Polycystin-1 dysfunction impairs electrolyte and water handling in a renal precystic mouse model for ADPKD.

The PKD1 gene encodes polycystin-1 (PC1), a mechanosensor triggering intracellular responses upon urinary flow sensing in kidney tubular cells. Mutations in PKD1 lead to autosomal dominant polycystic kidney disease (ADPKD). The involvement of PC1 in renal electrolyte handling remains unknown since renal electrolyte physiology in ADPKD patients has only been characterized in cystic ADPKD. We thus studied the renal electrolyte handling in inducible kidney-specific Pkd1 knockout (iKsp- Pkd1-/-) mice manifesting a precystic phenotype. Serum and urinary electrolyte determinations indicated that iKsp- Pkd1-/- mice display reduced serum levels of magnesium (Mg2+), calcium (Ca2+), sodium (Na+), and phosphate (Pi) compared with control ( Pkd1+/+) mice and renal Mg2+, Ca2+, and Pi wasting. In agreement with these electrolyte disturbances, downregulation of key genes for electrolyte reabsorption in the thick ascending limb of Henle's loop (TA;, Cldn16, Kcnj1, and Slc12a1), distal convoluted tubule (DCT; Trpm6 and Slc12a3) and connecting tubule (CNT; Calb1, Slc8a1, and Atp2b4) was observed in kidneys of iKsp- Pkd1-/- mice compared with controls. Similarly, decreased renal gene expression of markers for TAL ( Umod) and DCT ( Pvalb) was observed in iKsp- Pkd1-/- mice. Conversely, mRNA expression levels in kidney of genes encoding solute and water transporters in the proximal tubule ( Abcg2 and Slc34a1) and collecting duct ( Aqp2, Scnn1a, and Scnn1b) remained comparable between control and iKsp- Pkd1-/- mice, although a water reabsorption defect was observed in iKsp- Pkd1-/- mice. In conclusion, our data indicate that PC1 is involved in renal Mg2+, Ca2+, and water handling and its dysfunction, resulting in a systemic electrolyte imbalance characterized by low serum electrolyte concentrations.

Fluid shear stress-induced TGF-ß/ALK5 signaling in renal epithelial cells is modulated by MEK1/2.

Renal tubular epithelial cells are exposed to mechanical forces due to fluid flow shear stress within the lumen of the nephron. These cells respond by activation of mechano-sensors located at the plasma membrane or the primary cilium, having crucial roles in maintenance of cellular homeostasis and signaling. In this paper, we applied fluid shear stress to study TGF-ß signaling in renal epithelial cells with and without expression of the Pkd1-gene, encoding a mechano-sensor mutated in polycystic kidney disease. TGF-ß signaling modulates cell proliferation, differentiation, apoptosis, and fibrotic deposition, cellular programs that are altered in renal cystic epithelia. SMAD2/3-mediated signaling was activated by fluid flow, both in wild-type and Pkd1 -/- cells. This was characterized by phosphorylation and nuclear accumulation of p-SMAD2/3, as well as altered expression of downstream target genes and epithelial-to-mesenchymal transition markers. This response was still present after cilia ablation. An inhibitor of upstream type-I-receptors, ALK4/ALK5/ALK7, as well as TGF-ß-neutralizing antibodies effectively blocked SMAD2/3 activity. In contrast, an activin-ligand trap was ineffective, indicating that increased autocrine TGF-ß signaling is involved. To study potential involvement of MAPK/ERK signaling, cells were treated with a MEK1/2 inhibitor. Surprisingly, fluid flow-induced expression of most SMAD2/3 targets was further enhanced upon MEK inhibition. We conclude that fluid shear stress induces autocrine TGF-ß/ALK5-induced target gene expression in renal epithelial cells, which is partially restrained by MEK1/2-mediated signaling.

Meta-analysis of polycystic kidney disease expression profiles defines strong involvement of injury repair processes.

