Polycystin-1 Expression Research Articles

Revisiting the gene mutations and protein profile of WT 9-12: An autosomal dominant polycystic kidney disease cell line.

WT 9-12 is one of the cell lines commonly used for autosomal dominant polycystic kidney disease (ADPKD) studies. Previous studies had described the PKD gene mutations and polycystin expression in WT 9-12. Nonetheless, the mutations occurring in other ADPKD-associated genes have not been investigated. This study aims to revisit these mutations and protein profile of WT 9-12. Whole genome sequencing verified the presence of truncation mutation at amino acid 2556 (Q2556X) in PKD1 gene of WT 9-12. Besides, those variations with high impacts included single nucleotide polymorphisms (rs8054182, rs117006360, and rs12925771) and insertions and deletions (InDels) (rs145602984 and rs55980345) in PKD1L2; InDel (rs1296698195) in PKD1L3; and copy number variations in GANAB. Protein profiles generated from the total proteins of WT 9-12 and HK-2 cells were compared using isobaric tags for relative and absolute quantitation (iTRAQ) analysis. Polycystin-1 was absent in WT 9-12. The gene ontology enrichment and reactome pathway analyses revealed that the upregulated and downregulated proteins of WT 9-12 relative to HK-2 cell line leaded to signaling pathways related to immune response and amino acid metabolism, respectively. The ADPKD-related mutations and signaling pathways associated with differentially expressed proteins in WT 9-12 may help researchers in cell line selection for their studies.

Genetics of cystogenesis in base-edited human organoids reveal therapeutic strategies for polycystic kidney disease

In polycystic kidney disease (PKD), microscopic tubules expand into macroscopic cysts. Among the world's most common genetic disorders, PKD is inherited via heterozygous loss-of-function mutations but is theorized to require additional loss of function. To test this, we establish human pluripotent stem cells in allelic series representing four common nonsense mutations, using CRISPR base editing. When differentiated into kidney organoids, homozygous mutants spontaneously form cysts, whereas heterozygous mutants (original or base corrected) express no phenotype. Using these, we identify eukaryotic ribosomal selective glycosides (ERSGs) as PKD therapeutics enabling ribosomal readthrough of these same nonsense mutations. Two different ERSGs not only prevent cyst initiation but also limit growth of pre-formed cysts by partially restoring polycystin expression. Furthermore, glycosides accumulate in cyst epithelia in organoids and mice. Our findings define the human polycystin threshold as a surmountable drug target for pharmacological or gene therapy interventions, with relevance for understanding disease mechanisms and future clinical trials.

#4151 POLYCYSTINS ARE REQUIRED FOR RENAL TUBULOINTERSTITIAL FIBROSIS

Abstract Background and Aims Renal fibrosis is the common pathway of various chronic kidney diseases progressing to end stage renal failure. Polycystin-1 (encoded by PKD1 gene) and polycystin-2 (encoded by PKD2 gene) form a transmembrane complex and function as a stress sensor, which is located in the primary cilia. Polycystins are involved in the disease condition of different organs. Mutation of PKD genes causes autosomal dominant polycystic kidney disease and deletion of polycystin attenuates heart injury induced cardiac fibrosis. The role of polycystins in renal tubulointerstitial fibrosis is currently unclear. Method Unilateral ureteral obstruction (UUO), unilateral ischemia-reperfusion injury (UIRI), aristolochic acid or folic acid induced mouse models of renal fibrosis were established for this study. Results Here we showed that polycystin-2 is up-regulated in these three mouse models of renal fibrosis and tightly correlated with the expression of collagen-I in a time dependent manner. Treatment with triptolide inhibited the expression of polycystin-2 and pro-fibrotic markers in UUO and UIRI models. Moreover, triptolide or PKD2 siRNA inhibited the expression of polycystin-2 and pro-fibrotic markers in vitro. Using Pkd2 conditional knockout mice, we showed that genetic deletion of Pkd2 reduced the expression of pro-fibrotic markers in UUO kidneys. Polycystin-1 was also up-regulated in renal fibrotic models and conditional deletion of Pkd1 reduced the expression of pro-fibrotic markers in UUO or folic acid induced fibrotic kidneys. Furthermore, the expression of the methyltransferase EZH2 is positively correlated with the expression of polycystins in fibrotic kidneys. Conditional knockout of EZH2 attenuated the anti-fibrotic responses induced by Pkd1 deletion in UUO kidneys. Conclusion In conclusion, polycystins are up-regulated in fibrotic kidneys and promote renal tubulointerstitial fibrosis through up-regulation of EZH2, suggesting that primary cilia are required for renal tubulointerstitial fibrosis.

