Renal Cystogenesis Research Articles

Profiling renal sodium transporters in mice with nephron Ift88 disruption: Association with sex, cysts, and blood pressure.

Loss of nephron primary cilia due to disruption of the Ift88 gene results in sex‐ and age‐specific phenotypes involving renal cystogenesis, blood pressure (BP) and urinary Na+ excretion. Previous studies demonstrated that male mice undergoing induction of nephron‐specific Ift88 gene disruption at 2 months of age developed reduced BP and increased salt‐induced natriuresis when pre‐cystic (2 months post‐induction) and became hypertensive associated with frankly cystic kidneys by 9 months post‐induction; in contrast, female Ift88 KO mice manifested no unique phenotype 2 months post‐induction and had mildly reduced BP 9 months post‐induction. The current study utilized these Ift88 KO mice to investigate associated changes in renal Na+ transporter and channel protein expression. At 2 months post‐induction, pre‐cystic male Ift88 KO mice had reduced high salt diet associated total NKCC2 levels while female mice had no alterations in Na+ transporters or channels. At 9 months post‐induction, cystic male Ift88 KO mice had increased total and phosphorylated NHE3 levels together with reduced NKCC2, phosphorylated and/or total NCC, and ENaC‐α expression on normal and high salt diets. In contrast, female Ift88 KO mice at 9 months post‐induction had no changes in Na+ transporters or channels beyond an increase in phosphorylated‐NCC during high salt intake. Thus, reduced BP in pre‐cystic, and elevated BP in renal cystic, male Ift88 KO mice are associated with unique sex‐dependent changes in nephron Na+ transporter/channel expression.

Large deletion of Wdr19 in developing renal tubules disrupts primary ciliogenesis, leading to polycystic kidney disease in mice.

WD repeat domain 19 (Wdr19) is a major component of the intraflagellar transport (IFT) machinery, which is involved in the function of primary cilia. However, the effects of Wdr19 on primary cilia formation, cystogenesis, and polycystic kidney disease (PKD) progression remain unclear. To study these effects, we generated three lines of kidney-specific conditional knockout mice: Wdr19-knockout (Wdr19-KO, Wdr19f/- ::Cdh16-CreTg/0 ), Pkd1-knockout (Pkd1-KO, Pkd1f/- ::Cdh16-CreTg/0 ), and Wdr19/Pkd1-double knockout (Wdr19&Pkd1-dKO, Wdr19f/- ;Pkd1f/- ::Cdh16-CreTg/0 ) mice. Ultrastructural analysis using transmission electron microscopy (TEM) indicated that the primary cilia were almost absent at postnatal day 10 in Wdr19-KO mice compared with Pkd1-KO and wild-type (WT) mice. However, the primary cilia appeared structurally normal even if malfunctional in Pkd1-deficient cysts. The Pkd1-KO mice had the most severe PKD progression, including the shortest lifespan (14 days) and the largest renal cysts, among the three knockout lines. Thus, the molecular mechanism of renal cystogenesis in Wdr19-KO mice (primary cilia abrogation) was different from that in Pkd1-KO mice (primary cilia malfunction). In summary, Wdr19 deficiency leads to primary cilia abrogation and renal cyst formation. Wdr19 is primarily proposed to participate in retrograde IFT and to be crucial for the construction of primary cilia, which are critical organelles for tubulogenesis in the developing kidneys. © 2022 The Pathological Society of Great Britain and Ireland.

Novel zebrafish polycystic kidney disease models reveal functions of the Hippo pathway in renal cystogenesis.

ABSTRACTThe Hippo signaling pathway is a kinase cascade that plays an important role in organ size control. As the main effectors of the Hippo pathway, transcription coactivators Yap1/Wwtr1 are regulated by the upstream kinase Stk3. Recent studies in mammals have implicated the Hippo pathway in kidney development and kidney diseases. To further illustrate its roles in vertebrate kidney, we generated a series of zebrafish mutants targeting stk3, yap1 and wwtr1 genes. The stk3−/− mutant exhibited edema, formation of glomerular cysts and pronephric tubule dilation during the larval stage. Interestingly, disruption of wwtr1, but not yap1, significantly alleviated the renal phenotypes of the stk3−/− mutant, and overexpression of Wwtr1 with the CMV promoter also induced pronephric phenotypes, similar to those of the stk3−/− mutant, during larval stage. Notably, adult fish with Wwtr1 overexpression developed phenotypes similar to those of human polycystic kidney disease (PKD). Overall, our analyses revealed roles of Stk3 and Wwtr1 in renal cyst formation. Using a pharmacological approach, we further demonstrated that Stk3-deficient zebrafish could serve as a PKD model for drug development.

