Anti–microRNA-21 oligonucleotides prevent Alport nephropathy progression by stimulating metabolic pathways

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Abstract

MicroRNA-21 (miR-21) contributes to the pathogenesis of fibrogenic diseases in multiple organs, including the kidneys, potentially by silencing metabolic pathways that are critical for cellular ATP generation, ROS production, and inflammatory signaling. Here, we developed highly specific oligonucleotides that distribute to the kidney and inhibit miR-21 function when administered subcutaneously and evaluated the therapeutic potential of these anti–miR-21 oligonucleotides in chronic kidney disease. In a murine model of Alport nephropathy, miR-21 silencing did not produce any adverse effects and resulted in substantially milder kidney disease, with minimal albuminuria and dysfunction, compared with vehicle-treated mice. miR-21 silencing dramatically improved survival of Alport mice and reduced histological end points, including glomerulosclerosis, interstitial fibrosis, tubular injury, and inflammation. Anti–miR-21 enhanced PPARα/retinoid X receptor (PPARα/RXR) activity and downstream signaling pathways in glomerular, tubular, and interstitial cells. Moreover, miR-21 silencing enhanced mitochondrial function, which reduced mitochondrial ROS production and thus preserved tubular functions. Inhibition of miR-21 was protective against TGF-β–induced fibrogenesis and inflammation in glomerular and interstitial cells, likely as the result of enhanced PPARα/RXR activity and improved mitochondrial function. Together, these results demonstrate that inhibition of miR-21 represents a potential therapeutic strategy for chronic kidney diseases including Alport nephropathy.

Authors

Ivan G. Gomez, Deidre A. MacKenna, Bryce G. Johnson, Vivek Kaimal, Allie M. Roach, Shuyu Ren, Naoki Nakagawa, Cuiyan Xin, Rick Newitt, Shweta Pandya, Tai-He Xia, Xueqing Liu, Dorin-Bogdan Borza, Monica Grafals, Stuart J. Shankland, Jonathan Himmelfarb, Didier Portilla, Shiguang Liu, B. Nelson Chau, Jeremy S. Duffield

Altered trafficking and stability of polycystins underlie polycystic kidney disease

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Abstract

The most severe form of autosomal dominant polycystic kidney disease occurs in patients with mutations in the gene (PKD1) encoding polycystin-1 (PC1). PC1 is a complex polytopic membrane protein expressed in cilia that undergoes autoproteolytic cleavage at a G protein–coupled receptor proteolytic site (GPS). A quarter of PKD1 mutations are missense variants, though it is not clear how these mutations promote disease. Here, we established a cell-based system to evaluate these mutations and determined that GPS cleavage is required for PC1 trafficking to cilia. A common feature among a subset of pathogenic missense mutations is a resulting failure of PC1 to traffic to cilia regardless of GPS cleavage. The application of our system also identified a missense mutation in the gene encoding polycystin-2 (PC2) that prevented this protein from properly trafficking to cilia. Using a Pkd1-BAC recombineering approach, we developed murine models to study the effects of these mutations and confirmed that only the cleaved form of PC1 exits the ER and can rescue the embryonically lethal Pkd1-null mutation. Additionally, steady-state expression levels of the intramembranous COOH-terminal fragment of cleaved PC1 required an intact interaction with PC2. The results of this study demonstrate that PC1 trafficking and expression require GPS cleavage and PC2 interaction, respectively, and provide a framework for functional assays to categorize the effects of missense mutations in polycystins.

Authors

Yiqiang Cai, Sorin V. Fedeles, Ke Dong, Georgia Anyatonwu, Tamehito Onoe, Michihiro Mitobe, Jian-Dong Gao, Dayne Okuhara, Xin Tian, Anna-Rachel Gallagher, Zhangui Tang, Xiaoli Xie, Maria D. Lalioti, Ann-Hwee Lee, Barbara E. Ehrlich, Stefan Somlo

