Evidence for reduced expression of cyclin G associated kinase (GAK) in glomeruli of patients with chronic kidney disease was observed in the Nephroseq human database, and GAK was found to be associated with the decline in kidney function. To examine the role of GAK, a protein that functions to uncoat clathrin during endocytosis, we generated podocyte-specific Gak-knockout mice (Gak-KO), which developed progressive proteinuria and kidney failure with global glomerulosclerosis. We isolated glomeruli from the mice carrying the mutation to perform messenger RNA profiling and unearthed evidence for dysregulated podocyte calpain protease activity as an important contributor to progressive podocyte damage. Treatment with calpain inhibitor III specifically inhibited calpain-1/-2 activities, mitigated the degree of proteinuria and glomerulosclerosis, and led to a striking increase in survival in the Gak-KO mice. Podocyte-specific deletion of Capns1, essential for calpain-1 and calpain-2 activities, also improved proteinuria and glomerulosclerosis in Gak-KO mice. Increased podocyte calpain activity–mediated proteolysis of IκBα resulted in increased NF-κB p65–induced expression of growth arrest and DNA-damage-inducible 45 beta in the Gak-KO mice. Our results suggest that loss of podocyte-associated Gak induces glomerular injury secondary to calcium dysregulation and aberrant calpain activation, which when inhibited, can provide a protective role.
Xuefei Tian, Kazunori Inoue, Yan Zhang, Ying Wang, C. John Sperati, Christopher E. Pedigo, Tingting Zhao, Meihua Yan, Marwin Groener, Dennis G. Moledina, Karen Ebenezer, Wei Li, Zhenhai Zhang, Dan A. Liebermann, Lois Greene, Peter Greer, Chirag R. Parikh, Shuta Ishibe
Diabetic kidney disease (DKD) is the most common cause of severe renal disease worldwide and the single strongest predictor of mortality in diabetes patients. Kidney steatosis has emerged as a critical trigger in the pathogenesis of DKD; however, the molecular mechanism of renal lipotoxicity remains largely unknown. Our recent studies in genetic mouse models, human cell lines, and well-characterized patient cohorts have identified serine/threonine protein kinase (STK)25 as a critical regulator of ectopic lipid storage in several metabolic organs prone to diabetic damage. Here, we demonstrate that overexpression of STK25 aggravates renal lipid accumulation and exacerbates structural and functional kidney injury in a mouse model of DKD. Reciprocally, inhibiting STK25 signaling in mice ameliorates diet-induced renal steatosis and alleviates the development of DKD-associated pathologies. Further, we find that STK25 silencing in human kidney cells protects against lipid deposition as well as oxidative and endoplasmic reticulum stress. Together, our results suggest that STK25 regulates a critical node governing susceptibility to renal lipotoxicity and that STK25 antagonism could mitigate DKD progression.
Emmelie Cansby, Mara Caputo, Lei Gao, Nagaraj M. Kulkarni, Annika Nerstedt, Marcus Ståhlman, Jan Boren, Rando Porosk, Ursel Soomets, Matteo Pedrelli, Paolo Parini, Hanns-Ulrich Marschall, Jenny Nyström, Brian W. Howell, Margit Mahlapuu
Chronic kidney disease (CKD) induces the failure of arteriovenous fistulas (AVF) and promotes the differentiation of vascular adventitial GLI1+ mesenchymal stem cells (GMCs). However, the roles of GMCs in forming neointima in AVFs remains unknown. GMCs isolated from CKD mice showed increased potential capacity of differentiation into myofibroblast-like cells. Increased activation of expression of PDGFRA and hedgehog (HH) signaling were detected in adventitial cells of AVFs from ESRD patients and CKD mice. PDGFRA was translocated and accumulated in early endosome when hedgehog signaling stimulates was activated. In endosome, PDGFRA mediated activation of TGFB1/SMAD signaling promoting GMCs differentiation into myofibroblast, extracellular matrix deposition, and vascular fibrosis. These responses resulted in neointima formation and AVF failure. Knockout (KO) of Pdgfra or inhibition of HH signaling in GMCs suppressed the differentiation of GMCs into myofibroblasts. In vivo, specific KO of Pdgfra inhibited GMC activation and vascular fibrosis, resulting in suppression of neointima formation and improvement of AVF patency despite CKD. Our findings could yield strategies for maintaining AVF functions.
Ke Song, Ying Qing, Qunying Guo, Eric K. Peden, Changyi Chen, William E. Mitch, Luan Truong, Jizhong Cheng
Actin-associated nonmuscle myosin II (NM2) motor proteins play critical roles in a myriad of cellular functions including endocytosis and organelle transport pathways. Cell type-specific expression and unique subcellular localization of the NM2 proteins, encoded by the Myh9 and Myh10 genes, in the mouse kidney tubules led us to hypothesize that these proteins have specialized functional roles within the renal epithelium. Inducible, conditional knockout (cKO) of Myh9 and Myh10 in the renal tubules of adult mice resulted in progressive kidney disease. Prior to overt renal tubular injury, we observed intracellular accumulation of the GPI-anchored protein uromodulin and gradual loss of Na+ K+ 2Cl- cotransporter from the apical membrane of the thick ascending limb (TAL) epithelia. The UMOD accumulation coincided with expansion of endoplasmic reticulum (ER) tubules, activation of ER stress and unfolded protein response pathways in Myh9&10 cKO kidneys. We conclude that NM2 proteins are required for localization and transport of UMOD and loss of function results in accumulation of UMOD and ER stress mediated progressive renal tubulointerstitial disease. These observations establish cell type-specific role(s) for NM2 proteins in regulation of specialized renal epithelial transport pathways and reveal the possibility that human kidney disease associated with MYH9 mutations could be of renal epithelial origin..
