Multiscale and multimodal evaluation of autosomal dominant polycystic kidney disease development.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most prevalent kidney genetic disorder, producing structural abnormalities and impaired function. This research investigates its evolution on mouse models, utilizing a combination of histology imaging, Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) to evaluate its progression thoroughly. ADPKD has been induced in mice via PKD2 gene knockout, followed by image acquisition at different stages. Histology data provides two-dimensional details, like the cystic area ratio, whereas CT and MRI facilitate three-dimensional temporal monitoring. Our approach allows to quantify the affected tissue at different disease stages through multiple quantitative metrics. A pivotal point is shown at approximately ten weeks after induction, marked by a swift acceleration in disease advancement, and leading to a notable increase in cyst formation. This multimodal strategy augments our comprehension of ADPKD dynamics and suggests the possibility of employing higher-resolution imaging in the future for more accurate volumetric analyses.

[Genetic analysis and prenatal diagnosis for a Chinese pedigree affected with Autosomal dominant polycystic kidney disease].

OBJECTIVE: To explore the clinical phenotype and genetic etiology for a Chinese pedigree affected with Autosomal dominant polycystic kidney disease (ADPKD). METHODS: A pedigree with ADPKD diagnosed at the Department of Gynaecology of the First Affiliated Hospital of Zhengzhou University in December 2020 was selected as the study subject. Clinical data of the pedigree was collected, and whole exome sequencing (WES) was carried out for the proband. Candidate variants were verified by Sanger sequencing of the proband and her relatives. RESULTS: Fetal ultrasonography showed increased volume and parenchymal echogenicity in both kidneys. The fetus was found to harbor c.11098C>T (p.R3700C) and c.11039T>C (p.F3680S) compound heterozygous variants of the PKD1 gene, which were respectively inherited from its mother and father. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were predicted to be likely pathogenic (PM1+PM2_supporting+PP3). CONCLUSION: The c.11098C>T (p.R3700C) and c.11039T>C (p.F3680S) compound heterozygous variants of the PKD1 gene probably underlay the ADPKD in the fetus. Above finding has provided guidance for the genetic counseling and prenatal diagnosis for this pedigree.

Protecting the liver with PKD1.

Proliferation of somatic clones deficient in PKD1 prevents fatty liver disease without resulting in tumors.

[Characterization of 19 novel gene mutation sites associated with autosome-dominant polycystic kidney disease].

By analyzing the of genetic testing data of patients with renal polycystic kidney disease and their relatives, this study aims to identify unreported novel gene mutation sites associated with autosomal dominant polycystic kidney disease (ADPKD). Structural prediction software was employed to investigate protein structural changes before and after mutations, explore genotype-phenotype correlations, and enrich the ADPKD gene database. In this single-center retrospective study, patients with multiple renal cysts diagnosed from January 2019 to February 2023 at the Zhong Da Hospital Southeast University were included. Genetic and clinical data of patients and their families were collected. Unreported novel gene mutation sites associated with ADPKD were identified. The AlphaFold v2.3.1 software was used to predict protein structures. Changes in protein structure before and after mutations were compared to explore genotype-phenotype correlations and enrich the ADPKD gene database. Twelve mutated genes associated with renal cysts were detected in 52 families. Nineteen novel gene mutation sites associated with ADPKD were identified, including 17 mutations in the PKD1 gene (one splicing mutation, seven frameshift mutations, four nonsense mutations, one whole-codon insertion, and four missense mutations); one ALG9 missense mutation; and one chromosomal structural variation. Truncating mutations in the PKD1 gene were correlated with a more severe clinical phenotype, while non-truncating mutations were associated with greater clinical heterogeneity. Numerous novel gene mutation sites associated with ADPKD remain unreported. Therefore, it is essential to analyze the pathogenicity of these novel mutation sites, establish genotype-phenotype correlations, and enrich the ADPKD gene database.

