Background:Autosomal 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. Methods:Autophagy 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 PDGFRβ-expressing renal stromal cells in RC/RC (RC/RC;Atg5KO) and wild type (WT;Atg5KO) mice. Results:In human and mouse ADPKD kidneys, we detected LC3 puncta and autophagic organelles within αSMA-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-1α (HIF1α) in myofibroblasts, while pharmacological inhibition of HIF1α reduced the expression of autophagy-related genes and impaired autophagic flux. Conclusion:These findings reveal that autophagy in MFs is a previously unrecognized driver of cyst expansion and fibrosis in ADPKD. Lactate-mediated HIF1α 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.

Introduction:Sex influences susceptibility, recovery, and long-term outcomes after acute kidney injury (AKI) in humans. Rodent models have been invaluable for elucidating AKI mechanisms; however, most studies have focused on males, assuming direct applicability to females, an assumption that remains largely untested. In males, the transcription factor SOX9 regulates injury-associated proximal tubule cell states, supporting survival during injury but also contributing to maladaptive repair. Given the well-described role of SOX9 in sex determination during development and its emerging importance in AKI, we investigated whether it mediates sex-specific tubular responses in the adult kidney. Methods:We first optimized differential ischemic and cisplatin conditions in males and females to generate injury-matched models. We then used these models to ask whether proximal tubular cell responses, particularly Sox9 upregulation, are sex dependent. Tubule-specific knockout models of Sox9 , Sox4 , Sox11 , Vgf , Zfp24 , and the androgen receptor ( Ar ), along with gonadectomy and hormone replacement studies, combined with gene and protein analyses, were used to define regulatory networks. Results:In both ischemic and nephrotoxic AKI, SOX9 expression was markedly blunted in females, with levels more than fivefold lower than in males. While injury biomarkers such as NGAL and KIM1 were equally induced in both sexes, Sox9 and its downstream target Vgf showed markedly reduced induction in females, whereas the upstream regulator Zfp24 was functionally relevant only in males. Deletion of Sox9 , Zfp24 , or Vgf worsened injury in males but not in females. In contrast, Sox4 and Sox11 were equally upregulated and protective in both sexes. Castration or proximal tubule-specific deletion of Ar in males abolished Sox9 induction, establishing a testosterone-dependent regulatory axis. Conclusions:These findings define a hormone-driven, male-specific tubular repair program and demonstrate that injury and recovery pathways differ fundamentally between sexes, underscoring the need for sex-inclusive therapeutic strategies for AKI. Translational Statement:Sex influences susceptibility and outcomes after AKI, but the molecular basis remains unclear. SOX9, a transcription factor recently identified as one of the most highly upregulated genes in proximal tubular cells during AKI in both mice and humans, has been presumed to mediate a universal protective program. Using injury-matched murine models of AKI, we now demonstrate that this response is restricted to males, driven by testosterone and androgen receptor signaling, while SOX9 induction is markedly blunted and functionally dispensable in females. These findings reveal that tubular protective pathways differ fundamentally between sexes despite equivalent injury severity. Because SOX9 activation confers epithelial protection in males, therapeutic targeting of this pathway may have clinical relevance primarily in male patients. These data underscore the importance of incorporating biological sex into mechanistic, translational, and clinical studies of kidney injury and recovery.