Hypoxia induces polycystin-1 expression in the renal epithelium.

Mutations in polycystin-1 which is encoded by the PKD1 gene are the main causes for the development of autosomal dominant polycystic kidney disease. However, only little is known about the physiological function of polycystin-1 and even less about the regulation of its expression. Here, we show that expression of PKD1 is induced by hypoxia and compounds that stabilize the hypoxia-inducible transcription factor (HIF) 1α in primary human tubular epithelial cells. Knockdown of HIF subunits confirms HIF-1α-dependent regulation of polycystin-1 expression. Furthermore, HIF ChIP-seq reveals that HIF interacts with a regulatory DNA element within the PKD1 gene in renal tubule-derived cells. HIF-dependent expression of polycystin-1 can also be demonstrated in vivo in kidneys of mice treated with substances that stabilize HIF. Polycystin-1 and HIF-1α have been shown to promote epithelial branching during kidney development. In line with these findings, we show that expression of polycystin-1 within mouse embryonic ureteric bud branches is regulated by HIF. Our finding links expression of one of the main regulators of accurate renal development with the hypoxia signalling pathway and provides additional insight into the pathophysiology of polycystic kidney disease.

Polycystin-2 Associates With Malignancy in Meningiomas.

The involvement of polycystin-2 (PC2) in cell survival pathways raises questions about its role in carcinogenesis. Aberrant expression of PC2 has been associated with malignancy in various tumors. No evidence exists referring to PC2 expression in meningiomas. The aim of this study was to investigate the expression levels of PC2 in meningiomas and compare them with normal brain samples including leptomeninges. PC2 immunohistochemical expression was quantitatively analyzed in archival tissue from 60 patients with benign (WHO grade 1) and 22 patients with high-grade (21: WHO grade 2 and 1: grade 3) meningiomas. Specifically, the labeling index [the percentage of positive (labeled) cells out of the total number of tumor cells counted] was determined. PC2 mRNA levels were evaluated by quantitative real-time polymerase chain reaction. PC2 immunostaining was not detected in the leptomeninges. Gene expression analysis revealed increased levels of PC2 in WHO grade 1 ( P = 0.008) and WHO grade 2 ( P = 0.0007) meningiomas compared with that of normal brains. PC2 expression was significantly associated with an ascending grade of malignancy by both immunohistochemistry and quantitative real-time polymerase chain reaction ( P < 0.05). Recurrent meningiomas displayed higher levels of PC2 compared with primary meningiomas ( P = 0.008). Although no significant association of PC2 with the overall survival of the patients was found ( P > 0.05), it was noticed that the patients with WHO grade 2 meningiomas with low expression of PC2 survived longer compared with the patients with WHO grade 1 meningioma with high expression of PC2 (mean survival 49.5 and 28 months, respectively). The above results indicate a possible association of PC2 with malignancy in meningiomas. However, the mechanisms underlying PC2 implication in meningioma pathogenesis should be further elucidated.

Potential involvement of polycystins in the pathogenesis of ameloblastomas: Analysis based on bioinformatics and immunohistochemistry

ObjectiveTo perform an integrated analysis in identifying novel hub genes that could facilitate the diagnosis and targeted therapy of ameloblastoma. DesignThe expression profiling dataset, GSE38494, was obtained from the Gene Expression Omnibus database. Differentially expressed genes were identified through GEO2R online tool and characterised via Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The protein-protein interaction network and hub genes were screened using the STRING database and Cytoscape software. Subsequently, an upregulated gene was selected for further validation using the GSE132472 dataset. Further, immunohistochemistry was performed to assess the expression of the selected gene in ameloblastomas, odontogenic keratocysts, dentigerous cysts, and gingival tissues. The diagnostic and therapeutic utility of the selected hub genes were further verified by receiver operating characteristic analysis and the DGIdb database. ResultsWe identified six hub genes in ameloblastoma, among which the upregulated gene PKD2 and its related gene PKD1 were further validated. GO functional annotation revealed that PKD2 is involved in cell-cell junction, extracellular exosome, cytoplasm, endoplasmic reticulum, and calcium ion transport. The immunohistochemical analysis showed that the expression of polycystin-1 and polycystin-2, encoded by the PKD1 and PKD2 genes, respectively, was upregulated in ameloblastoma. PKD1 and PKD2 had a high diagnostic utility for ameloblastoma, and allopurinol interacted with the PKD2 gene. ConclusionOur research indicates that polycystins are highly expressed in ameloblastoma and might be involved in the oncogenesis of ameloblastoma, thus offering a new perspective on the molecular mechanisms and targeted therapies on ameloblastoma.