Is There a Functional Role of Mitochondrial Dysfunction in the Pathogenesis of ARPKD?

Polycystic kidney diseases (PKD) form a group of severe genetic kidney diseases that are characterized by fibrocystic changes of the kidneys and the liver. The main forms of PKD are autosomal dominant and autosomal recessive polycystic kidney disease (ADPKD, ARPKD). In general, cystic kidney diseases are the most common genetic cause of chronic kidney failure in both children and adults (1). ARPKD is a disorder that typically is diagnosed very early in life or even prenatally. Typically, the kidneys show bilateral massive enlargement although they keep their reniform shape. Kidney disease results in a need for kidney replacement therapy (KRT) in around 50% of patients in the first two decades of life (2, 3). Liver involvement is obligatory in ARPKD, typically presenting with congenital hepatic fibrosis and portal hypertension or with dilated bile ducts. Both are considered to be a consequence of a defect during development of the bile ducts (“ductal plate malformation”) (4). Adult patients with ARPKD have been described with variable disease manifestations. Hepatic disease may be the leading clinical issue in adults with ARPKD (5). In ADPKD renal cystogenesis starts early in life or even before birth but does not present with obvious clinical symptoms until adulthood in most patients (1, 6, 7). Liver involvement is typically characterized by liver cysts. Additional extrarenal symptoms in ADPKD include for example intracranial aneurysms, cardiac valve anomalies like mitral prolapse or abdominal wall diverticula in a subset of patients (1, 7). Extrarenal manifestations may be very helpful for clinical differential diagnosis and to distinguish ADPKD from ARPKD. ADPKD is mainly caused by variants in the genes PKD1 and PKD2 with the large majority of patients carrying PKD1 variants (1). Variants in other genes e.g., with atypical forms of ADPKD have been identified (7). Variants in PKHD1 are the main cause of ARPKD with phenotypical overlay e.g., of kidney disease in patients with other underlying genetic changes (1, 8), including biallelic hypomorphic PKD1 variants. Genotype-phenotype correlations in ADPKD show more rapid progression of kidney disease in patients with PKD1 variants and especially in those patients carrying variants resulting in protein truncation (7, 9). For ARPKD it was shown by various groups that biallelic truncating variants are associated with severe phenotypes. A recent study extended the findings and revealed that for missense variants the affected region in PKHD1 also seems to be important (10). In a study on 304 children with the clinical diagnosis of ARPKD and detected PKHD1 variants it was found that patients with either two missense variants affecting the amino acids 709–1837 or a null variant and a missense variant in this region less frequently showed progression to chronic kidney failure when being compared during their follow-up when compared to patients with variants affecting other regions of PKHD1. On the other hand patients with variants affecting the amino acids 2625–4074 showed less favorable hepatic outcome (10). The underlying molecular mechanisms of these associations remain to be explored.

Interactions Between TULP3 and ARL13B in Lipidated Protein Transport to Cilia and Regulation of Renal Cystogenesis

Interactions Between TULP3 and ARL13B in Lipidated Protein Transport to Cilia and Regulation of Renal Cystogenesis