Polycystic kidney disease (PKD) is a major cause of end-stage renal disease. The disease mechanisms are not well understood and the pathogenesis toward renal failure remains elusive. In this study, we present the first RNASeq analysis of a Pkd1-mutant mouse model in a combined meta-analysis with other published PKD expression profiles. We introduce the PKD Signature, a set of 1,515 genes that are commonly dysregulated in PKD studies. We show that the signature genes include many known and novel PKD-related genes and functions. Moreover, genes with a role in injury repair, as evidenced by expression data and/or automated literature analysis, were significantly enriched in the PKD Signature, with 35% of the PKD Signature genes being directly implicated in injury repair. NF-κB signaling, epithelial-mesenchymal transition, inflammatory response, hypoxia, and metabolism were among the most prominent injury or repair-related biological processes with a role in the PKD etiology. Novel PKD genes with a role in PKD and in injury were confirmed in another Pkd1-mutant mouse model as well as in animals treated with a nephrotoxic agent. We propose that compounds that can modulate the injury-repair response could be valuable drug candidates for PKD treatment.

STAT5 drives abnormal proliferation in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) leads to renal failure. The hallmark of ADPKD is increased epithelial proliferation, which has been proposed to be due to atypical signaling including abnormal JAK-STAT activity. However, the relative contribution of JAK-STAT family members in promoting proliferation in ADPKD is unknown. Here, we present siRNA JAK-STAT-focused screens discovering a previously unknown proliferative role for multiple JAK-STAT components (including STAT1, STAT2, STAT4, STAT5a, and STAT5b). Amongst these, we selected to study the growth hormone/growth hormone receptor/STAT5-axis because of its known role as a regulator of growth in nonrenal tissues. Loss of STAT5 function, facilitated by pharmacological inhibition or siRNAs, significantly reduced proliferation with an associated reduction in cyst growth in vitro. To study whether STAT5 is abnormally activated in vivo, we analyzed its expression using two independent mouse models of ADPKD. STAT5 was nuclear, thus activated, in renal epithelial cyst lining cells in both models. To test whether forced activation of STAT5 can modulate proliferation of renal cells in vivo, irrespective of the Pkd1 status, we overexpressed growth hormone. These mice showed increased STAT5 activity in renal epithelial cells, which correlated with de novo expression of cyclin D1, a STAT5 target gene. Chromatin immunoprecipitation experiments revealed that STAT5 transcriptionally activated cyclin D1 in a growth hormone-dependent fashion, thus providing a mechanism into how STAT5 enhances proliferation. Finally, we provide evidence of elevated serum growth hormone in Pkd1 mutant mice. Thus, the growth hormone/STAT5 signaling axis is a novel therapeutic target in ADPKD.

Variable Cyst Development in Autosomal Dominant Polycystic Kidney Disease: The Biologic Context.

Patients with autosomal dominant polycystic kidney disease (ADPKD) typically carry a mutation in either the PKD1 or PKD2 gene, which leads to massive cyst formation in both kidneys. However, the large intrafamilial variation in the progression rate of ADPKD suggests involvement of additional factors other than the type of mutation. The identification of these factors will increase our understanding of ADPKD and could ultimately help in the development of a clinically relevant therapy. Our review addresses the mechanisms by which various biologic processes influence cyst formation and cyst growth, thereby explaining an important part of the inter- and intrafamilial variability in ADPKD. Numerous studies from many laboratories provide compelling evidence for the influence on cyst formation by spatiotemporal gene inactivation, the genetic context, the metabolic status, the presence of existing cysts, and whether the kidneys were challenged by renal injury. Collectively, a solid basis is provided for the concept that the probability of cyst formation is determined by functional PKD protein levels and the biologic context. We model these findings in a graphic representation called the cystic probability landscape, providing a robust conceptual understanding of why cells sometimes do or do not form cysts.

Proteomics of Urinary Vesicles Links Plakins and Complement to Polycystic Kidney Disease.

Novel therapies in autosomal dominant polycystic kidney disease (ADPKD) signal the need for markers of disease progression or response to therapy. This study aimed to identify disease-associated proteins in urinary extracellular vesicles (uEVs), which include exosomes, in patients with ADPKD. We performed quantitative proteomics on uEVs from healthy controls and patients with ADPKD using a labeled approach and then used a label-free approach with uEVs of different subjects (healthy controls versus patients with ADPKD versus patients with non-ADPKD CKD). In both experiments, 30 proteins were consistently more abundant (by two-fold or greater) in ADPKD-uEVs than in healthy- and CKD-uEVs. Of these proteins, we selected periplakin, envoplakin, villin-1, and complement C3 and C9 for confirmation because they were also significantly overrepresented in pathway analysis and were previously implicated in ADPKD pathogenesis. Immunoblotting confirmed higher abundances of the selected proteins in uEVs from three independent groups of patients with ADPKD. Whereas uEVs of young patients with ADPKD and preserved kidney function already had higher levels of complement, only uEVs of patients with advanced stages of ADPKD had increased levels of villin-1, periplakin, and envoplakin. Furthermore, all five proteins correlated positively with total kidney volume. Analysis in kidney tissue from mice with kidney-specific, tamoxifen-inducible Pkd1 deletion demonstrated higher expression in more severe stages of the disease and correlation with kidney weight for each protein of interest. In summary, proteomic analysis of uEVs identified plakins and complement as disease-associated proteins in ADPKD. These proteins are new candidates for evaluation as biomarkers or targets for therapy in ADPKD.