Karla L. Otterpohl, Brook W. Busselman, Ishara Ratnayake, Ryan G. Hart, Kimberly Hart, Claire Evans, Carrie L. Phillips, Jordan R. Beach, Phil Ahrenkiel, Bruce Molitoris, Kameswaran Surendran, Indra Chandrasekar
Fibrosis is the final common pathway in the pathophysiology of most forms of chronic kidney disease (CKD). As treatment of renal fibrosis still remains largely supportive, a refined understanding of the cellular and molecular mechanisms of kidney fibrosis and the development of novel compounds are urgently needed. Whether arginases play a role in development of fibrosis in CKD is unclear. We hypothesize that endothelial-arginase-2 (Arg2) promotes the development of kidney fibrosis induced by unilateral ureteral obstruction (UUO). Arg2 expression and arginase activity significantly increased following renal fibrosis. Pharmacological blockade or genetic deficiency of Arg2 conferred kidney protection following renal fibrosis as reflected by a reduction in kidney interstitial fibrosis and fibrotic markers. Selective deletion of Arg2 in endothelial cells (Tie2Cre/Arg2flox/flox) reduced the level of fibrosis after UUO. In contrast, selective deletion of Arg2 specifically in proximal tubular cells (Ggt1Cre/Arg2flox/flox) failed to reduce renal fibrosis after UUO. Furthermore, arginase inhibition restored kidney nitric oxide (NO) levels, oxidative stress, and mitochondrial function following UUO.These findings indicate that endothelial-Arg2 plays a major role in renal fibrosis via its action on NO and mitochondrial function. Blocking Arg2 activity or expression could be a novel therapeutic approach for prevention of CKD.
Michael Wetzel, Kristen Stanley, Wei Wei Wang, Soumya Maity, Muniswamy Madesh, W. Brian Reeves, Alaa S. Awad
Lupus Nephritis (LN) is a major organ complication and cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). There is an unmet medical need for developing more efficient and specific, mechanism-based therapies, which depends on improved understanding of the underlying LN pathogenesis. Here we present direct visual evidence from high-power intravital imaging of the local kidney tissue microenvironment in new mouse models showing that activated memory T cells originated in immune organs and the LN-specific robust accumulation of the glomerular endothelial glycocalyx play central roles in LN development. The glomerular homing of T cells was mediated via the direct binding of their CD44 to the hyaluronic acid (HA) component of the endothelial glycocalyx, and glycocalyx-degrading enzymes efficiently disrupted it. Short-course treatment with either hyaluronidase or heparinase III provided long-term organ protection as evidenced by vastly improved albuminuria and survival rate. This glycocalyx/HA/memory T cell interaction is present in multiple SLE-affected organs, and may be therapeutically targeted for SLE complications including LN.
Hiroyuki Kadoya, Ning Yu, Ina Maria Schiessl, Anne Riquier-Brison, Georgina Gyarmati, Dorinne Desposito, Kengo Kidokoro, Matthew J. Butler, Chaim O. Jacob, János Peti-Peterdi
Using the global Hipk2-null mice in various models of kidney disease, we previously demonstrated the central role of homeodomain interacting protein kinase 2 (HIPK2) in the development of renal fibrosis. However, whether the renal tubular epithelial cell (RTEC)-specific HIPK2 function significantly contributed to renal fibrogenesis had not been established. Herein, using RTEC-specific HIPK2 null mice and transgenic mice with RTEC-specific overexpression of the wildtype (WT) or kinase-dead (KD) mutant of HIPK2, we now show that the modulation of tubular HIPK2 expression and activity can profoundly affect renal fibrosis development in vivo. The loss of HIPK2 expression in RTECs resulted in a marked diminution of renal fibrosis in mouse models of unilateral obstruction (UUO) and HIV-associated nephropathy (HIVAN) in Tg26 mice, which was associated with the reduction of Smad3 activation and downstream expression of profibrotic markers. Conversely, the overexpression of WT HIPK2 in RTECs accentuated the extent of renal fibrosis in the setting of UUO, HIVAN, and folic acid nephropathy (FAN) in mice. Importantly, the overexpression of HIPK2 KD mutant or administration of BT173, an allosteric inhibitor of HIPK2-Smad3 interaction, markedly attenuated the renal fibrosis in these mouse models of kidney disease, indicating that HIPK2 requires both the kinase activity and its interaction with Smad3 to promote TGF--mediated renal fibrosis. Together, these results establish an important RTEC-specific role of HIPK2 in kidney fibrosis and further substantiates the inhibition of HIPK2 as a therapeutic approach toward renal fibrosis.