Mechanisms of Tethered-Ligand Mediated Polycystin-1 GPCR Signaling

Polycystin-1 (PC1) is the protein product of the most commonly mutated gene in autosomal dominant polycystic kidney disease (ADPKD). PC1 functions as an atypical G protein-coupled receptor (GPCR) and is a subunit of the PC1-PC2 ion channel complex. In vivo studies using different models of ADPKD demonstrated that PC1-regulated G protein signaling is a critical function for preventing renal cystogenesis. ADPKD resulting from non-truncating mutations of PC1 may be amenable to mutation-specific therapies. However, this type of therapeutic approach will not be feasible without an in-depth understanding of the functional mechanisms of PC1. In this study, we have combined functional-biochemical analyses, mutation experiments and accelerated molecular simulations to investigate the dynamic mechanisms of PC1 signaling. All-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method are consistent with mutation experimental data on the PC1 C-terminal fragment (CTF) for the wildtype, ADPKD mutants and CTF without the stalk region (∆CTF). The stalk region is identified to be an important tethered ligand, which mediates PC1-G protein signaling. Key residue interactions predicted from the GaMD simulations are further validated with newly designed mutation experiments. Complementary experiments and simulations have provided important insights into the mechanisms of PC1 GPCR signaling at an atomic level. This is expected to facilitate future drug design efforts targeting PC1 for more effective treatments of ADPKD.

PKD1-Dependent Renal Cystogenesis in Human Induced Pluripotent Stem Cell-Derived Ureteric Bud/Collecting Duct Organoids.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease leading to renal failure, wherein multiple cysts form in renal tubules and collecting ducts derived from distinct precursors: the nephron progenitor and ureteric bud (UB), respectively. Recent progress in induced pluripotent stem cell (iPSC) biology has enabled cyst formation in nephron progenitor-derived human kidney organoids in which PKD1 or PKD2, the major causative genes for ADPKD, are deleted. However, cysts have not been generated in UB organoids, despite the prevalence of collecting duct cysts in patients with ADPKD. CRISPR-Cas9 technology deleted PKD1 in human iPSCs and the cells induced to differentiate along pathways leading to formation of either nephron progenitor or UB organoids. Cyst formation was investigated in both types of kidney organoid derived from PKD1-deleted iPSCs and in UB organoids generated from iPSCs from a patient with ADPKD who had a missense mutation. Cysts formed in UB organoids with homozygous PKD1 mutations upon cAMP stimulation and, to a lesser extent, in heterozygous mutant organoids. Furthermore, UB organoids generated from iPSCs from a patient with ADPKD who had a heterozygous missense mutation developed cysts upon cAMP stimulation. Cysts form in PKD1 mutant UB organoids as well as in iPSCs derived from a patient with ADPKD. The organoids provide a robust model of the genesis of ADPKD.

Rapamycin and dexamethasone during pregnancy prevent tuberous sclerosis complex-associated cystic kidney disease.

Chronic kidney disease is the main cause of mortality in patients with tuberous sclerosis complex (TSC) disease. The mechanisms underlying TSC cystic kidney disease remain unclear, with no available interventions to prevent cyst formation. Using targeted deletion of TSC1 in nephron progenitor cells, we showed that cysts in TSC1-null embryonic kidneys originate from injured proximal tubular cells with high mTOR complex 1 activity. Injection of rapamycin to pregnant mice inhibited the mTOR pathway and tubular cell proliferation in kidneys of TSC1-null offspring. Rapamycin also prevented renal cystogenesis and prolonged the life span of TSC newborns. Gene expression analysis of proximal tubule cells identified sets of genes and pathways that were modified secondary to TSC1 deletion and rescued by rapamycin administration during nephrogenesis. Inflammation with mononuclear infiltration was observed in the cystic areas of TSC1-null kidneys. Dexamethasone administration during pregnancy decreased cyst formation by not only inhibiting the inflammatory response, but also interfering with the mTORC1 pathway. These results reveal mechanisms of cystogenesis in TSC disease and suggest interventions before birth to ameliorate cystic disease in offspring.

Integrin-β1 is required for the renal cystogenesis caused by ciliary defects.

Defects in the function of primary cilia are commonly associated with the development of renal cysts. On the other hand, the intact cilium appears to contribute a cystogenic signal whose effectors remain unclear. As integrin-β1 is required for the cystogenesis caused by the deletion of the polycystin 1 gene, we asked whether it would be similarly important in the cystogenetic process caused by other ciliary defects. We addressed this question by investigating the effect of integrin-β1 deletion in a ciliopathy genetic model in which the Ift88 gene, a component of complex B of intraflagellar transport that is required for the proper assembly of cilia, is specifically ablated in principal cells of the collecting ducts. We showed that the renal cystogenesis caused by loss of Ift88 is prevented when integrin-β1 is simultaneously depleted. In parallel, pathogenetic manifestations of the disease, such as increased inflammatory infiltrate and fibrosis, were also significantly reduced. Overall, our data indicate that integrin-β1 is also required for the renal cystogenesis caused by ciliary defects and point to integrin-β1-controlled pathways as common drivers of the disease and as possible targets to interfere with the cystogenesis caused by ciliary defects.