Polycystin-1 Is a Crucial Regulator of BIN1 Expression and T-Tubule Remodeling Associated with the Development of Dilated Cardiomyopathy

Cardiomyopathy is commonly observed in patients with autosomal dominant polycystic kidney disease (ADPKD), even when they have normal renal function and arterial pressure. The role of cardiomyocyte polycystin-1 (PC1) in cardiovascular pathophysiology remains unknown. PC1 is a potential regulator of BIN1 that maintains T-tubule structure, and alterations in BIN1 expression induce cardiac pathologies. We used a cardiomyocyte-specific PC1-silenced (PC1-KO) mouse model to explore the relevance of cardiomyocyte PC1 in the development of heart failure (HF), considering reduced BIN1 expression induced T-tubule remodeling as a potential mechanism. PC1-KO mice exhibited an impairment of cardiac function, as measured by echocardiography, but no signs of HF until 7-9 months of age. Of the PC1-KO mice, 43% died suddenly at 7 months of age, and 100% died after 9 months with dilated cardiomyopathy. Total BIN1 mRNA, protein levels, and its localization in plasma membrane-enriched fractions decreased in PC1-KO mice. Moreover, the BIN1 + 13 isoform decreased while the BIN1 + 13 + 17 isoform was overexpressed in mice without signs of HF. However, BIN1 + 13 + 17 overexpression was not observed in mice with HF. T-tubule remodeling and BIN1 score measured in plasma samples were associated with decreased PC1-BIN1 expression and HF development. Our results show that decreased PC1 expression in cardiomyocytes induces dilated cardiomyopathy associated with diminished BIN1 expression and T-tubule remodeling. In conclusion, positive modulation of BIN1 expression by PC1 suggests a novel pathway that may be relevant to understanding the pathophysiological mechanisms leading to cardiomyopathy in ADPKD patients.

Concurrent Reduced Expression of Contiguous PKD1, TSC2 and NTHL1 Leading to Kidney Diseases and Multiple Diverse Renal Cancers.

Several cases of concurrent reduction of expression of polycystin 1 (PKD1) and Tuberous Sclerosis Complex 2 (TSC2) that are contiguous in chromosome 16p13 have been previously reported. This study newly addresses the concurrent reduction of expression of PKD1, TSC2 and NTHL1, which is adjacent to TSC2 and is a tumor suppressor gene. We investigated the mRNA expression levels of PKD1, TSC2, PKD2, TSC1 and NTHL1 in blood and renal cell carcinoma (RCC) tissues in a proband with autosomal dominant polycystic kidney disease (ADPKD), tuberous sclerosis complex (TSC) and multiple pathologically diverse RCCs, including clear cell, papillary and chromophobe types. Additionally, we investigated germline variants in blood using whole exome sequencing (WES) in the proband and her four siblings. mRNA expression levels of PKD1, TSC2 and NTHL1 were reduced in the proband's blood and RCCs, compared with control groups. WES identified one novel variant with amino acid changes in the PKD1 exon in the three subjects with ADPKD, including the proband. Moreover, two variants in the TSC2 intron specific to the proband were also identified. In this study, we report a novel pathogenic variant in the PKD1 exon which likely led to ADPKD, and two variants in the TSC2 intron, which might have led to reduction in the expression of both TSC2 and NTHL1, consequently leading to TSC and multiple pathologically diverse RCCs.

Low-frequency pulsed electromagnetic fields promote osteoblast mineralization and maturation of rats through the PC2/sAC/PKA/CREB signaling pathway