Introduction:Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid filled cysts, progressive fibrosis and chronic inflammation, often leading to kidney failure. Renal fibrosis in ADPKD is primarily driven by myofibroblast activation and excessive extracellular matrix (ECM) accumulation, which contribute to disease progression. Here we investigated the therapeutic potential of pirfenidone, an antifibrotic drug, on myofibroblast activity, ECM production, and ADPKD progression. Methods:Primary cultures of myofibroblasts from human ADPKD kidneys were treated with pirfenidone in vitro, and cell proliferation, migration, contractility and changes in ECM production were measured. In vivo, the effect of pirfenidone on cyst growth, fibrosis and renal function were determined in the Pkd1RC/RC male mouse model of ADPKD and wild type controls. Results:Analysis of single-nucleus RNA sequencing data of human ADPKD kidneys revealed that fibroblasts are a primary source of fibrous and cell-adhesive ECM, with higher ECM gene expression compared to normal human kidneys. Treatment of human ADPKD renal myofibroblasts with pirfenidone led to reduced ECM gene expression, cell proliferation, migration and contractility. In vivo, pirfenidone treatment in Pkd1RC/RC mice reduced renal fibrosis, collagen deposition, myofibroblast accumulation, pro-fibrotic gene expression and decreased TGF-β/SMAD3 and mTOR signaling. While kidney cyst number remained unchanged, kidney size and cyst area were reduced, leading to improved kidney morphology and improved renal function in RC/RC mice. Conclusion:These findings suggest that pirfenidone mitigates renal fibrosis and preserves renal architecture in ADPKD, supporting its potential as a therapeutic strategy to inhibit fibrosis in ADPKD.

Arginine vasopressin (AVP) and its type-2 receptor (V2R) play an essential role in the regulation of salt and water homeostasis by the kidneys. V2R activation also stimulates proliferation of renal cell carcinoma (RCC) cell lines in vitro. The current studies investigated V2R expression and activity in human RCC tumors, and its role in RCC tumor growth. Examination of the cancer genome atlas (TCGA) database, and analysis of human RCC tumor tissue microarrays, cDNA arrays and tumor biopsy samples demonstrated V2R expression and activity in clear cell RCC (ccRCC). In vitro, V2R antagonists OPC31260 and Tolvaptan, or V2R gene silencing reduced wound closure and cell viability of 786-O and Caki-1 human ccRCC cell lines. Similarly in mouse xenograft models, Tolvaptan and OPC31260 decreased RCC tumor growth by reducing cell proliferation and angiogenesis, while increasing apoptosis. In contrast, the V2R agonist dDAVP significantly increased tumor growth. High intracellular cAMP levels and ERK1/2 activation were observed in human ccRCC tumors. In mouse tumors and Caki-1 cells, V2R agonists reduced cAMP and ERK1/2 activation, while dDAVP treatment had the reverse effect. V2R gene silencing in Caki-1 cells also reduced cAMP and ERK1/2 activation. These results provide novel evidence for a pathogenic role of V2R signaling in ccRCC, and suggest that inhibitors of the AVP-V2R pathway, including the FDA-approved drug Tolvaptan, could be utilized as novel ccRCC therapeutics.