Interferon Regulatory Factor-5 in Resident Macrophage Promotes Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease is caused by genetic mutations in PKD1 or PKD2. Macrophages and their associated inflammatory cytokines promote cyst progression; however, transcription factors within macrophages that control cytokine production and cystic disease are unknown. In these studies, we used conditional Pkd1 mice to test the hypothesis that macrophage-localized interferon regulatory factor-5 (IRF5), a transcription factor associated with production of cyst-promoting cytokines (TNFα, IL-6), is required for accelerated cyst progression in a unilateral nephrectomy (1K) model. Analyses of quantitative real-time PCR (qRT-PCR) and flow-cytometry data 3 weeks post nephrectomy, a time point before the onset of severe cystogenesis, indicate an accumulation of inflammatory infiltrating and resident macrophages in 1K Pkd1 mice compared with controls. qRT-PCR data from FACS cells at this time demonstrate that macrophages from 1K Pkd1 mice have increased expression of Irf5 compared with controls. To determine the importance of macrophage-localized Irf5 in cyst progression, we injected scrambled or IRF5 antisense oligonucleotide (ASO) in 1K Pkd1 mice and analyzed the effect on macrophage numbers, cytokine production, and renal cystogenesis 6 weeks post nephrectomy. Analyses of qRT-PCR and IRF5 ASO treatment significantly reduced macrophage numbers, Irf5 expression in resident-but not infiltrating-macrophages, and the severity of cystic disease. In addition, IRF5 ASO treatment in 1K Pkd1 mice reduced Il6 expression in resident macrophages, which was correlated with reduced STAT3 phosphorylation and downstream p-STAT3 target gene expression. These data suggest that Irf5 promotes inflammatory cytokine production in resident macrophages resulting in accelerated cystogenesis.

Informatics-guided drug repurposing for Autosomal Dominant Polycystic Kidney Disease (ADPKD).

Informatics-guided drug repurposing for Autosomal Dominant Polycystic Kidney Disease (ADPKD).

TMEM33 regulates intracellular calcium homeostasis in renal tubular epithelial cells

Mutations in the polycystins cause autosomal dominant polycystic kidney disease (ADPKD). Here we show that transmembrane protein 33 (TMEM33) interacts with the ion channel polycystin-2 (PC2) at the endoplasmic reticulum (ER) membrane, enhancing its opening over the whole physiological calcium range in ER liposomes fused to planar bilayers. Consequently, TMEM33 reduces intracellular calcium content in a PC2-dependent manner, impairs lysosomal calcium refilling, causes cathepsins translocation, inhibition of autophagic flux upon ER stress, as well as sensitization to apoptosis. Invalidation of TMEM33 in the mouse exerts a potent protection against renal ER stress. By contrast, TMEM33 does not influence pkd2-dependent renal cystogenesis in the zebrafish. Together, our results identify a key role for TMEM33 in the regulation of intracellular calcium homeostasis of renal proximal convoluted tubule cells and establish a causal link between TMEM33 and acute kidney injury.

New targets for renal and hepatic cystogenesis. The help of proteomic in the understanding of ADPKD

New targets for renal and hepatic cystogenesis. The help of proteomic in the understanding of ADPKD

Dysregulation of metalloproteinase complex ADAM10‐MMP14 plays the key role in preservation of adherens junction integrity at renal cystogenesis