TRPM2 mediates ischemic kidney injury and oxidant stress through RAC1

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Abstract

Ischemia is a leading cause of acute kidney injury. Kidney ischemia is associated with loss of cellular ion homeostasis; however, the pathways that underlie ion homeostasis dysfunction are poorly understood. Here, we evaluated the nonselective cation channel transient receptor potential melastatin 2 (TRPM2) in a murine model of kidney ischemia/reperfusion (I/R) injury. TRPM2-deficient mice were resistant to ischemic injury, as reflected by improved kidney function, reduced histologic damage, suppression of proapoptotic pathways, and reduced inflammation. Moreover, pharmacologic TRPM2 inhibition was also protective against I/R injury. TRPM2 was localized mainly in kidney proximal tubule epithelial cells, and studies in chimeric mice indicated that the effects of TRPM2 are due to expression in parenchymal cells rather than hematopoietic cells. TRPM2-deficient mice had less oxidative stress and lower levels of NADPH oxidase activity after ischemia. While RAC1 is a component of the NADPH oxidase complex, its relation to TRPM2 and kidney ischemic injury is unknown. Following kidney ischemia, TRPM2 promoted RAC1 activation, with active RAC1 physically interacting with TRPM2 and increasing TRPM2 expression at the cell membrane. Finally, inhibition of RAC1 reduced oxidant stress and ischemic injury in vivo. These results demonstrate that TRPM2-dependent RAC1 activation increases oxidant stress and suggest that therapeutic approaches targeting TRPM2 and/or RAC1 may be effective in reducing ischemic kidney injury.

Authors

Guofeng Gao, Weiwei Wang, Raghu K. Tadagavadi, Nicole E. Briley, Michael I. Love, Barbara A. Miller, W. Brian Reeves

Hyperkalemic hypertension–associated cullin 3 promotes WNK signaling by degrading KLHL3

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Abstract

Familial hyperkalemic hypertension (FHHt) is a monogenic disease resulting from mutations in genes encoding WNK kinases, the ubiquitin scaffold protein cullin 3 (CUL3), or the substrate adaptor kelch-like 3 (KLHL3). Disease-associated CUL3 mutations abrogate WNK kinase degradation in cells, but it is not clear how mutant forms of CUL3 promote WNK stability. Here, we demonstrated that an FHHt-causing CUL3 mutant (CUL3 Δ403–459) not only retains the ability to bind and ubiquitylate WNK kinases and KLHL3 in cells, but is also more heavily neddylated and activated than WT CUL3. In cells, activated CUL3 Δ403–459 depleted KLHL3, preventing WNK degradation, despite increased CUL3-mediated WNK ubiquitylation; therefore, CUL3 loss in kidney should phenocopy FHHt in murine models. As predicted, nephron-specific deletion of Cul3 in mice did increase WNK kinase levels and the abundance of phosphorylated Na-Cl cotransporter (NCC). Over time, however, Cul3 deletion caused renal dysfunction, including hypochloremic alkalosis, diabetes insipidus, and salt-sensitive hypotension, with depletion of sodium potassium chloride cotransporter 2 and aquaporin 2. Moreover, these animals exhibited renal inflammation, fibrosis, and increased cyclin E. These results indicate that FHHt-associated CUL3 Δ403–459 targets KLHL3 for degradation, thereby preventing WNK degradation, whereas general loss of CUL3 activity — while also impairing WNK degradation — has widespread toxic effects in the kidney.

Authors

James A. McCormick, Chao-Ling Yang, Chong Zhang, Brittney Davidge, Katharina I. Blankenstein, Andrew S. Terker, Bethzaida Yarbrough, Nicholas P. Meermeier, Hae J. Park, Belinda McCully, Mark West, Aljona Borschewski, Nina Himmerkus, Markus Bleich, Sebastian Bachmann, Kerim Mutig, Eduardo R. Argaiz, Gerardo Gamba, Jeffrey D. Singer, David H. Ellison

Integrin-mediated type II TGF-β receptor tyrosine dephosphorylation controls SMAD-dependent profibrotic signaling

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Abstract

Tubulointerstitial fibrosis underlies all forms of end-stage kidney disease. TGF-β mediates both the development and the progression of kidney fibrosis through binding and activation of the serine/threonine kinase type II TGF-β receptor (TβRII), which in turn promotes a TβRI-mediated SMAD-dependent fibrotic signaling cascade. Autophosphorylation of serine residues within TβRII is considered the principal regulatory mechanism of TβRII-induced signaling; however, there are 5 tyrosine residues within the cytoplasmic tail that could potentially mediate TβRII-dependent SMAD activation. Here, we determined that phosphorylation of tyrosines within the TβRII tail was essential for SMAD-dependent fibrotic signaling within cells of the kidney collecting duct. Conversely, the T cell protein tyrosine phosphatase (TCPTP) dephosphorylated TβRII tail tyrosine residues, resulting in inhibition of TβR-dependent fibrotic signaling. The collagen-binding receptor integrin α1β1 was required for recruitment of TCPTP to the TβRII tail, as mice lacking this integrin exhibited impaired TCPTP-mediated tyrosine dephosphorylation of TβRII that led to severe fibrosis in a unilateral ureteral obstruction model of renal fibrosis. Together, these findings uncover a crosstalk between integrin α1β1 and TβRII that is essential for TβRII-mediated SMAD activation and fibrotic signaling pathways.