Cilia-enriched oxysterol 7ß,27-DHC is required for polycystin ion channel activation.

Polycystin-1 (PC-1) and PC-2 form a heteromeric ion channel complex that is abundantly expressed in primary cilia of renal epithelial cells. This complex functions as a non-selective cation channel, and mutations within the polycystin complex cause autosomal dominant polycystic kidney disease (ADPKD). The spatial and temporal regulation of the polycystin complex within the ciliary membrane remains poorly understood. Using both whole-cell and ciliary patch-clamp recordings, we identify a cilia-enriched oxysterol, 7ß,27-dihydroxycholesterol (DHC), that serves as a necessary activator of the polycystin complex. We further identify an oxysterol-binding pocket within PC-2 and showed that mutations within this binding pocket disrupt 7ß,27-DHC-dependent polycystin activation. Pharmacologic and genetic inhibition of oxysterol synthesis reduces channel activity in primary cilia. In summary, our findings reveal a regulator of the polycystin complex. This oxysterol-binding pocket in PC-2 may provide a specific target for potential ADPKD therapeutics.

Rapamycin-encapsulated nanoparticle delivery in polycystic kidney disease mice.

Rapamycin slows cystogenesis in murine models of polycystic kidney disease (PKD) but failed in clinical trials, potentially due to insufficient drug dosing. To improve drug efficiency without increasing dose, kidney-specific drug delivery may be used. Mesoscale nanoparticles (MNP) selectively target the proximal tubules in rodents. We explored whether MNPs can target cystic kidney tubules and whether rapamycin-encapsulated-MNPs (RapaMNPs) can slow cyst growth in Pkd1 knockout (KO) mice. MNP was intravenously administered in adult Pkd1KO mice. Serum and organs were harvested after 8, 24, 48 or 72 h to measure MNP localization, mTOR levels, and rapamycin concentration. Pkd1KO mice were then injected bi-weekly for 6 weeks with RapaMNP, rapamycin, or vehicle to determine drug efficacy on kidney cyst growth. Single MNP injections lead to kidney-preferential accumulation over other organs, specifically in tubules and cysts. Likewise, one RapaMNP injection resulted in higher drug delivery to the kidney compared to the liver, and displayed sustained mTOR inhibition. Bi-weekly injections with RapaMNP, rapamycin or vehicle for 6 weeks resulted in inconsistent mTOR inhibition and little change in cyst index, however. MNPs serve as an effective short-term, kidney-specific delivery system, but long-term RapaMNP failed to slow cyst progression in Pkd1KO mice.

Reprogramming of Energy Metabolism in Human PKD1 Polycystic Kidney Disease: A Systems Biology Analysis.

Multiple alterations of cellular metabolism have been documented in experimental studies of autosomal dominant polycystic kidney disease (ADPKD) and are thought to contribute to its pathogenesis. To elucidate the molecular pathways and transcriptional regulators associated with the metabolic changes of renal cysts in ADPKD, we compared global gene expression data from human PKD1 renal cysts, minimally cystic tissues (MCT) from the same patients, and healthy human kidney cortical tissue samples. We found gene expression profiles of PKD1 renal cysts were consistent with the Warburg effect with gene pathway changes favoring increased cellular glucose uptake and lactate production, instead of pyruvate oxidation. Additionally, mitochondrial energy metabolism was globally depressed, associated with downregulation of gene pathways related to fatty acid oxidation (FAO), branched-chain amino acid (BCAA) degradation, the Krebs cycle, and oxidative phosphorylation (OXPHOS) in renal cysts. Activation of mTORC1 and its two target proto-oncogenes, HIF-1α and MYC, was predicted to drive the expression of multiple genes involved in the observed metabolic reprogramming (e.g., GLUT3, HK1/HK2, ALDOA, ENO2, PKM, LDHA/LDHB, MCT4, PDHA1, PDK1/3, MPC1/2, CPT2, BCAT1, NAMPT); indeed, their predicted expression patterns were confirmed by our data. Conversely, we found AMPK inhibition was predicted in renal cysts. AMPK inhibition was associated with decreased expression of PGC-1α, a transcriptional coactivator for transcription factors PPARα, ERRα, and ERRγ, all of which play a critical role in regulating oxidative metabolism and mitochondrial biogenesis. These data provide a comprehensive map of metabolic pathway reprogramming in ADPKD and highlight nodes of regulation that may serve as targets for therapeutic intervention.