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is one of the most common lifeth‐reatening genetic diseases and is characterized by early cystogenesis in kidney, which eventually results in end‐stage renal disease. We previously reported that the inhibition of A Disintegrin and metalloproteinase domain‐containing protein 10 (ADAM10) can block the cystogenesis in renal tubular epithelial cells through the preservation of E‐Cadherin. In addition, recent studies also indicate that another major matrix metalloproteinase, Matrix metalloproteinase‐14 (MMP14) promotes cystogenesis in kidney epithelial cells, which was blocked by its inhibitors. In our study, we investigate these two major sheddases association and their roles in preservation of adherens junctions in renal cystogenesisMethodsImmunoprecipitation, immunostaining and 3‐D cell culture technologies are used to analyze the interaction between metalloproteinase ADAM10 and MMP14 in Madin‐Darby Canine Kidney (MDCK) cells.ResultsOur data shows that ADAM10 and MMP14 form a stable complex on the membrane of MDCK cells at hemopexin domain of MMP14. The activation of MMP14 is required for ADAM10 in shedding of E‐Cadherin. Either an enzyme‐inactive mutant MMP14E240A (Glu240 to Ala) or the deletion of the catalytic domain of MMP14 (MMP14ΔCAT) significantly decreases the sheddase activity of ADAM10. In addition, knockdown of MMP14 decreases the cleavage of ECadherin by ADAM10, and knockdown of ADAM10 enhances the activation of proMMP2 by MMP14. Furthermore, ectopic expression of MMP14 increases the proliferation of renal epithelial cells, alters the cell‐cell adhesion and promotes the cystogenesis of renal epithelial cells in 3‐dimensional collagen‐1 gels. Ectopic expression of the MMP14E240A mutant promotes strong contact inhibition of the cells, and results in tubular rather than cystic growth in collagen gels.ConclusionOur study shows for the first time that ADAM10 form a unique, stable complex at hemopexin domain of MMP14 on the surface of epithelial cells, and the ADAM10‐MMP14 complex regulates their sheddase activity on many cell junctional proteins. Furthermore, ADAM10 is a negative regulator of MMP14 that ADAM10 inhibits the shaddase activity of MMP14. The functional interaction between ADAM10 and MMP14 as a complex could play a pivotal role in the cystogenesis of renal epithelial cells. Our data provide a potential therapeutic target in ADPKD through the modulation of ADAM10‐MMP14 complex.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ezrin is a novel target in cyst initiation in ADPKD

Autosomal dominant polycystic kidney disease (ADPKD), characterized by progressive renal cystogenesis and other extrarenal manifestations, is one of the most common monogenetic diseases, and is caused by loss of function mutations in PKD1 and PKD2, which encode transmembrane proteins polycystin 1 and 2 (PC1/2). The polycystin proteins have been hypothesized to function as a complex that has been localized to the plasma membrane, cilia, endoplasmic reticulum, and junctions of epithelial cells. The downstream effects of either PC1 or PC2 function loss have not been elucidated. Previously, an investigation of smaller cysts from nephrectomized human ADPKD kidneys revealed a pattern of increased cell width and an altered apical compartment. Subsequently, we observed a decreased abundance and disrupted localization of ezrin, a master scaffold and apical compartment regulator, in similarly characterized cysts. Following this observation, we explored the role of ezrin disruption in a three‐dimensional in vitro model of cystogenesis. Acute inactivation of Pkd2 lead to significant loss of ezrin protein. We confirmed the regulatory relationship between PC2 and ezrin with immunohistochemistry in a genetically inducible in vivo short induction model of Pkd2 inactivation (Pkd2fl/fl Pax8 rtTA TetOCre) that revealed a kidney tubule wide decrease in ezrin at the apical membrane. Lipid protein overlay assays and immunoprecipitation experiments demonstrate both PC2 and ezrin interact with similar phosphoinositides, and possibly with each other, suggesting a role for PC2 in apical compartment and junctional assembly. Functionally, we found that PC2 is critical in junctional integrity, with acute inactivation of Pkd2 causing decreases in transepithelial electrical resistance in inducible primary renal epithelial cells (Pkd2fl/fl Pax8 rtTA TetOCre). Ezrin, expressed in all segments of the kidney, is a regulator of many components of the junctions and apical compartment of renal epithelial cells. The disruption of ezrin following inactivation of Pkd2 in both in vitro and in vivo models, and shared interacting lipids, suggests that PC2 may play a role in the regulation of ezrin in renal epithelial cells and that ezrin may be involved in the initiation of cystogenesis following loss of PC2.Support or Funding InformationNIDDK F31 NRSA, PKD Foundation, NIDDK Baltimore Polycystic Kidney Disease Research and Clinical Core CenterThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tulp3 Regulates Renal Cystogenesis by Trafficking of Cystoproteins to Cilia