To explore whether the effect of low-frequency pulsed electromagnetic fields (PEMFs) in promoting osteoblast mineralization and maturation is related to the primary cilia, polycystin2 (PC2) and sAC/PKA/CREB signaling pathway. We detected the expression levels of PC2, sAC, PKA, CREB and their phosphorylated proteins in primary rat calvarial osteoblasts exposed to 50 Hz 0.6 mT PEMFs for 0, 5, 15, 30, 60, 90, and 120 min. We blocked PC2 function with amiloride hydrochloride and detected the changes in the activity of sAC/PKA/CREB signal pathway and the mineralization and maturation of the osteoblasts. These examinations were repeated in the osteoblasts after specific knockdown of PC2 via RNA interference and were the co-localization of PC2, sAC, PKA, CREB and their phosphorylated proteins with the primary cilia were using immunofluorescence staining. The expressions of PC2 and the signaling proteins of sAC/PKA/CREB pathway were detected after inhibition of primary ciliation by RNA interference. The expression levels of PC2, sAC, p-PKA and p- CREB were significantly increased in the osteoblasts after exposure to PEMFs for different time lengths (P < 0.01). Blocking PC2 function or PC2 knockdown in the osteoblasts resulted in failure of sAC/PKA/CREB signaling pathway activation and arrest of osteoblast mineralization and maturation. PC2, sAC, p-PKA and p-CREB were localized to the entire primary cilia or its roots, but PKA and CREB were not detected in the primary cilia. After interference of the primary cilia, PEMFs exposure no longer caused increase of PC2 expression and failed to activate the sAC/PKA/CREB signaling pathway or promote osteoblast mineralization and maturation. PC2, located on the surface of the primary cilia of osteoblasts, can perceive and transmit the physical signals from PEMFs and promote the mineralization and maturation of osteoblasts by activating the PC2/ sAC/PKA/CREB signaling pathway.

MO449: Polycystins are Upregulated in Fibrotic Kidneys and Promote Renal Fibrosis

Abstract BACKGROUND AND AIMS Mutations in PKD1 or PKD2 gene, which encodes polycystin-1 or polycystin-2, cause autosomal polycystic kidney disease (ADPKD). The development of ADPKD is associated with the progression of renal fibrosis. Whether renal fibrosis in ADPKD is a direct effect of PKD mutation or a consequence of cyst growth-induced tubular obstruction is currently unknown. Polycystin-2 has been identified as a direct target of triptolide and we used triptolide as a probe to study the role of polycystin-2 in renal fibrosis. METHOD Unilateral ureteral obstruction (UUO), unilateral ischemia-reperfusion injury (UIRI) and aristolochic acid nephropathy mouse models were established. Mice were treated with vehicle or triptolide. Western blotting and Masson's trichrome staining were performed to evaluate renal fibrosis. Moreover, human kidney 2 (HK2) cells and normal rat kidney 49F (NRK-49F) cells were used as in vitro models. RESULTS Here we showed that polycystin-2 is up-regulated in these three mouse models and tightly correlated with the expression of collagen-I in a time-dependent manner. Treatment with triptolide inhibited the expression of polycystin-2 and pro-fibrotic markers in UUO and UIRI models. Moreover, triptolide dose-dependently inhibited the expression of polycystin-2 and pro-fibrotic markers in rat renal fibroblasts or in TGF-β stimulated human renal epithelial (HK2) cells. Knockdown of PKD2 reduced the expression of pro-fibrotic markers in TGF-β stimulated or unstimulated HK2 cells. Moreover, we showed that knockdown of PKD2 attenuated the inhibitory effect of triptolide on the expression of pro-fibrotic markers in TGF-β stimulated HK2 cells. Finally, using Pkd2 conditional knockout mice, we showed that heterozygous or homozygous deletion of Pkd2 reduced the expression of pro-fibrotic markers in UUO kidneys. Polycystin-1 was also up-regulated in three renal fibrotic models and conditional deletion of Pkd1 reduced the expression of pro-fibrotic markers in UUO or folic acid-induced fibrotic kidneys. Furthermore, conditional knockout of the methyltransferase EZH2 attenuated the anti-fibrotic responses induced by PKD1 deletion in UUO kidneys. CONCLUSION Polycystins are pro-fibrotic proteins in injured kidneys suggesting that renal fibrosis in ADPKD kidneys is not a direct effect of PKD mutation.

Effect of polycystin2 on differentiation and maturation of osteoblasts promoted by low-frequency pulsed electromagnetic fields

It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro. However, the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown. In this study, we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2 (PC2) located on the primary cilia on the surface of ROBs. First, immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs, and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting. Subsequently, we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP), as well as the expression of Runx-2, Bmp-2, Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI), a PC2 blocker. Moreover, we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference. The results showed that PC2 was localized on the primary cilia of ROBs, and PEMFs treatment increased the expression of PC2 protein. When PC2 was blocked by AMI, PEMFs could no longer increase PC2 protein expression and ALP activity, and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset. After inhibiting the expression of PC2 using RNA interference, PEMFs can no longer increase the expression of genes related to bone formation. The results showed that PC2, located on the surface of primary cilia of osteoblasts, plays an indispensable role in perceiving and transmitting the physical signals from PEMFs, and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2. This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.