Background:Autosomal 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. Methods:To disrupt the circadian clock, we deleted Bmal1 gene in the renal collecting ducts of the Pkd1 RC/RC (RC/RC) mouse model of ADPKD (RC/RC; Bmal1 f/f ; Pkhd1 cre , called DKO mice), and in Pkd1 knockout mouse inner medullary collecting duct cells ( Pkd1Bmal1 KO mIMCD3 cells). Only male mice were used. Results:Human nephrectomy ADPKD kidneys and Pkd1 KO 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, Pkd1Bmal1 KO 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 Pkd1Bmal1 KO cells showed increased cell proliferation compared to Pkd1KO cells, which was rescued by pharmacological inhibition of lipogenesis. Conclusion:Renal collecting duct specific Bmal1 gene deletion disrupts the circadian clock and triggers accelerated ADPKD progression by altering lipid metabolism-related gene expression. Key points:Lack of BMAL1, a circadian clock protein in renal collecting ducts disrupted the clock and increased cyst growth and fibrosis in an ADPKD mouse model.BMAL1 gene deletion increased cell proliferation by increasing lipogenesis in kidney cells.Thus, circadian clock disruption could be a risk factor for accelerated disease progression in patients with ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) involves the development and persistent growth of fluid filled kidney cysts. In a recent study, we showed that ADPKD kidney cyst epithelial cells can stimulate the proliferation and differentiation of peri-cystic myofibroblasts. Although dense myofibroblast populations are often found surrounding kidney cysts, their role in cyst enlargement or fibrosis in ADPKD is unclear. To clarify this, we examined the effect of myofibroblast depletion in the Pkd1RC/RC (RC/RC) mouse model of ADPKD. RC/RC;αSMAtk mice that use the ganciclovir-thymidine kinase system to selectively deplete α-smooth muscle actin expressing myofibroblasts were generated. Ganciclovir treatment for four weeks depleted myofibroblasts, reduced kidney fibrosis and preserved kidney function in these mice. Importantly, myofibroblast depletion significantly reduced cyst growth and cyst epithelial cell proliferation in RC/RC;αSMAtk mouse kidneys. Similar ganciclovir treatment did not alter cyst growth or fibrosis in wild-type or RC/RC littermates. In vitro, co-culture with myofibroblasts from the kidneys of patients with ADPKD increased 3D microcyst growth of human ADPKD cyst epithelial cells. Treatment with conditioned culture media from ADPKD kidney myofibroblasts increased microcyst growth and cell proliferation of ADPKD cyst epithelial cells. Further examination of ADPKD myofibroblast conditioned media showed high levels of protease inhibitors including PAI1, TIMP1 and 2, NGAL and TFPI-2, and treatment with recombinant PAI1 and TIMP1 increased ADPKD cyst epithelial cell proliferation in vitro. Thus, our findings show that myofibroblasts directly promote cyst epithelial cell proliferation, cyst growth and fibrosis in ADPKD kidneys, and their targeting could be a novel therapeutic strategy to treat PKD.

The expression of the angiotensin-converting enzyme 2 (ACE2) is altered in multiple chronic kidney diseases like hypertension and renal fibrosis, where the signaling from the basal membrane proteins is critical for the development and progression of the various pathologies. Integrins are heterodimeric cell surface receptors that have important roles in the progression of these chronic kidney diseases by altering various cell signaling pathways in response to changes in the basement membrane proteins. It is unclear whether integrin or integrin-mediated signaling affects the ACE2 expression in the kidney. The current study tests the hypothesis that integrin β1 regulates the expression of ACE2 in kidney epithelial cells. The role of integrin β1 in ACE2 expression in renal epithelial cells was investigated by shRNA-mediated knockdown and pharmacological inhibition. In vivo studies were carried out using epithelial cell-specific deletion of integrin β1 in the kidneys. Deletion of integrin β1 from the mouse renal epithelial cells reduced the expression of ACE2 in the kidney. Furthermore, the downregulation of integrin β1 using shRNA decreased ACE2 expression in human renal epithelial cells. ACE2 expression levels were also decreased in renal epithelial cells and cancer cells when treated with an integrin α2β1 antagonist, BTT 3033. SARS-CoV-2 viral entry to human renal epithelial cells and cancer cells was also inhibited by BTT 3033. This study demonstrates that integrin β1 positively regulates the expression of ACE2, which is required for the entry of SARS-CoV-2 into kidney cells.

India is the highest TB burden country in the world. The burden however is not uniform in different strata including tribal population - one of the key affected populations in the country. As the evidences from tribal population are hardly available, most of the policies and strategies implemented under National Tuberculosis Elimination Programme (NTEP) are usually based on the evidences from general populations. NTEP is continuously taking steps to strengthen TB services in tribal areas. The Social Action Plan including Tribal Action Plan is in place and the appropriate strategies are incorporated in the National Strategic Plan (NSP) to ensure universal access to quality TB services to vulnerable population groups. However, its implementation becomes challenging especially in tribal areas as different tribal groups have their own unique ways of dealing with health issues. These issues are therefore required to be addressed holistically involving all the stakeholders. In view of this a symposium was jointly organized by the Central TB Division (CTD), Govt. of India and ICMR - National Institute of Research in Tribal Health (NIRTH), Jabalpur on 17th and 18th December, 2019 at ICMR - NIRTH, Jabalpur. It provided an excellent platform for all the stakeholders from different parts of the country to share their experiences in tuberculosis particularly among marginalized populations. The recommendations emerged out of this interactive symposium highlight the sincere effort of NTEP to tackle TB situation in tribal population and show the way forward towards India's TB elimination goal by 2025 especially in hard to reach tribal areas.

