Biology subjects

Varghese, M. M.

Publications and source records attributed to Varghese, M. M..

4 recordsLinked to original sources

bioRxiv2026

Myofibroblast- specific autophagy drives cyst growth in autosomal dominant polycystic kidney disease

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive cyst expansion, fibrosis and inflammation, leading to kidney failure. Myofibroblasts (MFs) often accumulate around cysts and promote fibrosis and cyst growth, but the cellular mechanisms enabling their pro-cystogenic activity remain unclear. Here we examined the role of autophagy within MFs, on their paracrine stimulation of cyst expansion in ADPKD. MethodsAutophagy was assessed in human ADPKD nephrectomy tissue, primary human ADPKD renal myofibroblasts (ADPKD-MFs) and male RC/RC mouse model of ADPKD using immunostaining, LC3/p62 analyses, and transmission electron microscopy. Autophagy in MFs was inhibited pharmacologically in ADPKD-MFs, or by conditional Atg5 deletion in PDGFR{beta}-expressing renal stromal cells in RC/RC (RC/RC;Atg5KO) and wild type (WT;Atg5KO) mice. ResultsIn human and mouse ADPKD kidneys, we detected LC3 puncta and autophagic organelles within SMA-expressing MFs. Inhibition of autophagy in ADPKD-MFs blocked their paracrine stimulation of cyst epithelial cell proliferation in vitro. RC/RC;Atg5KO mice showed significantly reduced cystic growth, fibrosis, MF abundance, and improved kidney function. WT;Atg5KO mice showed no abnormalities in kidney structure or function. Targeted metabolomics performed on ADPKD cyst epithelial-cell conditioned media (ADPKD-ECs CM) revealed moderate increase in lactate levels compared to normal human kidney epithelial-cell conditioned media. Furthermore, lactate treatment stabilized hypoxia-inducible factor-1 (HIF1) in myofibroblasts, while pharmacological inhibition of HIF1 reduced the expression of autophagy-related genes and impaired autophagic flux. ConclusionThese findings reveal that autophagy in MFs is a previously unrecognized driver of cyst expansion and fibrosis in ADPKD. Lactate-mediated HIF1 stabilization in MFs promotes autophagy that is required for their paracrine stimulation of cyst epithelial growth. Targeting MF-specific autophagy or its upstream regulators may represent a therapeutic strategy to limit cyst growth and fibrosis in ADPKD.

molecular biology↗

bioRxiv2026

CRISPR activation of endogenous PKD1 increases polycystin-1 levels and suppresses cellular features of ADPKD

Most cases of autosomal dominant polycystic kidney disease (ADPKD) are caused by mutations in PKD1, which reduce polycystin-1 (PC1) levels below a critical functional threshold. Normalizing PC1 dosage mitigates disease progression; therefore, we sought to develop a CRISPR activation (CRISPRa) strategy to transcriptionally upregulate endogenous PKD1. We systematically screened multiple single-guide RNAs using an EGFP-reporter platform and identified potent candidates targeting the proximal PKD1 promoter in mouse and human cell models. Our results demonstrate that CRISPRa effectively increased endogenous Pkd1 mRNA in the mouse collecting duct-derived Pkd1RC/-cell model and in the primary renal epithelial cells from PKD mice. In Pkd1RC/- cells, CRISPRa of Pkd1 increased PC1 protein levels and significantly reduced cell proliferation and in vitro cyst formation in 3D cultures. Mechanistically, Pkd1 activation improved mitochondrial membrane potential, reduced dependency on aerobic glycolysis, and corrected signaling pathways involved in cystogenesis, specifically reducing intracellular cAMP, cMyc, pCreb, and pErk levels, while increasing pYap1 levels. We confirmed the translational potential of this platform by successfully activating PKD1 in primary renal epithelial cells from human kidneys. We observed a heterogeneous response across both normal and ADPKD patient-derived donor lines, with significant upregulation achieved in two of the tested cell preparations. These findings provide a compelling proof-of-concept that CRISPRa-mediated gene augmentation can increase PC1 levels, establishing a foundation for promising gene therapies aimed at successfully suppressing the pathogenic features of ADPKD.