[The application value of whole exome sequencing technology in diagnosis of hereditary renal cysts].

The data of 57 renal cyst patients who visited the First Affiliated Hospital of Zhengzhou University from January 2023 to March 2024 were retrospectively analyzed. The age of patients ranged from three months to 60 years old, with 31 males and 26 females. The whole exome sequencing (WES) detected pathogenic or suspected pathogenic (P/LP) variants in 48 renal cystic probands, with a detection rate of 84.2% (48/57), including PKD1, PKD2, PKHD1, LRP5, COL4A4 and ALG8 gene variants as well as copy number variations (CNV). In addition, four PKD1 gene variants of uncertain significance (VUS) were detected. In five WES negative families, one PKD1 nonsense variation was detected through long-range PCR (LR-PCR)+Oxford nanopore technologies, and one heterozygous deletion in exon 22 of PKD1 gene was detected through multiplex ligation-dependent probe amplification (MLPA). In summary, WES can detect multiple types of variations, which is helpful for early diagnosis and prognosis prediction of renal cyst patients. However, there is still a risk of failing to detect PKD1 gene by WES, therefore, healthcare practitioners should beware of the negative results of WES.

Identification and Properties of TRPV4 Mutant Channels Present in Polycystic Kidney Disease Patients.

Polycystic kidney disease (PKD), a disease characterized by the enlargement of the kidney through cystic growth is the fourth leading cause of end-stage kidney disease world-wide. Transient receptor potential Vanilloid 4 (TRPV4), a calcium-permeable TRP, channel participates in kidney cell physiology and since TRPV4 forms complexes with another channel whose malfunction is associated to PKD, TRPP2 (or PKD2), we sought to determine whether patients with PKD, exhibit previously unknown mutations in TRPV4. Here, we report the presence of mutations in the TRPV4 gene in patients diagnosed with PKD and determine that they produce gain-of-function (GOF). Mutations in the sequence of the TRPV4 gene have been associated to a broad spectrum of neuropathies and skeletal dysplasias but not PKD, and their biophysical effects on channel function have not been elucidated. We identified and examined the functional behavior of a novel E6K mutant and of the previously known S94L and A217S mutant TRVP4 channels. The A217S mutation has been associated to mixed neuropathy and/or skeletal dysplasia phenotypes, however, the PKD carriers of these variants had not been diagnosed with these reported clinical manifestations. The presence of certain mutations in TRPV4 may influence the progression and severity of PKD through GOF mechanisms. PKD patients carrying TRVP4 mutations are putatively more likely to require dialysis or renal transplant as compared to those without these mutations.

Disease-associated missense mutations in the pore loop of polycystin-2 alter its ion channel function in a heterologous expression system.