Polycystic kidney disease proteins, polycystin-1 and polycystin-2, localize to primary cilia. Polycystin knockouts have severe cystogenesis compared to ciliary disruption, whereas simultaneous ciliary loss suppresses excessive cyst growth. These data suggest the presence of a cystogenic activator that is inhibited by polycystins and an independent but relatively minor cystogenic inhibitor, either of which are cilia dependent. However, current genetic models targeting cilia completely ablate the compartment, making it difficult to uncouple cystoprotein function from ciliary localization. Thus, the role of cilium-generated signaling in cystogenesis is unclear. We recently demonstrated that the tubby family protein Tulp3 determines ciliary trafficking of polycystins in kidney collecting duct cells without affecting protein levels or cilia. Here, we demonstrate that embryonic-stage, nephron-specific Tulp3 knockout mice developed cystic kidneys, while retaining intact cilia. Cystic kidneys showed increased mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), mTOR, and persistently high cyclic AMP (cAMP) signaling, suggesting contribution of multiple factors to cystogenesis. Based on kidney-to-body-weight ratio, cystic index, and epithelial proliferation in developing tubules or cysts, the severity of cystogenesis upon Tulp3 deletion was intermediate between that caused by loss of polycystin-1 or cilia. However, concomitant Tulp3 loss did not inhibit cystogenesis in polycystin-1 knockouts, unlike ciliary disruption. Interestingly, ciliary trafficking of the small guanosine triphosphatase (GTPase) Arl13b, loss of which causes cystogenic severity similar to ciliary loss, was reduced prior to cyst initiation. Thus, we propose that cystogenesis in Tulp3 mutants results from a reduction of ciliary levels of polycystins, Arl13b, and Arl13b-dependent lipidated cargoes. Arl13b might be the ciliary factor that represses cystogenesis distinct from polycystins.

Tsc1 ablation in Prx1 and Osterix lineages causes renal cystogenesis in mouse

Tuberous Sclerosis Complex (TSC) is caused by mutations in TSC1 or TSC2, which encode negative regulators of the mTOR signaling pathway. The renal abnormalities associated with TSC include angiomyolipoma, cysts, and renal cell carcinoma. Here we report that specific ablation of Tsc1 using the mesenchymal stem cell-osteoblast lineage markers induced cystogenesis in mice. Using Rosa-tdTomato mice, we found that Prx1- or Dermo1-labeled cells were present in the nephron including glomerulus but they were not stained by markers for podocytes, mesangial cells, endothelial cells, or proximal or loop of Henle tubular cells, while Osx is known to label tubular cells. Tsc1 deficiency in Prx1 lineage cells caused development of mild cysts that were positive only for Tamm-Horsfall protein (THP), a loop of Henle marker, while Tsc1 deficiency in Osx lineage cells caused development of cysts that were positive for Villin, a proximal tubular cell marker. On the other hand, Tsc1 deficiency in the Dermo1 lineage did not produce detectable phenotypical changes in the kidney. Cyst formation in Prx1-Cre; Tsc1f/f and Osx-Cre; Tsc1f/f mice were associated with increase in both proliferative and apoptotic cells in the affected tissue and were largely suppressed by rapamycin. These results suggest that Prx1 and Osx lineages cells may contribute to renal cystogenesis in TSC patients.

Effect of Trehalose Supplementation on Autophagy and Cystogenesis in a Mouse Model of Polycystic Kidney Disease.