Wenzhen Xiao, Jing E, Li Bao, Ying Fan, Yuanmeng Jin, Andrew Wang, David Bauman, Zhengzhe Li, Ya-Li Zheng, Ruijie Liu, Kyung Lee, John Cijiang He
TLR7 has been linked to the pathogenesis of glomerulonephritis, but its precise roles are not clear. In this study, we evaluated the roles of TLR7 in IgA nephropathy (IgAN). TLR7 proteins were abundant in CD19+ B cells infiltrated in the kidneys of patients with IgAN. The intensities of both intrarenal TLR7 and CD19 proteins were closely associated with kidney function (estimated glomerular filtration rate [eGFR] and serum creatinine concentration) and renal histopathology (tubular atrophy, leukocyte infiltration, tubulointerstitial fibrosis, and global glomerulosclerosis) in patients with IgAN. Meanwhile, TLR7 mRNA levels were significantly increased in peripheral blood B cells of patients with IgAN. TLR7+CD19+ B cells expressed inflammatory cytokines (IL-6 and IL-12) in kidneys and produced high levels of IgA1 and galactose deficient-IgA1 (Gd-IgA1) in peripheral blood of patients with IgAN. Mechanistically, TLR7 activated B cells to produce high levels of Gd-IgA1 via the TLR7-GALNT2 axis in IgAN. Protein levels of GALNT2 were increased by overexpression of TLR7, while they were reduced by TLR7 knockdown in B cells. GALNT2 overexpression augmented Gd-IgA1 production in B cells derived from patients with IgAN. Taken together, high TLR7 expression in B cells has dual roles in the development and progression of IgAN, by facilitating renal inflammation and Gd-IgA1 antibody synthesis.
Nuoyan Zheng, Kaifeng Xie, Hongjian Ye, Yu Dong, Bing Wang, Ning Luo, Jinjin Fan, Jiaqing Tan, Wei Chen, Xueqing Yu
Histone deacetylase (HDAC) enzymes regulate transcription through epigenetic modification of chromatin structure, but their specific functions in the kidney remain elusive. We discovered that the human kidney expresses class I HDACs. Kidney medullary-specific inhibition of class I HDACs in the rat during high salt feeding results in hypertension, polyuria, hypokalemia, and nitric oxide (NO) deficiency. Three new, inducible murine models were used to determine that HDAC1 and HDAC2 in the kidney epithelium are necessary for maintaining epithelial integrity and maintaining fluid-electrolyte balance during increased dietary sodium intake. Moreover, single nucleus RNA sequencing determined that epithelial HDAC1 and HDAC2 are necessary for expression of many sodium or water transporters and channels. In performing a systematic review and meta-analysis of serious adverse events associated with clinical HDAC inhibitor use, we found that HDAC inhibitors increased the odds ratio of experiencing fluid-electrolyte disorders such as hypokalemia. This study provides insight on the mechanisms of potential serious adverse events with HDAC inhibitors, which may be fatal to critically ill patients. In conclusion, kidney tubular HDACs provide a link between the environment, such as consumption of high salt diets, with regulation of homeostatic mechanisms to remain in fluid-electrolyte balance.
Kelly A. Hyndman, Joshua S. Speed, Luciano D. Mendoza, John Allan, Jackson Colson, Randee Sedaka, Chunhua Jin, Hyun Jun Jung, Samir El-Dahr, David Pollock, Jennifer Pollock.
Mutations in PKD1 (encoding for Polycystin-1, PC1) are found in 80-85% of patients with ADPKD. We tested the hypothesis that changes in actin dynamics result from PKD1 mutations through dysregulation of compartmentalised centrosomal RhoA signalling mediated by specific RhoGAP (ARHGAP) proteins resulting in the complex cellular cystic phenotype. Initial studies revealed that the actin cytoskeleton was highly disorganised in PKD1 patient-derived cells and was associated with an increase in total and centrosomal RhoA activation and ROCK signalling. Using cilia length as a phenotypic readout for centrosomal RhoA activity, we identified ARHGAP5, 29, 35 as essential regulators of ciliation in normal human renal tubular cells. Importantly, a specific decrease in centrosomal ARHGAP35 was observed in PKD1 null cells using a centrosome-targeted proximity ligation assay and by immunofluorescence labelling. Finally, we demonstrate that another ROCK inhibitor (hydroxyfasudil) reduced cyst expansion in both human PKD1 3D cyst assays and an inducible Pkd1 mouse model. In summary, we report a novel interaction between PC1 and ARHGAP35 in the regulation of centrosomal RhoA activation and ROCK signalling. Targeting the RhoA/ROCK pathway inhibited cyst formation in vitro and in vivo indicating its relevance to ADPKD pathogenesis and for developing new therapies to inhibit cyst initiation.
Andrew J. Streets, Philipp P. Prosseda, Albert C.M. Ong
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