Polycystin-2 (PC2) is mutated in ∼15% of patients with autosomal dominant polycystic kidney disease (ADPKD). PC2 belongs to the family of transient receptor potential (TRP) channels and can function as a homotetramer. We investigated whether three disease-associated mutations (F629S, C632R, or R638C) localized in the channel's pore loop alter ion channel properties of human PC2 expressed in Xenopus laevis oocytes. Expression of wild-type (WT) PC2 typically resulted in small but measurable Na+ inward currents in the absence of extracellular divalent cations. These currents were no longer observed when individual pore mutations were introduced in WT PC2. Similarly, Na+ inward currents mediated by the F604P gain-of-function (GOF) PC2 construct (PC2 F604P) were abolished by each of the three pore mutations. In contrast, when the mutations were introduced in another GOF construct, PC2 L677A N681A, only C632R had a complete loss-of-function effect, whereas significant residual Na+ inward currents were observed with F629S (∼15%) and R638C (∼30%). Importantly, the R638C mutation also abolished the Ca2+ permeability of PC2 L677A N681A and altered its monovalent cation selectivity. To elucidate the molecular mechanisms by which the R638C mutation affects channel function, molecular dynamics (MD) simulations were used in combination with functional experiments and site-directed mutagenesis. Our findings suggest that R638C stabilizes ionic interactions between Na+ ions and the selectivity filter residue D643. This probably explains the reduced monovalent cation conductance of the mutant channel. In summary, our data support the concept that altered ion channel properties of PC2 contribute to the pathogenesis of ADPKD.

Ciliary intrinsic mechanisms regulate dynamic ciliary extracellular vesicle release from sensory neurons.

Extracellular vesicles (EVs) are submicron membranous structures and key mediators of intercellular communication.1,2 Recent research has highlighted roles for cilia-derived EVs in signal transduction, underscoring their importance as bioactive extracellular organelles containing conserved ciliary signaling proteins.3,4 Members of the transient receptor potential (TRP) channel polycystin-2 (PKD-2) family are found in ciliary EVs of the green algae Chlamydomonas and the nematode Caenorhabditis elegans5,6 and in EVs in the mouse embryonic node and isolated from human urine.7,8 In C. elegans, PKD-2 is expressed in male-specific EV-releasing sensory neurons, which extend ciliary tips to ciliary pore and directly release EVs into the environment.6,9 Males release EVs in a mechanically stimulated manner, regulate EV cargo content in response to mating partners, and deposit PKD-2::GFP-labeled EVs on the vulval cuticle of hermaphrodites during mating.9,10 Combined, our findings suggest that ciliary EV release is a dynamic process. Herein, we identify mechanisms controlling dynamic EV shedding using time-lapse imaging. Cilia can sustain the release of PKD-2-labeled EVs for 2 h. This extended release doesn't require neuronal transmission. Instead, ciliary intrinsic mechanisms regulate PKD-2 ciliary membrane replenishment and dynamic EV release. The kinesin-3 motor kinesin-like protein 6 (KLP-6) is necessary for initial and extended EV release, while the transition zone protein NPHP-4 is required only for sustained EV release. The dynamic replenishment of PKD-2 at the ciliary tip is key to sustained EV release. Our study provides a comprehensive portrait of real-time ciliary EV release and mechanisms supporting cilia as proficient EV release platforms.

Increased Expression of Orexin-A in Patients Affected by Polycystic Kidney Disease.

Orexin-A is a neuropeptide product of the lateral hypothalamus that acts on two receptors, OX1R and OX2R. The orexinergic system is involved in feeding, sleep, and pressure regulation. Recently, orexin-A levels have been found to be negatively correlated with renal function. Here, we analyzed orexin-A levels as well as the incidence of SNPs in the hypocretin neuropeptide precursor (HCRT) and its receptors, HCRTR1 and HCRTR2, in 64 patients affected by autosomal dominant polycystic kidney disease (ADPKD) bearing truncating mutations in the PKD1 or PKD2 genes. Twenty-four healthy volunteers constituted the control group. Serum orexin-A was assessed by ELISA, while the SNPs were investigated through Sanger sequencing. Correlations with the main clinical features of PKD patients were assessed. PKD patients showed impaired renal function (mean eGFR 67.8 ± 34.53) and a statistically higher systolic blood pressure compared with the control group (p < 0.001). Additionally, orexin-A levels in PKD patients were statistically higher than those in healthy controls (477.07 ± 69.42 pg/mL vs. 321.49 ± 78.01 pg/mL; p < 0.001). Furthermore, orexin-A inversely correlated with blood pressure (p = 0.0085), while a direct correlation with eGFR in PKD patients was found. None of the analyzed SNPs showed any association with orexin-A levels in PKD. In conclusion, our data highlights the emerging role of orexin-A in renal physiology and its potential relevance to PKD. Further research is essential to elucidate the intricate mechanisms underlying orexin-A signaling in renal function and its therapeutic implications for PKD and associated cardiovascular complications.