Inhibition of Activin Signaling Slows Progression of Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD), characterized by the formation of numerous kidney cysts, is caused by PKD1 or PKD2 mutations and affects 0.1% of the population. Although recent clinical studies indicate that reduction of cAMP levels slows progression of PKD, this finding has not led to an established safe and effective therapy for patients, indicating the need to find new therapeutic targets. The role of TGF-ß in PKD is not clearly understood, but nuclear accumulation of phosphorylated SMAD2/3 in cyst-lining cells suggests the involvement of TGF-ß signaling in this disease. In this study, we ablated the TGF-ß type 1 receptor (also termed activin receptor-like kinase 5) in renal epithelial cells of PKD mice, which had little to no effect on the expression of SMAD2/3 target genes or the progression of PKD. Therefore, we investigated whether alternative TGF-ß superfamily ligands account for SMAD2/3 activation in cystic epithelial cells. Activins are members of the TGF-ß superfamily and drive SMAD2/3 phosphorylation via activin receptors, but activins have not been studied in the context of PKD. Mice with PKD had increased expression of activin ligands, even at early stages of disease. In addition, treatment with a soluble activin receptor IIB fusion (sActRIIB-Fc) protein, which acts as a soluble trap to sequester activin ligands, effectively inhibited cyst formation in three distinct mouse models of PKD. These data point to activin signaling as a key pathway in PKD and a promising target for therapy.

Scattered Deletion of PKD1 in Kidneys Causes a Cystic Snowball Effect and Recapitulates Polycystic Kidney Disease.

In total, 1 in 1000 individuals carries a germline mutation in the PKD1 or PKD2 gene, which leads to autosomal dominant polycystic kidney disease (ADPKD). Cysts can form early in life and progressively increase in number and size during adulthood. Extensive research has led to the presumption that somatic inactivation of the remaining allele initiates the formation of cysts, and the progression is further accelerated by renal injury. However, this hypothesis is primarily on the basis of animal studies, in which the gene is inactivated simultaneously in large percentages of kidney cells. To mimic human ADPKD in mice more precisely, we reduced the percentage of Pkd1-deficient kidney cells to 8%. Notably, no pathologic changes occurred for 6 months after Pkd1 deletion, and additional renal injury increased the likelihood of cyst formation but never triggered rapid PKD. In mildly affected mice, cysts were not randomly distributed throughout the kidney but formed in clusters, which could be explained by increased PKD-related signaling in not only cystic epithelial cells but also, healthy-appearing tubules near cysts. In the majority of mice, these changes preceded a rapid and massive onset of severe PKD that was remarkably similar to human ADPKD. Our data suggest that initial cysts are the principal trigger for a snowball effect driving the formation of new cysts, leading to the progression of severe PKD. In addition, this approach is a suitable model for mimicking human ADPKD and can be used for preclinical testing.

Urine Fetuin-A is a biomarker of autosomal dominant polycystic kidney disease progression.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by numerous fluid-filled cysts that frequently result in end-stage renal disease. While promising treatment options are in advanced clinical development, early diagnosis and follow-up remain a major challenge. We therefore evaluated the diagnostic value of Fetuin-A as a new biomarker of ADPKD in human urine. RESULTS: We found that renal Fetuin-A levels are upregulated in both Pkd1 and Bicc1 mouse models of ADPKD. Measurement by ELISA revealed that urinary Fetuin-A levels were significantly higher in 66 ADPKD patients (17.5 ± 12.5 µg/mmol creatinine) compared to 17 healthy volunteers (8.5 ± 3.8 µg/mmol creatinine) or 50 control patients with renal diseases of other causes (6.2 ± 2.9 µg/mmol creatinine). Receiver operating characteristics (ROC) analysis of urinary Fetuin-A levels for ADPKD rendered an optimum cut-off value of 12.2 µg/mmol creatinine, corresponding to 94% of sensitivity and 60% of specificity (area under the curve 0.74 ; p = 0.0019). Furthermore, urinary Fetuin-A levels in ADPKD patients correlated with the degree of renal insufficiency and showed a significant increase in patients with preserved renal function followed for two years. CONCLUSIONS: Our findings establish urinary Fetuin-A as a sensitive biomarker of the progression of ADPKD. Further studies are required to examine the pathogenic mechanisms of elevated renal and urinary Fetuin-A in ADPKD.