Vasopressin type-2 receptor (V2R) is ectopically expressed and plays a pathogenic role in clear cell renal cell carcinoma (ccRCC) tumor cells. Here we examined how V2R signaling within human ccRCC tumor cells (Caki1 cells) stimulates stromal cancer-associated fibroblasts (CAFs). We found that cell culture conditioned media from Caki1 cells increased activation, migration, and proliferation of fibroblasts in vitro, which was inhibited by V2R gene silencing in Caki1 cells. Analysis of the conditioned media and mRNA of the V2R gene silenced and control Caki1 cells showed that V2R regulates the production of CAF-activating factors. Some of these factors were also found to be regulated by YAP in these Caki1 cells. YAP expression colocalized and correlated with V2R expression in ccRCC tumor tissue. V2R gene silencing or V2R antagonist significantly reduced YAP in Caki1 cells. Moreover, the V2R antagonist reduced YAP expression and myofibroblasts in mouse xenograft tumors. These results suggest that V2R plays an important role in secreting pro-fibrotic factors that stimulate fibroblast activation by a YAP-dependent mechanism in ccRCC tumors. Our results demonstrate a novel role for the V2R-YAP axis in the regulation of myofibroblasts in ccRCC and a potential therapeutic target.

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and is characterized by progressive growth of fluid-filled cysts. Growth factors binding to receptor tyrosine kinases (RTKs) stimulate cell proliferation and cyst growth in PKD. Nintedanib, a triple RTK inhibitor, targets the vascular endothelial growth-factor receptor (VEGFR), platelet-derived growth-factor receptor (PDGFR), and fibroblast growth-factor receptor (FGFR), and is an approved drug for the treatment of non-small-cell lung carcinoma and idiopathic lung fibrosis. To determine if RTK inhibition using nintedanib can slow ADPKD progression, we tested its effect on human ADPKD renal cyst epithelial cells and myofibroblasts in vitro, and on Pkd1f/fPkhd1Cre and Pkd1RC/RC, orthologous mouse models of ADPKD. Nintedanib significantly inhibited cell proliferation and in vitro cyst growth of human ADPKD renal cyst epithelial cells, and cell viability and migration of human ADPKD renal myofibroblasts. Consistently, nintedanib treatment significantly reduced kidney-to-body-weight ratio, renal cystic index, cystic epithelial cell proliferation, and blood-urea nitrogen levels in both the Pkd1f/fPkhd1Cre and Pkd1RC/RC mice. There was a corresponding reduction in ERK, AKT, STAT3, and mTOR activity and expression of proproliferative factors, including Yes-associated protein (YAP), c-Myc, and Cyclin D1. Nintedanib treatment significantly reduced fibrosis in Pkd1RC/RC mice, but did not affect renal fibrosis in Pkd1f/fPkhd1Cre mice. Overall, these results suggest that nintedanib may be repurposed to effectively slow cyst growth in ADPKD.

The tumor microenvironment plays a critical role in defining the growth and malignancy of solid tumors. Extracellular matrix (ECM) proteins such as collagen, vitronectin, and fibronectin are major components of the tumor microenvironment. Tumor growth-promoting reciprocal interaction between ECM and cytoplasmic proteins is regulated by the cell surface receptors called integrins. This study investigated the mechanism by which integrin β1 promotes pancreatic tumor growth. In MIA PaCa-2 pancreatic cancer cell line, the loss of integrin β1 protein reduced the ability of cells to proliferate in a 3D matrix and compromised the ability to form a focal adhesion complex. Decreased expression of integrin α5 was observed in KO cells, which resulted in impaired cell spreading and adhesion on vitronectin and fibronectin. Reduced expression of the integrin-associated protein, kindlin-2 was also recorded. The downregulation of kindlin-2 decreased the phosphorylation of Smad2/3 by reducing the expression of TGF-β receptor 2. These results unravel a new mechanism of integrin β1 in tumor growth by modifying the expression of kindlin-2 and TGF-β receptor 2 signaling.