Myocardin-Related Transcription Factor Mediates Epithelial Fibrogenesis in Polycystic Kidney Disease.

Polycystic kidney disease (PKD) is characterized by extensive cyst formation and progressive fibrosis. However, the molecular mechanisms whereby the loss/loss-of-function of Polycystin 1 or 2 (PC1/2) provokes fibrosis are largely unknown. The small GTPase RhoA has been recently implicated in cystogenesis, and we identified the RhoA/cytoskeleton/myocardin-related transcription factor (MRTF) pathway as an emerging mediator of epithelium-induced fibrogenesis. Therefore, we hypothesized that MRTF is activated by PC1/2 loss and plays a critical role in the fibrogenic reprogramming of the epithelium. The loss of PC1 or PC2, induced by siRNA in vitro, activated RhoA and caused cytoskeletal remodeling and robust nuclear MRTF translocation and overexpression. These phenomena were also manifested in PKD1 (RC/RC) and PKD2 (WS25/-) mice, with MRTF translocation and overexpression occurring predominantly in dilated tubules and the cyst-lining epithelium, respectively. In epithelial cells, a large cohort of PC1/PC2 downregulation-induced genes was MRTF-dependent, including cytoskeletal, integrin-related, and matricellular/fibrogenic proteins. Epithelial MRTF was necessary for the paracrine priming of the fibroblast-myofibroblast transition. Thus, MRTF acts as a prime inducer of epithelial fibrogenesis in PKD. We propose that RhoA is a common upstream inducer of both histological hallmarks of PKD: cystogenesis and fibrosis.

DLG1 functions upstream of SDCCAG3 and IFT20 to control ciliary targeting of polycystin-2.

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney causes ciliary elongation and cystogenesis, and cell-based proximity labeling proteomics and fluorescence microscopy show alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20, and polycystin-2 (PC2) are reduced in the cilia of DLG1-deficient cells compared to control cells. This phenotype is recapitulated in vivo and rescuable by re-expression of wild-type DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggest that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.

Discovery of putative inhibitors of human Pkd1 enzyme: Molecular docking, dynamics and simulation, QSAR, and MM/GBSA.

Polycystic kidney disease is the most prevalent hereditary kidney disease globally and is mainly linked to the overexpression of a gene called PKD1. To date, there is no effective treatment available for polycystic kidney disease, and the practicing treatments only provide symptomatic relief. Discovery of the compounds targeting the PKD1 gene by inhibiting its expression under the disease condition could be crucial for effective drug development. In this study, a molecular docking and molecular dynamic simulation, QSAR, and MM/GBSA-based approaches were used to determine the putative inhibitors of the Pkd1 enzyme from a library of 1379 compounds. Initially, fourteen compounds were selected based on their binding affinities with the Pkd1 enzyme using MOE and AutoDock tools. The selected drugs were further investigated to explore their properties as drug candidates and the stability of their complex formation with the Pkd1 enzyme. Based on the physicochemical and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties, and toxicity profiling, two compounds including olsalazine and diosmetin were selected for the downstream analysis as they demonstrated the best drug-likeness properties and highest binding affinity with Pkd1 in the docking experiment. Molecular dynamic simulation using Gromacs further confirmed the stability of olsalazine and diosmetin complexes with Pkd1 and establishing interaction through strong bonding with specific residues of protein. High biological activity and binding free energies of two complexes calculated using 3D QSAR and Schrodinger module, respectively further validated our results. Therefore, the molecular docking and dynamics simulation-based in-silico approach used in this study revealed olsalazine and diosmetin as potential drug candidates to combat polycystic kidney disease by targeting Pkd1 enzyme.