Polycystin-1 regulates cell proliferation and migration through AKT/mTORC2 pathway in a human craniosynostosis cell model.

Craniosynostosis is the premature fusion of skull sutures and has a severe pathological impact on childrens’ life. Mechanical forces are capable of triggering biological responses in bone cells and regulate osteoblastogenesis in cranial sutures, leading to premature closure. The mechanosensitive proteins polycystin‐1 (PC1) and polycystin‐2 (PC2) have been documented to play an important role in craniofacial proliferation and development. Herein, we investigated the contribution of PC1 to the pathogenesis of non‐syndromic craniosynostosis and the associated molecular mechanisms. Protein expression of PC1 and PC2 was detected in bone fragments derived from craniosynostosis patients via immunohistochemistry. To explore the modulatory role of PC1 in primary cranial suture cells, we further abrogated the function of PC1 extracellular mechanosensing domain using a specific anti‐PC1 IgPKD1 antibody. Effect of IgPKD1 treatment was evaluated with cell proliferation and migration assays. Activation of PI3K/AKT/mTOR pathway components was further detected via Western blot in primary cranial suture cells following IgPKD1 treatment. PC1 and PC2 are expressed in human tissues of craniosynostosis. PC1 functional inhibition resulted in elevated proliferation and migration of primary cranial suture cells. PC1 inhibition also induced activation of AKT, exhibiting elevated phospho (p)‐AKT (Ser473) levels, but not 4EBP1 or p70S6K activation. Our findings indicate that PC1 may act as a mechanosensing molecule in cranial sutures by modulating osteoblastic cell proliferation and migration through the PC1/AKT/mTORC2 cascade with a potential impact on the development of non‐syndromic craniosynostosis.

Interdependent Regulation of Polycystin Expression Influences Starvation-Induced Autophagy and Cell Death.

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by deficiency of polycystin-1 (PC1) or polycystin-2 (PC2). Altered autophagy has recently been implicated in ADPKD progression, but its exact regulation by PC1 and PC2 remains unclear. We therefore investigated cell death and survival during nutritional stress in mouse inner medullary collecting duct cells (mIMCDs), either wild-type (WT) or lacking PC1 (PC1KO) or PC2 (PC2KO), and human urine-derived proximal tubular epithelial cells (PTEC) from early-stage ADPKD patients with PC1 mutations versus healthy individuals. Basal autophagy was enhanced in PC1-deficient cells. Similarly, following starvation, autophagy was enhanced and cell death reduced when PC1 was reduced. Autophagy inhibition reduced cell death resistance in PC1KO mIMCDs to the WT level, implying that PC1 promotes autophagic cell survival. Although PC2 expression was increased in PC1KO mIMCDs, PC2 knockdown did not result in reduced autophagy. PC2KO mIMCDs displayed lower basal autophagy, but more autophagy and less cell death following chronic starvation. This could be reversed by overexpression of PC1 in PC2KO. Together, these findings indicate that PC1 levels are partially coupled to PC2 expression, and determine the transition from renal cell survival to death, leading to enhanced survival of ADPKD cells during nutritional stress.

Polycystin-1 dependent regulation of polycystin-2 via GRP94, a member of HSP90 family that resides in the endoplasmic reticulum.

Autosomal dominant polycystic kidney disease is a common inherited renal disorder that results from mutations in either PKD1 or PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Downregulation or overexpression of PKD1 or PKD2 in mouse models results in renal cyst formation, suggesting that the quantity of PC1 and PC2 needs to be maintained within a tight functional window to prevent cystogenesis. Here we show that enhanced PC2 expression is a common feature of PKD1 mutant tissues, in part due to an increase in Pkd2 mRNA. However, our data also suggest that more effective protein folding contributes to the augmented levels of PC2. We demonstrate that the unfolded protein response is activated in Pkd1 knockout kidneys and in Pkd1 mutant cells and that this is coupled with increased levels of GRP94, an endoplasmic reticulum protein that is a member of the HSP90 family of chaperones. GRP94 was found to physically interact with PC2 and depletion or chemical inhibition of GRP94 led to a decrease in PC2, suggesting that GRP94 serves as its chaperone. Moreover, GRP94 is acetylated and binds to histone deacetylase 6 (HDAC6), a known deacetylase and activator of HSP90 proteins. Inhibition of HDAC6 decreased PC2 suggesting that HDAC6 and GRP94 work together to regulate PC2 levels. Lastly, we showed that inhibition of GRP94 prevents cAMP-induced cyst formation in vitro. Taken together our data uncovered a novel HDAC6-GRP94-related axis that likely participates in maintaining elevated PC2 levels in Pkd1 mutant cells.