Autophagy impairment has been demonstrated in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) and could be a new target of treatment. Trehalose is a natural, nonreducing disaccharide that has been shown to enhance autophagy. Therefore, we investigated whether trehalose treatment reduces renal cyst formation in a Pkd1-hypomorphic mouse model. Pkd1 miRNA transgenic (Pkd1 miR Tg) mice and wild-type littermates were given drinking water supplemented with 2% trehalose from postnatal day 35 to postnatal day 91. The control groups received pure water or 2% sucrose for the control of hyperosmolarity. The effect on kidney weights, cystic indices, renal function, cell proliferation, and autophagic activities was determined. We found that Pkd1 miR Tg mice had a significantly lower renal mRNA expression of autophagy-related genes, including atg5, atg12, ulk1, beclin1, and p62, compared with wild-type control mice. Furthermore, immunohistochemical analysis showed that cystic lining cells had strong positive staining for the p62 protein, indicating impaired degradation of the protein by the autophagy-lysosome pathway. However, trehalose treatment did not improve reduced autophagy activities, nor did it reduce relative kidney weights, plasma blood urea nitrogen levels, or cystatin C levels in Pkd1 miR Tg mice. Histomorphological analysis revealed no significant differences in the renal cyst index, fibrosis score, or proliferative score among trehalose-, sucrose-, and water-treated groups. Our results demonstrate that adding trehalose to drinking water does not modulate autophagy activities and renal cystogenesis in Pkd1-deficient mice, suggesting that an oral supplement of trehalose may not affect the progression of ADPKD.

Bexarotene - a novel modulator of AURKA and the primary cilium in VHL-deficient cells.

ABSTRACTLoss of the gene von Hippel–Lindau (VHL) is associated with loss of primary cilia and is causally linked to elevated levels of Aurora kinase A (AURKA). We developed an image-based high-throughput screening (HTS) assay using a dual-labeling image analysis strategy that identifies both the cilium and the basal body. By using this strategy, we screened small-molecule compounds for the targeted rescue of cilia defects associated with VHL deficiency with high accuracy and reproducibility. Bexarotene was identified and validated as a positive regulator of the primary cilium. Importantly, the inability of an alternative retinoid X receptor (RXR) agonist to rescue ciliogenesis, in contrast to bexarotene, suggested that multiple bexarotene-driven mechanisms were responsible for the rescue. We found that bexarotene decreased AURKA expression in VHL-deficient cells, thereby restoring the ability of these cells to ciliate in the absence of VHL. Finally, bexarotene treatment reduced the propensity of subcutaneous lesions to develop into tumors in a mouse xenograft model of renal cell carcinoma (RCC), with a concomitant decrease in activated AURKA, highlighting the potential of bexarotene treatment as an intervention strategy in the clinic to manage renal cystogenesis associated with VHL deficiency and elevated AURKA expression.

Effect of celastrol on the progression of polycystic kidney disease in a Pkd1-deficient mouse model

AimsCelastrol, a naturally occurring pentacyclic triterpene, has attracted considerable interest because it exhibits potent anti-inflammatory and anti-tumor properties. However, the effects of celastrol in autosomal dominant polycystic kidney disease (ADPKD) remain uninvestigated. Main methodsWe determined the effects of celastrol on ADPKD progression in a novel Pkd1-hypomorphic mouse model by intraperitoneal injection (postnatal day 35–63). Key findingsPkd1 miRNA transgenic (Pkd1 miR TG) mice treated with 1 mg/kg/day of celastrol exhibited a lower renal cystic index (by 21.5%) than the vehicle-treated controls, but the fractional kidney weights and blood urea nitrogen levels were not significantly affected with celastrol treatment. At a high dose (2 mg/kg/day), celastrol caused marginal weight loss in the treated mice and had no significant effect on renal cystogenesis, thus indicating a potential toxic effect. We further identified that celastrol increased the phosphorylation level of adenosine monophosphate-activated protein kinase (AMPK) in the cystic kidneys. Moreover, celastrol reduced the renal mRNA expression levels of tumor necrosis factor-α, interleukin-1β, P2RX7, F4/80, CD68, transforming growth factor-β, collagen-1, and fibronectin, which were high in the Pkd1 miR TG mice. Immunohistological analysis revealed that celastrol suppressed macrophage infiltration in the cystic kidneys; however, the renal fibrosis scores and proliferation indices remained high. SignificanceThese results indicate that celastrol could be a potent anti-inflammatory agent and a natural AMPK enhancer. However, celastrol has only modest effects on renal cystogenesis and has a narrow therapeutic window. Further studies are needed to clarify whether celastrol has the potential for the treatment of ADPKD.