PKD1 mutant clones within cirrhotic livers inhibit steatohepatitis without promoting cancer.

Somatic mutations in non-malignant tissues are selected for because they confer increased clonal fitness. However, it is uncertain whether these clones can benefit organ health. Here, ultra-deep targeted sequencing of 150 liver samples from 30 chronic liver disease patients revealed recurrent somatic mutations. PKD1 mutations were observed in 30% of patients, whereas they were only detected in 1.3% of hepatocellular carcinomas (HCCs). To interrogate tumor suppressor functionality, we perturbed PKD1 in two HCC cell lines and six in vivo models, in some cases showing that PKD1 loss protected against HCC, but in most cases showing no impact. However, Pkd1 haploinsufficiency accelerated regeneration after partial hepatectomy. We tested Pkd1 in fatty liver disease, showing that Pkd1 loss was protective against steatosis and glucose intolerance. Mechanistically, Pkd1 loss selectively increased mTOR signaling without SREBP-1c activation. In summary, PKD1 mutations exert adaptive functionality on the organ level without increasing transformation risk.

Reduced decay-accelerating factor expression promotes complement-mediated cystogenesis in murine ADPKD.

Patients with autosomal dominant polycystic kidney disease (ADPKD), a genetic disease due to mutations of the PKD1 or PKD2 gene, show signs of complement activation in the urine and cystic fluid, but their pathogenic role in cystogenesis is unclear. We tested the causal relationship between complement activation and cyst growth using a Pkd1KO renal tubular cell line and newly generated conditional Pkd1-/- C3-/- mice. Pkd1-deficient tubular cells have increased expression of complement-related genes (C3, C5, CfB, C3ar, and C5ar1), while the gene and protein expression of complement regulators DAF, CD59, and Crry is decreased. Pkd1-/- C3-/- mice are unable to fully activate the complement cascade and are characterized by a significantly slower kidney cystogenesis, preserved renal function, and reduced intrarenal inflammation compared with Pkd1-/- C3+/+ controls. Transgenic expression of the cytoplasmic C-terminal tail of Pkd1 in Pkd1KO cells lowered C5ar1 expression, restored Daf levels, and reduced cell proliferation. Consistently, both DAF overexpression and pharmacological inhibition of C5aR1 (but not C3aR) reduced Pkd1KO cell proliferation. In conclusion, the loss of Pkd1 promotes unleashed activation of locally produced complement by downregulating DAF expression in renal tubular cells. Increased C5a formation and C5aR1 activation in tubular cells promotes cyst growth, offering a new therapeutic target.

Runx2 and Polycystins in Bone Mechanotransduction: Challenges for Therapeutic Opportunities.

Bone mechanotransduction is a critical process during skeletal development in embryogenesis and organogenesis. At the same time, the type and level of mechanical loading regulates bone remodeling throughout the adult life. The aberrant mechanosensing of bone cells has been implicated in the development and progression of bone loss disorders, but also in the bone-specific aspect of other clinical entities, such as the tumorigenesis of solid organs. Novel treatment options have come into sight that exploit the mechanosensitivity of osteoblasts, osteocytes, and chondrocytes to achieve efficient bone regeneration. In this regard, runt-related transcription factor 2 (Runx2) has emerged as a chief skeletal-specific molecule of differentiation, which is prominent to induction by mechanical stimuli. Polycystins represent a family of mechanosensitive proteins that interact with Runx2 in mechano-induced signaling cascades and foster the regulation of alternative effectors of mechanotransuction. In the present narrative review, we employed a PubMed search to extract the literature concerning Runx2, polycystins, and their association from 2000 to March 2024. The keywords stated below were used for the article search. We discuss recent advances regarding the implication of Runx2 and polycystins in bone remodeling and regeneration and elaborate on the targeting strategies that may potentially be applied for the treatment of patients with bone loss diseases.