Polycystin-1 regulates cardiomyocyte mitophagy.

Polycystin-1 (PC1) is a transmembrane protein found in different cell types, including cardiomyocytes. Alterations in PC1 expression have been linked to mitochondrial damage in renal tubule cells and in patients with autosomal dominant polycystic kidney disease. However, to date, the regulatory role of PC1 in cardiomyocyte mitochondria is not well understood. The analysis of mitochondrial morphology from cardiomyocytes of heterozygous PC1 mice (PDK1+/- ) using transmission electron microscopy showed that cardiomyocyte mitochondria were smaller with increased mitochondria density and circularity. These parameters were consistent with mitochondrial fission. We knocked-down PC1 in cultured rat cardiomyocytes and human-induced pluripotent stem cells (iPSC)-derived cardiomyocytes to evaluate mitochondrial function and morphology. The results showed that downregulation of PC1 expression results in reduced protein levels of sub-units of the OXPHOS complexes and less functional mitochondria (reduction of mitochondrial membrane potential, mitochondrial respiration, and ATP production). This mitochondrial dysfunction activates the elimination of defective mitochondria by mitophagy, assessed by an increase of autophagosome adapter protein LC3B and the recruitment of the Parkin protein to the mitochondria. siRNA-mediated PC1 knockdown leads to a loss of the connectivity of the mitochondrial network and a greater number of mitochondria per cell, but of smaller sizes, which characterizes mitochondrial fission. PC1 silencing also deregulates the AKT-FoxO1 signaling pathway, which is involved in the regulation of mitochondrial metabolism, mitochondrial morphology, and processes that are part of cell quality control, such as mitophagy. Together, these data provide new insights about the controls that PC1 exerts on mitochondrial morphology and function in cultured cardiomyocytes dependent on the AKT-FoxO1 signaling pathway.

MO020AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE, CYTOPENIA AND POSTTRANSPLANT OUTCOMES: A RETROSPECTIVE ANALYSIS

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD), the most common monogenetic inherited kidney disease, has been reported to be associated with distinct cytopenias. The pathophysiological mechanism behind this association and its clinical implications are unknown. Sparse and conflicting data have suggested a tendency to greater vulnerability for cancer and infections in ADPKD renal transplant recipients as compared to their non-ADPKD counterparts. Furthermore polycystin expression has been demonstrated in lymphocytes in vitro with impact on lymphocyte function. From this background, cytopenia could be hypothesized to be directly induced by the molecular PKD defects and to impact on outcome in affected patients. The main interest of the current study is to confirm the association between ADPKD and cytopenia in a well-defined clinical cohort of patients at the time of kidney transplantation. Furthermore, the impact of ADPKD and cytopenia on posttransplant outcomes is studied. Method Baseline (pre-transplantation) and follow-up data from all patients who underwent a first renal transplantation between 01/04/1964 and 01/09/2019 at the Leuven University Hospitals were retrieved from the renal transplantation database. Results 4103 patients were included: 611 ADPKD and 3492 non-ADPKD. Immediately before transplantation, significant differences in total white blood cell (6.33 +/- 2.59 ADPKD versus 7.17 +/- 2.21 non-ADPKD; p&amp;lt;0.0001), neutrophil (4.04 +/- 1.59 versus 4.54 +/- 1.88; p&amp;lt;0.0001), lymphocyte (1.45 +/- 0.05 versus 1.57 +/- 0.68; p&amp;lt;0.0001), basophil (0.041 +/- 0.084 versus 0.046 +/- 0.048; p&amp;lt;0.0001), eosinophil (0.23 +/- 0.24 versus 0.29 +/- 0.33; p&amp;lt;0.0001) and thrombocyte counts (203.00 +/- 67.21 versus 230.18 +/- 76.00; p&amp;lt;0.0001) between ADPKD and non-ADPKD patients were observed. After multiple linear regression analysis, ADPKD remained significantly associated with total white blood cell, neutrophil, monocyte and thrombocyte counts. In terms of post-transplant outcomes, significant univariate differences between ADPKD and non-ADPKD transplant recipients in favor of the ADPKD patients were observed for overall survival, time to first rejection and time to transplant failure. On the contrary, a significantly faster onset of the first posttransplant malignancy and infection was observed in ADPKD as compared to non-ADPKD. Furthermore, the numbers of posttransplant infections and malignancies tended to be higher in the ADPKD patient cohort, the number of transplant rejections was significantly lower. While pre-transplantation lymphopenia was univariately associated with lower overall survival, none of the other post-transplant outcomes was associated with lymphopenia, nor with any of the other blood cell counts. After multivariate Cox proportional hazard regression analysis, ADPKD remained significantly associated with time to first rejection (p=0.0310; HR 0.632) and overall survival (p&amp;lt;0.0001; HR 0.512). None of the blood cell count variables retained significance in the multivariate outcome models. Conclusion This large retrospective single center study confirmed the association between cytopenia and ADPKD immediately before kidney transplantation. Furthermore, ADPKD was also associated with differences in posttransplant outcomes. In multivariate survival analysis, the impact of ADPKD on outcomes outweighed that of other variables, amongst others the blood cell counts. From this, it is tempting to speculate that ADPKD influences outcome through a disease-related impact on blood cells, however many more factors are most probably involved. The hypothesis that cytopenia could be an extra-renal manifestation of ADPKD, directly linked to its genetic basis, and its impact on outcome warrant further investigation.