molecular biology↗

bioRxiv2025

Pirfenidone treatment attenuates fibrosis in autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a leading genetic cause of kidney failure, marked by progressive cyst expansion, inflammation and fibrosis. Renal fibrosis, characterized by myofibroblast activation and excessive extracellular matrix (ECM) deposition is a central driver of disease progression in ADPKD, yet targeted anti-fibrotic therapies remain limited. Here, we evaluated the therapeutic potential of pirfenidone to suppress fibrosis and disease progression in ADPKD. To define the ECM in human ADPKD kidneys, we analyzed snRNA-seq data and found that fibroblasts are the principal source of fibrous and adhesive ECM in ADPKD kidneys, exhibiting higher ECM gene expression than normal controls. In vitro, primary culture human ADPKD renal myofibroblasts showed a similar profibrotic gene expression profile, and pirfenidone treatment suppressed ECM gene expression, cell proliferation, migration and contractility. In the Pkd1RC/RC mouse model of ADPKD, pirfenidone treatment significantly reduced renal fibrosis, myofibroblast accumulation, ECM deposition, pro-fibrotic gene expression and associated cell signaling pathways. Pirfenidone also decreased kidney-to-body weight ratio and improved kidney function in Pkd1RC/RC mice, without altering cyst burden. Collectively, these findings demonstrate that pirfenidone attenuates renal fibrosis and improves kidney function in ADPKD by targeting myofibroblast activation and ECM production, supporting a complementary therapeutic approach to cyst-directed therapies.

molecular biology↗

bioRxiv2024

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

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 and PKD2 genes, and often progresses to kidney failure. ADPKD progression is not uniform among patients, suggesting that factors secondary to the PKD1/2 gene mutation could regulate the rate of disease progression. Here we tested the effect of circadian clock disruption on ADPKD progression. Circadian rhythms are regulated by cell-autonomous circadian clocks composed of clock proteins. BMAL1 is a core constituent of the circadian clock. MethodsTo disrupt the circadian clock, we deleted Bmal1 gene in the renal collecting ducts of the Pkd1RC/RC (RC/RC) mouse model of ADPKD (RC/RC;Bmal1f/f;Pkhd1cre, called DKO mice), and in Pkd1 knockout mouse inner medullary collecting duct cells (Pkd1Bmal1KO mIMCD3 cells). Only male mice were used. ResultsHuman nephrectomy ADPKD kidneys and Pkd1KO mIMCD3 cells showed reduced Bmal1 gene expression compared to normal controls. When compared to RC/RC kidneys, DKO kidneys showed significantly altered clock gene expression, increased cyst growth, cell proliferation, apoptosis and fibrosis. DKO kidneys also showed increased lipogenesis and cholesterol synthesis-related gene expression, and increased tissue triglyceride levels compared to RC/RC kidneys. Similarly, in vitro, Pkd1Bmal1KO cells showed altered clock genes, increased lipogenesis and cholesterol synthesis-related genes, and reduced fatty-acid oxidation-related gene expression compared to Pkd1KO cells. The Pkd1Bmal1KO cells showed increased cell proliferation compared to Pkd1KO cells, which was rescued by pharmacological inhibition of lipogenesis. ConclusionRenal collecting duct specific Bmal1 gene deletion disrupts the circadian clock and triggers accelerated ADPKD progression by altering lipid metabolism-related gene expression. Key pointsO_LILack of BMAL1, a circadian clock protein in renal collecting ducts disrupted the clock and increased cyst growth and fibrosis in an ADPKD mouse model. C_LIO_LIBMAL1 gene deletion increased cell proliferation by increasing lipogenesis in kidney cells. C_LIO_LIThus, circadian clock disruption could be a risk factor for accelerated disease progression in patients with ADPKD. C_LI

molecular biology↗