Authors

Xiwu Chen, Hongtao Wang, Hong-Jun Liao, Wen Hu, Leslie Gewin, Glenda Mernaugh, Sheng Zhang, Zhong-Yin Zhang, Lorenzo Vega-Montoto, Roberto M. Vanacore, Reinhard Fässler, Roy Zent, Ambra Pozzi

KLF4-dependent epigenetic remodeling modulates podocyte phenotypes and attenuates proteinuria

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Abstract

The transcription factor Kruppel-like factor 4 (KLF4) has the ability, along with other factors, to reprogram somatic cells into induced pluripotent stem (iPS) cells. Here, we determined that KLF4 is expressed in kidney glomerular podocytes and is decreased in both animal models and humans exhibiting a proteinuric. Transient restoration of KLF4 expression in podocytes of diseased glomeruli in vivo, either by gene transfer or transgenic expression, resulted in a sustained increase in nephrin expression and a decrease in albuminuria. In mice harboring podocyte-specific deletion of Klf4, adriamycin-induced proteinuria was substantially exacerbated, although these animals displayed minimal phenotypical changes prior to adriamycin administration. KLF4 overexpression in cultured human podocytes increased expression of nephrin and other epithelial markers and reduced mesenchymal gene expression. DNA methylation profiling and bisulfite genomic sequencing revealed that KLF4 expression reduced methylation at the nephrin promoter and the promoters of other epithelial markers; however, methylation was increased at the promoters of genes encoding mesenchymal markers, suggesting selective epigenetic regulation of podocyte gene expression. Together, these results suggest that KLF4 epigenetically modulates podocyte phenotype and function and that the podocyte epigenome can be targeted for direct intervention and reduction of proteinuria.

Authors

Kaori Hayashi, Hiroyuki Sasamura, Mari Nakamura, Tatsuhiko Azegami, Hideyo Oguchi, Yusuke Sakamaki, Hiroshi Itoh

Endothelial HIF-2 mediates protection and recovery from ischemic kidney injury

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Abstract

The hypoxia-inducible transcription factors HIF-1 and HIF-2 mediate key cellular adaptions to hypoxia and contribute to renal homeostasis and pathophysiology; however, little is known about the cell type–specific functions of HIF-1 and HIF-2 in response to ischemic kidney injury. Here, we used a genetic approach to specifically dissect the roles of endothelial HIF-1 and HIF-2 in murine models of hypoxic kidney injury induced by ischemia reperfusion or ureteral obstruction. In both models, inactivation of endothelial HIF increased injury-associated renal inflammation and fibrosis. Specifically, inactivation of endothelial HIF-2α, but not endothelial HIF-1α, resulted in increased expression of renal injury markers and inflammatory cell infiltration in the postischemic kidney, which was reversed by blockade of vascular cell adhesion molecule-1 (VCAM1) and very late antigen-4 (VLA4) using monoclonal antibodies. In contrast, pharmacologic or genetic activation of HIF via HIF prolyl-hydroxylase inhibition protected wild-type animals from ischemic kidney injury and inflammation; however, these same protective effects were not observed in HIF prolyl-hydroxylase inhibitor–treated animals lacking endothelial HIF-2. Taken together, our data indicate that endothelial HIF-2 protects from hypoxia-induced renal damage and represent a potential therapeutic target for renoprotection and prevention of fibrosis following acute ischemic injury.

Authors

Pinelopi P. Kapitsinou, Hideto Sano, Mark Michael, Hanako Kobayashi, Olena Davidoff, Aihua Bian, Bing Yao, Ming-Zhi Zhang, Raymond C. Harris, Kevin J. Duffy, Connie L. Erickson-Miller, Timothy A. Sutton, Volker H. Haase

Type 1 angiotensin receptors on macrophages ameliorate IL-1 receptor–mediated kidney fibrosis

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Abstract

In a wide array of kidney diseases, type 1 angiotensin (AT1) receptors are present on the immune cells that infiltrate the renal interstitium. Here, we examined the actions of AT1 receptors on macrophages in progressive renal fibrosis and found that macrophage-specific AT1 receptor deficiency exacerbates kidney fibrosis induced by unilateral ureteral obstruction (UUO). Macrophages isolated from obstructed kidneys of mice lacking AT1 receptors solely on macrophages had heightened expression of proinflammatory M1 cytokines, including IL-1. Evaluation of isolated AT1 receptor–deficient macrophages confirmed the propensity of these cells to produce exaggerated levels of M1 cytokines, which led to more severe renal epithelial cell damage via IL-1 receptor activation in coculture compared with WT macrophages. A murine kidney crosstransplantation concomitant with UUO model revealed that augmentation of renal fibrosis instigated by AT1 receptor–deficient macrophages is mediated by IL-1 receptor stimulation in the kidney. This study indicates that a key role of AT1 receptors on macrophages is to protect the kidney from fibrosis by limiting activation of IL-1 receptors in the kidney.