Cyst expansion and regression in a mouse model of polycystic kidney disease.

Autosomal-dominant polycystic kidney disease is characterized by progressive cyst formation and fibrosis in the kidneys. Here we describe an orthologous Pkd1(nl,nl) mouse model, with reduced expression of the normal Pkd1 transcript, on a fixed genetic background of equal parts C57Bl/6 and 129Ola/Hsd mice (B6Ola-Pkd1(nl,nl)). In these mice, the first cysts develop from mature proximal tubules around birth. Subsequently, larger cysts become visible at day 7, followed by distal tubule and collecting duct cyst formation, and progressive cystic enlargement to develop into large cystic kidneys within 4 weeks. Interestingly, cyst expansion was followed by renal volume regression due to cyst collapse. This was accompanied by focal formation of fibrotic areas, an increased expression of genes involved in matrix remodeling and subsequently an increase in infiltrating immune cells. After an initial increase in blood urea within the first 4 weeks, renal function remained stable over time and the mice were able to survive up to a year. Also, in kidneys of ADPKD patients collapsed cysts were observed, in addition to massive fibrosis and immune infiltrates. Thus, B6Ola-Pkd1(nl,nl) mice show regression of cysts and renal volume that is not accompanied by a reduction in blood urea levels.

Dose-dependent effects of sirolimus on mTOR signaling and polycystic kidney disease.

Inhibition of the mammalian target of rapamycin (mTOR) shows beneficial effects in animal models of polycystic kidney disease (PKD); however, two clinical trials in patients with autosomal dominant PKD failed to demonstrate a short-term benefit in either the early or progressive stages of disease. The stage of disease during treatment and the dose of mTOR inhibitors may account for these differing results. Here, we studied the effects of a conventional low dose and a higher dose of sirolimus (blood levels of 3 ng/ml and 30-60 ng/ml, respectively) on mTOR activity and renal cystic disease in two Pkd1-mutant mouse models at different stages of the disease. When initiated at early but not late stages of disease, high-dose treatment strongly reduced mTOR signaling in renal tissues, inhibited cystogenesis, accelerated cyst regression, and abrogated fibrosis and the infiltration of immune cells. In contrast, low-dose treatment did not significantly reduce renal cystic disease. Levels of p-S6Rp(Ser240/244), which marks mTOR activity, varied between kidneys; severity of the renal cystic phenotype correlated with the level of mTOR activity. Taken together, these data suggest that long-term treatment with conventional doses of sirolimus is insufficient to inhibit mTOR activity in renal cystic tissue. Mechanisms to increase bioavailability or to target mTOR inhibitors more specifically to kidneys, alone or in combination with other compounds, may improve the potential for these therapies in PKD.

Preclinical evaluation of tolvaptan and salsalate combination therapy in a Pkd1-mouse model.

Background: Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder and an important cause of end stage renal disease (ESRD). Tolvaptan (a V2R antagonist) is the first disease modifier drug for treatment of ADPKD, but also causes severe polyuria. AMPK activators have been shown to attenuate cystic kidney disease. Methods: In this study, we tested the efficacy of the combined administration of salsalate (a direct AMPK activator) and tolvaptan using clinically relevant doses in an adult-onset conditional Pkd1 knock-out (KO) mouse model. Results: Compared to untreated Pkd1 mutant mice, the therapeutic effects of salsalate were similar to that of tolvaptan. The combined treatment tended to be more effective than individual drugs used alone, and was associated with improved kidney survival (p < 0.0001) and reduced kidney weight to body weight ratio (p < 0.0001), cystic index (p < 0.001) and blood urea levels (p < 0.001) compared to untreated animals, although the difference between combination and single treatments was not statistically significant. Gene expression profiling and protein expression and phosphorylation analyses support the mild beneficial effects of co-treatment, and showed that tolvaptan and salsalate cooperatively attenuated kidney injury, cell proliferation, cell cycle progression, inflammation and fibrosis, and improving mitochondrial health, and cellular antioxidant response. Conclusion: These data suggest that salsalate-tolvaptan combination, if confirmed in clinical testing, might represent a promising therapeutic strategy in the treatment of ADPKD.