FC 008INTERDEPENDENT REGULATION OF POLYCYSTIN EXPRESSION INFLUENCES STARVATION-INDUCED AUTOPHAGY AND CELL DEATH

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by mutations in either PKD1 (ca. 78%) or PKD2 (ca. 15%), encoding for the proteins polycystin-1 (PC1) or polycystin-2 (PC2), respectively. Mutations in PKD1 generally lead to a more severe disease progression compared to PKD2 patients. The exact function of the polycystins in cyst formation remains unclear, but it is clear that the levels of PC1 and PC2 are inversely correlated to cyst formation. Moreover, renal stress has been proposed to enhance cystogenesis. We therefore aimed to investigate the cellular response towards nutritional stress in mouse inner medullary collecting duct cells (mIMCDs), either wild-type (WT) or lacking PC1 or PC2 (PC1KO or PC2KO), and unique human urine-derived proximal tubular epithelial cells (PTECs) of early-stage ADPKD patients with truncating PKD1 mutations versus healthy individuals, with a focus on cell survival (autophagy) and cell death. Method Cell death was assessed with Cytotox green-based live cell imaging in the Incucyte, by trypane blue exclusion and by analyzing the levels of cleaved Caspase 3. Autophagy was measured by LC3 immunoblotting and by counting GFP-LC3 punctae. Autophagy was blocked with Bafilomycin A1. To modulate the levels of PC1 and PC2, transient overexpression of human PC1 or siRNA-mediated knockdown of PC2 was performed. Results During chronic starvation, cell death was reduced and autophagy was increased in mouse PC1- and PC2-deficient mIMCDs. This was validated in human cells from early-stage ADPKD patients. Autophagy inhibition restored cell death resistance in KO cells, implying that decrease in cell death was caused by autophagy upregulation in PC1- and PC2KO cells. Interestingly, PC2 expression was increased in PC1KO cells, while PC2KO displayed a downregulation of PC1. Although PC2 is known to regulate autophagy, PC2 knockdown did not reduce autophagy in PC1KO cells, while the effect in PC2KO could be reversed by overexpression of PC1. Conclusion These findings indicate that PC1 levels determine the transition from renal cell survival to death, leading to enhanced survival of ADPKD cells during nutritional stress. Moreover, PC1 also indirectly influences this process by regulating PC1 levels during starvation. Our findings imply that in early stage ADPKD, cells with the lowest polycystin levels (which are most prone to form cysts) are more resistant to stress by autophagy upregulation. This is important, as renal stress is inherent to the cystic environment and has been proposed as an additional trigger in cystogenesis.

MiR-4787-5p Regulates Vascular Smooth Muscle Cell Apoptosis by Targeting PKD1 and Inhibiting the PI3K/Akt/FKHR Pathway.