Acute kidney injury (AKI) is a common clinical syndrome that involves renal tubular epithelial cell death and leads to acute decline in renal function. Improper tubular regeneration following AKI often leads to CKD. We discuss the role of a serine/threonine protein kinase called glycogen synthase kinase-3 (GSK3) in renal tubular injury and renal fibrosis. We also highlight the importance of GSK3 as a potential drug target in AKI patients and molecular mechanisms promoting tissue regeneration.

Acute kidney injury (AKI) is characterized by injury to the tubular epithelium that leads to the sudden loss of renal function. Proper tubular regeneration is essential to prevent progression to chronic kidney disease. In this study, we examined the role of FoxM1, a forkhead box family member transcription factor in tubular repair after AKI. Renal FoxM1 expression increased after renal ischemia/reperfusion (I/R)‐induced AKI in mouse kidneys. Treatment with thiostrepton, a FoxM1 inhibitor, reduced FoxM1 regulated pro‐proliferative factors and cell proliferation in vitro, and tubular regeneration in mouse kidneys after AKI. Glycogen synthase kinase‐3 (GSK3) was found to be an upstream regulator of FoxM1 because GSK3 inhibition or renal tubular GSK3β gene deletion significantly increased FoxM1 expression, and improved tubular repair and renal function. GSK3 inactivation increased β‐catenin, Cyclin D1, and c‐Myc, and reduced cell cycle inhibitors p21 and p27. Importantly, thiostrepton treatment abolished the improved tubular repair in GSK3β knockout mice following AKI. These results demonstrate that FoxM1 is important for renal tubular regeneration following AKI and that GSK3β suppresses tubular repair by inhibiting FoxM1.

BACKGROUND Fibrosis is a major cause of loss of renal function in autosomal dominant polycystic kidney disease (ADPKD). In this study, we examined whether vasopressin type-2 receptor (V2R) activity in cystic epithelial cells can stimulate interstitial myofibroblasts and fibrosis in ADPKD kidneys. METHODS We treated Pkd1 gene knockout (Pkd1KO) mice with dDAVP, a V2R agonist, for 3 days and evaluated the effect on myofibroblast deposition of extracellular matrix (ECM). We also analyzed the effects of conditioned media from primary cultures of human ADPKD cystic epithelial cells on myofibroblast activation. Because secretion of the profibrotic connective tissue growth factor (CCN2) increased significantly in dDAVP-treated Pkd1KO mouse kidneys, we examined its role in V2R-dependent fibrosis in ADPKD as well as that of yes-associated protein (YAP). RESULTS V2R stimulation using dDAVP increased the renal interstitial myofibroblast population and ECM deposition. Similarly, conditioned media from human ADPKD cystic epithelial cells increased myofibroblast activation in vitro, suggesting a paracrine mechanism. Renal collecting duct-specific gene deletion of CCN2 significantly reduced cyst growth and myofibroblasts in Pkd1KO mouse kidneys. We found that YAP regulates CCN2, and YAP inhibition or gene deletion reduces renal fibrosis in Pkd1KO mouse kidneys. Importantly, YAP inactivation blocks the dDAVP-induced increase in myofibroblasts in Pkd1KO kidneys. Further in vitro studies showed that V2R regulates YAP by an ERK1/2-dependent mechanism in human ADPKD cystic epithelial cells. CONCLUSIONS Our results demonstrate a novel mechanism by which cystic epithelial cells stimulate myofibroblasts in the pericystic microenvironment, leading to fibrosis in ADPKD. The V2R-YAP-CCN2 cell signaling pathway may present a potential therapeutic target for fibrosis in ADPKD.