Short salsalate administration affects cell proliferation, metabolism, and inflammation in polycystic kidney disease.

Metabolic reprogramming is a driver of autosomal dominant polycystic kidney disease (ADPKD) progression and a potential therapeutic intervention route. We showed before that the AMP-associated protein kinase (AMPK) activator salsalate attenuates cystic disease progression. Here, we aim to study the early, direct effects of short salsalate treatment in adult-onset conditional Pkd1 deletion mice. Cystic mice were treated with salsalate for two weeks, after which NMR metabolomics and RNA sequencing analyses were performed. Pkd1 deletion resulted in clear metabolomic dysregulation. Short salsalate treatment has small, but significant, effects, reverting acetylcarnitine and phosphocholine concentrations back to wildtype levels, and showing associations with altered purine metabolism. RNA sequencing revealed that short salsalate treatment, next to restoring energy metabolism toward wildtype levels, also affects cell proliferation and inflammation, in PKD. We show that salsalate positively affects major dysregulated processes in ADPKD: energy metabolism, cell proliferation, and inflammation, providing more insights into its working mechanisms.

Altered Hippo signalling in polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive deterioration of renal function and formation of cysts, and is an important cause of end-stage renal disease. Previously we showed that tubular epithelial injury accelerates cyst formation in inducible Pkd1-deletion mice. In these mice, expression of the planar cell polarity (PCP) component Four-jointed (Fjx1) is decreased during epithelial repair, while in control mice Fjx1 expression is increased and may be required during tissue regeneration. In cystic kidneys, however, Fjx1 expression is also increased. Besides a PCP component, Four-jointed is also implicated in the Hippo-signalling pathway. This pathway is involved in organ size control by regulating proliferation and apoptosis. The role of Hippo signalling, together with the opposing expression pattern of Fjx1 during epithelial repair and at cystic stages, triggered us to investigate the activity of the Hippo pathway during these processes. Therefore, we examined its final effector molecule, the transcriptional co-activator Yes-associated protein (YAP) and observed that during tissue repair, YAP expression was not different between Pkd1-deletion mice and controls, ie during tissue regeneration YAP expression was increased and predominantly localized in the cytoplasm but normalized after tissue repair. At a later stage, however, in cystic epithelia and epithelia of dilated tubules, strong nuclear YAP accumulation was observed, accompanied by up-regulation of the YAP transcriptional targets Birc-3, Ctgf, InhbA, and Fjx1. Altered activity of the Hippo pathway was confirmed in renal tissues from human ADPKD and ARPKD patients, as well as in cystic renal tumours. Our data strengthen the concept that during epithelial repair Four-jointed is involved in PCP signalling, while in cystic kidneys it is related to Hippo signalling and cyst growth.

Effects of Hydrochlorothiazide and Metformin on Aquaresis and Nephroprotection by a Vasopressin V2 Receptor Antagonist in ADPKD: A Randomized Crossover Trial.

BACKGROUND AND OBJECTIVES: The vasopressin V2 receptor antagonist tolvaptan is the only drug that has been proven to be nephroprotective in autosomal dominant polycystic kidney disease (ADPKD). Tolvaptan also causes polyuria, limiting tolerability. We hypothesized that cotreatment with hydrochlorothiazide or metformin may ameliorate this side effect. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We performed a clinical study and an animal study. In a randomized, controlled, double-blind, crossover trial, we included 13 tolvaptan-treated patients with ADPKD. Patients were treated for three 2-week periods with hydrochlorothiazide, metformin, or placebo in random order. Primary outcome was change in 24-hour urine volume. We also measured GFR and a range of metabolic and kidney injury markers. RESULTS: Patients (age 45±8 years, 54% women, measured GFR of 55±11 ml/min per 1.73 m2) had a baseline urine volume on tolvaptan of 6.9±1.4 L/24 h. Urine volume decreased to 5.1 L/24 h (P<0.001) with hydrochlorothiazide and to 5.4 L/24 h (P<0.001) on metformin. During hydrochlorothiazide treatment, plasma copeptin (surrogate for vasopressin) decreased, quality of life improved, and several markers of kidney damage and glucose metabolism improved. Metformin did not induce changes in these markers or in quality of life. Given these results, the effect of adding hydrochlorothiazide to tolvaptan was investigated on long-term kidney outcome in an animal experiment. Water intake in tolvaptan-hydrochlorothiazide cotreated mice was 35% lower than in mice treated with tolvaptan only. Combination treatment was superior to "no treatment" on markers of disease progression (kidney weight, P=0.003 and cystic index, P=0.04) and superior or equal to tolvaptan alone. CONCLUSIONS: Both metformin and hydrochlorothiazide reduced tolvaptan-caused polyuria in a short-term study. Hydrochlorothiazide also reduced polyuria in a long-term animal model without negatively affecting nephroprotection. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2022_03_21_CJN11260821.mp3.

