is one of the genetic diseases with the highest prevalence in humans. In cats, this disease also has a high prevalence, mainly in the Persian breed, being one of the most common feline genetic diseases. Imaging tests seem to be the most reliable method for diagnosis of the disease, although more genetic tests are being developed to detect the presence of the responsible mutation. In this review, we summarize the current knowledge about feline PKD to guide future research related to an adequate diagnosis and early detection of causal mutations. It can allow the establishment of selection programs to reduce or eliminate this pathology in feline breeds.

Metabolic derangement in polycystic kidney disease mouse models is ameliorated by mitochondrial-targeted antioxidants

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressively enlarging cysts. Here we elucidate the interplay between oxidative stress, mitochondrial dysfunction, and metabolic derangement using two mouse models of PKD1 mutation, PKD1RC/null and PKD1RC/RC. Mouse kidneys with PKD1 mutation have decreased mitochondrial complexes activity. Targeted proteomics analysis shows a significant decrease in proteins involved in the TCA cycle, fatty acid oxidation (FAO), respiratory complexes, and endogenous antioxidants. Overexpressing mitochondrial-targeted catalase (mCAT) using adeno-associated virus reduces mitochondrial ROS, oxidative damage, ameliorates the progression of PKD and partially restores expression of proteins involved in FAO and the TCA cycle. In human ADPKD cells, inducing mitochondrial ROS increased ERK1/2 phosphorylation and decreased AMPK phosphorylation, whereas the converse was observed with increased scavenging of ROS in the mitochondria. Treatment with the mitochondrial protective peptide, SS31, recapitulates the beneficial effects of mCAT, supporting its potential application as a novel therapeutic for ADPKD.