Vascular smooth muscle cell (VSMC) dysfunction is the main cause of aortic dissection (AD). In this study, we focused on the role and mechanism of miR-4787-5p in regulating VSMC apoptosis. Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of miR-4787-5p in aorta tissues of AD (n = 10) and normal aortic tissues of donors (n = 10). Cell apoptosis was tested by TUNEL assay and Annexin V FITC/PI staining flow cytometry. The expression of PC1 and the PI3K/Akt/FKHR signaling pathway associated proteins in VSMCs was measured by Western blot. We found that the miR-4787-5p was highly expressed in aorta tissues of AD compared with 10 healthy volunteers. Meanwhile, PI3K/Akt/FKHR signaling pathway was inactive in the aortic tissue of AD. The overexpression of miR-4787-5p significantly induced VSMC apoptosis, and miR-4787-5p knockdown showed the opposite results. In addition, polycystic kidney disease 1 gene, which encodes polycystin-1 (PC1), was found to be a direct target of miR-4787-5p in the VSMCs and this was validated using a luciferase reporter assay. Overexpression of PC1 by a lentivirus packaging PC1-overexpression plasmid (LV-PC1) plasmids markedly eliminated the promotion of miR-4787-5p overexpression on VSMC apoptosis. Finally, it was found that miR-4787-5p deactivated the PI3K/Akt/FKHR pathway, as demonstrated by the down-regulation of phosphorylated (p-)PI3K, p-Akt, and p-FKHR. In conclusion, these findings confirm an important role for the miR-4787-5p/polycystic kidney disease 1 axis in AD pathobiology.

Polycystin-2 Is Required for Chondrocyte Mechanotransduction and Traffics to the Primary Cilium in Response to Mechanical Stimulation.

Primary cilia and associated intraflagellar transport are essential for skeletal development, joint homeostasis, and the response to mechanical stimuli, although the mechanisms remain unclear. Polycystin-2 (PC2) is a member of the transient receptor potential polycystic (TRPP) family of cation channels, and together with Polycystin-1 (PC1), it has been implicated in cilia-mediated mechanotransduction in epithelial cells. The current study investigates the effect of mechanical stimulation on the localization of ciliary polycystins in chondrocytes and tests the hypothesis that they are required in chondrocyte mechanosignaling. Isolated chondrocytes were subjected to mechanical stimulation in the form of uniaxial cyclic tensile strain (CTS) in order to examine the effects on PC2 ciliary localization and matrix gene expression. In the absence of strain, PC2 localizes to the chondrocyte ciliary membrane and neither PC1 nor PC2 are required for ciliogenesis. Cartilage matrix gene expression (Acan, Col2a) is increased in response to 10% CTS. This response is inhibited by siRNA-mediated loss of PC1 or PC2 expression. PC2 ciliary localization requires PC1 and is increased in response to CTS. Increased PC2 cilia trafficking is dependent on the activation of transient receptor potential cation channel subfamily V member 4 (TRPV4) activation. Together, these findings demonstrate for the first time that polycystins are required for chondrocyte mechanotransduction and highlight the mechanosensitive cilia trafficking of PC2 as an important component of cilia-mediated mechanotransduction.

Polycystin-1 Downregulation Induced Vascular Smooth Muscle Cells Phenotypic Alteration and Extracellular Matrix Remodeling in Thoracic Aortic Dissection

ObjectivePolycystin-1 (PC-1) is a protein encoded by the gene of polycystic kidney disease-1 (PKD-1). This study was designed to investigate the regulatory mechanisms of PC-1 on phenotypes of aortic vascular smooth muscle cells (VSMCs) and functions of extracellular matrix (ECM) in thoracic aortic dissection (TAD).MethodsAortic tissues from patients with TAD and healthy controls were collected, primary aortic VSMCs were also isolated. Immunohistochemistry, immunofluorescence, and immunocytochemistry was used to visualize the target proteins. Western blot and RT-qPCR were used to examine the expression of mRNA and proteins. Lentivirus infection was used to downregulate or overexpress PC-1.ResultsCompared with the control group, expression of PC-1 and the contractile phenotypic markers of VSMCs were decreased in TAD group, whereas expression of the synthetic markers of VSMCs, matrix metalloproteinase (MMP)-2, collagen I and collagen III were increased. The phosphorylation of mTOR, S6K and S6 were also elevated in TAD group. PC-1 downregulation of aortic VSMCs inhibited the expression of the contractile markers, but elevated the expression of the synthetic markers, MMP-2, collagen I and collagen III compared with the control group. The phosphorylation of mTOR, S6K and S6 were also increased in PKD-1-knockdown VSMCs. PC-1 upregulation reversed all these expression characteristics in aortic VSMCs. Furthermore, rapamycin treatment to PKD-1-knockdown VSMCs inhibited the effects caused by PC-1 downregulation.ConclusionOur study revealed PC-1 downregulation induces aortic VSMCs phenotypic alteration and ECM remodeling via activation of mTOR/S6K/S6 signaling pathway. Downregulation of PC-1 might be a potential mechanism for the development and progression of TAD. Rapamycin might be a potential inhibitor to attenuate the development and progression of TAD.