Curcumin inhibits cystogenesis by simultaneous interference of multiple signaling pathways: in vivo evidence from a Pkd1-deletion model.

Autosomal dominant polycystic kidney disease (ADPKD) caused by mutations in either the PKD1 or PKD2 gene is a major cause of end-stage renal failure. A number of compounds targeting specific signaling pathways were able to inhibit cystogenesis in rodent models and are currently being tested in clinical trials. However, given the complex signaling in ADPKD, an ideal therapy would likely have to comprise several pathways at once. Therefore, multitarget compounds may provide promising therapeutic interventions for the treatment of ADPKD. To test this hypothesis, we treated Pkd1-deletion mice with diferuloylmethane (curcumin), a compound without appreciable side effects and known to modulate several pathways that are also altered in ADPKD, e.g., mammalian target of rapamycin (mTOR) and Wnt signaling. After conditional inactivation of Pkd1, mTOR signaling was indeed elevated in cystic kidneys. Interestingly, also activation of signal transducers and activator of transcription 3 (STAT3) strongly correlated with cyst progression. Both pathways were effectively inhibited in vitro by curcumin. Importantly, Pkd1-deletion mice that were treated with curcumin and killed at an early stage of PKD displayed improved renal histology and reduced STAT3 activation, proliferation index, cystic index, and kidney weight/body weight ratios. In addition, renal failure was significantly postponed in mice with severe PKD. These data suggest that multitarget compounds hold promising potential for safe and effective treatment of ADPKD.

Toxic tubular injury in kidneys from Pkd1-deletion mice accelerates cystogenesis accompanied by dysregulated planar cell polarity and canonical Wnt signaling pathways.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by large fluid-filled cysts and progressive deterioration of renal function necessitating renal replacement therapy. Previously, we generated a tamoxifen-inducible, kidney epithelium-specific Pkd1-deletion mouse model and showed that inactivation of the Pkd1 gene induces rapid cyst formation in developing kidneys and a slow onset of disease in adult mice. Therefore, we hypothesized that injury-induced tubular epithelial cell proliferation may accelerate cyst formation in the kidneys of adult Pkd1-deletion mice. Mice were treated with the nephrotoxicant 1,2-dichlorovinyl-cysteine (DCVC) after Pkd1-gene inactivation, which indeed accelerated cyst formation significantly. After the increased proliferation during tissue regeneration, proliferation decreased to basal levels in Pkd1-deletion mice just as in DCVC-treated controls. However, in severe cystic kidneys, 10-14 weeks after injury, proliferation increased again. This biphasic response suggests that unrestricted cell proliferation after injury is not the underlying mechanism for cyst formation. Aberrant planar cell polarity (PCP) signaling and increased canonical Wnt signaling are suggested to be involved in cyst formation. Indeed, we show here that in Pkd1 conditional deletion mice expression of the PCP component Four-jointed (Fjx1) is decreased while its expression is required during tissue regeneration. In addition, we show that altered centrosome position and the activation of canonical Wnt signaling are early effects of Pkd1-gene disruption. This suggests that additional stimuli or events are required to trigger the process of cyst formation. We propose that during tissue repair, the integrity of the newly formed Pkd1-deficient cells is modified rendering them susceptible to subsequent cyst formation.