Authors

Jian-dong Zhang, Mehul B. Patel, Robert Griffiths, Paul C. Dolber, Phillip Ruiz, Matthew A. Sparks, Johannes Stegbauer, Huixia Jin, Jose A. Gomez, Anne F. Buckley, William S. Lefler, Daian Chen, Steven D. Crowley

Intravital imaging of podocyte calcium in glomerular injury and disease

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Abstract

Intracellular calcium ([Ca²⁺]_i) signaling mediates physiological and pathological processes in multiple organs, including the renal podocyte; however, in vivo podocyte [Ca²⁺]_i dynamics are not fully understood. Here we developed an imaging approach that uses multiphoton microscopy (MPM) to directly visualize podocyte [Ca²⁺]_i dynamics within the intact kidneys of live mice expressing a fluorescent calcium indicator only in these cells. [Ca²⁺]_i was at a low steady-state level in control podocytes, while Ang II infusion caused a minor elevation. Experimental focal podocyte injury triggered a robust and sustained elevation of podocyte [Ca²⁺]_i around the injury site and promoted cell-to-cell propagating podocyte [Ca²⁺]_i waves along capillary loops. [Ca²⁺]_i wave propagation was ameliorated by inhibitors of purinergic [Ca²⁺]_i signaling as well as in animals lacking the P2Y2 purinergic receptor. Increased podocyte [Ca²⁺]_i resulted in contraction of the glomerular tuft and increased capillary albumin permeability. In preclinical models of renal fibrosis and glomerulosclerosis, high podocyte [Ca²⁺]_i correlated with increased cell motility. Our findings provide a visual demonstration of the in vivo importance of podocyte [Ca²⁺]_i in glomerular pathology and suggest that purinergic [Ca²⁺]_i signaling is a robust and key pathogenic mechanism in podocyte injury. This in vivo imaging approach will allow future detailed investigation of the molecular and cellular mechanisms of glomerular disease in the intact living kidney.

Authors

James L. Burford, Karie Villanueva, Lisa Lam, Anne Riquier-Brison, Matthias J. Hackl, Jeffrey Pippin, Stuart J. Shankland, János Peti-Peterdi

Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis

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Abstract

Focal segmental glomerular sclerosis (FSGS) is a primary kidney disease that is commonly associated with proteinuria and progressive loss of glomerular function, leading to development of chronic kidney disease (CKD). FSGS is characterized by podocyte injury and depletion and collapse of glomerular capillary segments. Progression of FSGS is associated with TGF-β activation in podocytes; however, it is not clear how TGF-β signaling promotes disease. Here, we determined that podocyte-specific activation of TGF-β signaling in transgenic mice and BALB/c mice with Adriamycin-induced glomerulosclerosis is associated with endothelin-1 (EDN1) release by podocytes, which mediates mitochondrial oxidative stress and dysfunction in adjacent endothelial cells via paracrine EDN1 receptor type A (EDNRA) activation. Endothelial dysfunction promoted podocyte apoptosis, and inhibition of EDNRA or scavenging of mitochondrial-targeted ROS prevented podocyte loss, albuminuria, glomerulosclerosis, and renal failure. We confirmed reciprocal crosstalk between podocytes and endothelial cells in a coculture system. Biopsies from patients with FSGS exhibited increased mitochondrial DNA damage, consistent with EDNRA-mediated glomerular endothelial mitochondrial oxidative stress. Our studies indicate that segmental glomerulosclerosis develops as a result of podocyte-endothelial crosstalk mediated by EDN1/EDNRA-dependent mitochondrial dysfunction and suggest that targeting the reciprocal interaction between podocytes and endothelia may provide opportunities for therapeutic intervention in FSGS.

Authors

Ilse Daehn, Gabriella Casalena, Taoran Zhang, Shaolin Shi, Franz Fenninger, Nicholas Barasch, Liping Yu, Vivette D’Agati, Detlef Schlondorff, Wilhelm Kriz, Borje Haraldsson, Erwin P. Bottinger