Elevated TGFbeta-Smad signalling in experimental Pkd1 models and human patients with polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited renal disease characterized by many fluid-filled cysts and interstitial fibrosis in the kidneys, leading to chronic renal failure. During cystogenesis the renal tubules undergo extensive structural alterations that are accompanied by altered cellular signalling, directly and/or indirectly regulated by the PKD1 and PKD2 proteins. Since transforming growth factor (TGF)-beta signalling modulates cell proliferation, differentiation, apoptosis, adhesion and migration of various cell types, we studied the activation of this signalling pathway in Pkd1-mutant mouse models at different stages of the disease. Therefore, we analysed expression of the TGFbeta-Smad signalling pathway and its target genes in different Pkd1 mutant mouse models in various stages of polycystic disease. Nuclear accumulation of P-Smad2 in cyst lining epithelial cells was not observed in the initiation phase but was observed at mild and more advanced stages of PKD. This coincides with mild fibrosis and increased mRNA levels of TGFbeta target genes, such as fibronectin, collagen type I, plasminogen activator inhibitor 1 and matrix metalloproteinase-2. At this stage many interstitial fibroblasts were found around cysts, which also showed nuclear localization for P-Smad2. However, bone morphogenetic protein (BMP) signalling, which can antagonize TGFbeta signalling, is not affected, since nuclear expression of P-Smad1/5/8 and expression of the BMP target gene, inhibitor of DNA binding/differential-1 (ID-1) is not altered compared to wild-type controls. Also, human kidneys with progressive ADPKD showed increased nuclear localization of P-Smad2, while in general expression of P-Smad1/5/8 was weak. These results exclude TGFbeta signalling at the initiation of cystogenesis, but indicate an important role during cyst progression and in fibrogenesis of progressive ADPKD.

Renal cyst growth is attenuated by a combination treatment of tolvaptan and pioglitazone, while pioglitazone treatment alone is not effective.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common monogenic disorders, characterized by the progressive formation of fluid-filled cysts. Tolvaptan is an approved drug for ADPKD patients, but is also associated with multiple side effects. The peroxisome proliferator-activator receptor gamma (PPARγ) agonist pioglitazone slows disease progression in the PCK rat model for PKD. Here, we tested whether a combination treatment of relevant doses of tolvaptan and pioglitazone leads to improved efficacy in an adult-onset PKD mouse model. Tolvaptan indeed slowed PKD progression, but the combination treatment was not more effective than tolvaptan alone. In addition, although pioglitazone raised plasma levels of its surrogate drug marker adiponectin, the drug unexpectedly failed to slow PKD progression. The pioglitazone target PPARγ was expressed at surprisingly low levels in mouse, rat and human kidneys. Other pioglitazone targets were more abundantly expressed, but this pattern was comparable across various species. The data suggest that several potential pharmacokinetic and pharmacodynamic (PK/PD) differences between different species may underlie whether or not pioglitazone is able to slow PKD progression. The ongoing phase II clinical trial with low-dose pioglitazone treatment (NCT02697617) will show whether pioglitazone is a suitable drug candidate for ADPKD treatment.

Prioritization of novel ADPKD drug candidates from disease-stage specific gene expression profiles.

BACKGROUND: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common causes of end-stage renal failure, caused by mutations in PKD1 or PKD2 genes. Tolvaptan, the only drug approved for ADPKD treatment, results in serious side-effects, warranting the need for novel drugs. METHODS: In this study, we applied RNA-sequencing of Pkd1cko mice at different disease stages, and with/without drug treatment to identify genes involved in ADPKD progression that were further used to identify novel drug candidates for ADPKD. We followed an integrative computational approach using a combination of gene expression profiling, bioinformatics and cheminformatics data. FINDINGS: We identified 1162 genes that had a normalized expression after treating the mice with drugs proven effective in preclinical models. Intersecting these genes with target affinity profiles for clinically-approved drugs in ChEMBL, resulted in the identification of 116 drugs targeting 29 proteins, of which several are previously linked to Polycystic Kidney Disease such as Rosiglitazone. Further testing the efficacy of six candidate drugs for inhibition of cyst swelling using a human 3D-cyst assay, revealed that three of the six had cyst-growth reducing effects with limited toxicity. INTERPRETATION: Our data further establishes drug repurposing as a robust drug discovery method, with three promising drug candidates identified for ADPKD treatment (Meclofenamic Acid, Gamolenic Acid and Birinapant). Our strategy that combines multiple-omics data, can be extended for ADPKD and other diseases in the future. FUNDING: European Union's Seventh Framework Program, Dutch Technology Foundation Stichting Technische Wetenschappen and the Dutch Kidney Foundation.