Gq signaling causes glomerular injury by activating TRPC6

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Abstract

Familial forms of focal segmental glomerulosclerosis (FSGS) have been linked to gain-of-function mutations in the gene encoding the transient receptor potential channel C6 (TRPC6). GPCRs coupled to Gq signaling activate TRPC6, suggesting that Gq-dependent TRPC6 activation underlies glomerular diseases. Here, we developed a murine model in which a constitutively active Gq α subunit (Gq^Q209L, referred to herein as GqQ>L) is specifically expressed in podocytes and examined the effects of this mutation in response to puromycin aminonucleoside (PAN) nephrosis. We found that compared with control animals, animals expressing GqQ>L exhibited robust albuminuria, structural features of FSGS, and reduced numbers of glomerular podocytes. Gq activation stimulated calcineurin (CN) activity, resulting in CN-dependent upregulation of TRPC6 in murine kidneys. Deletion of TRPC6 in GqQ>L-expressing mice prevented FSGS development and inhibited both tubular damage and podocyte loss induced by PAN nephrosis. Similarly, administration of the CN inhibitor FK506 reduced proteinuria and tubular injury but had more modest effects on glomerular pathology and podocyte numbers in animals with constitutive Gq activation. Moreover, these Gq-dependent effects on podocyte injury were generalizable to diabetic kidney disease, as expression of GqQ>L promoted albuminuria, mesangial expansion, and increased glomerular basement membrane width in diabetic mice. Together, these results suggest that targeting Gq/TRPC6 signaling may have therapeutic benefits for the treatment of glomerular diseases.

Authors

Liming Wang, Grant Jirka, Paul B. Rosenberg, Anne F. Buckley, Jose A. Gomez, Timothy A. Fields, Michelle P. Winn, Robert F. Spurney

KIM-1–mediated phagocytosis reduces acute injury to the kidney

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Abstract

Kidney injury molecule 1 (KIM-1, also known as TIM-1) is markedly upregulated in the proximal tubule after injury and is maladaptive when chronically expressed. Here, we determined that early in the injury process, however, KIM-1 expression is antiinflammatory due to its mediation of phagocytic processes in tubule cells. Using various models of acute kidney injury (AKI) and mice expressing mutant forms of KIM-1, we demonstrated a mucin domain–dependent protective effect of epithelial KIM-1 expression that involves downregulation of innate immunity. Deletion of the mucin domain markedly impaired KIM-1–mediated phagocytic function, resulting in increased proinflammatory cytokine production, decreased antiinflammatory growth factor secretion by proximal epithelial cells, and a subsequent increase in tissue macrophages. Mice expressing KIM-1^Δmucin had greater functional impairment, inflammatory responses, and mortality in response to ischemia- and cisplatin-induced AKI. Compared with primary renal proximal tubule cells isolated from KIM-1^Δmucin mice, those from WT mice had reduced proinflammatory cytokine secretion and impaired macrophage activation. The antiinflammatory effect of KIM-1 expression was due to the interaction of KIM-1 with p85 and subsequent PI3K-dependent downmodulation of NF-κB. Hence, KIM-1–mediated epithelial cell phagocytosis of apoptotic cells protects the kidney after acute injury by downregulating innate immunity and inflammation.

Authors

Li Yang, Craig R. Brooks, Sheng Xiao, Venkata Sabbisetti, Melissa Y. Yeung, Li-Li Hsiao, Takaharu Ichimura, Vijay Kuchroo, Joseph V. Bonventre

Lymphocyte adaptor protein LNK deficiency exacerbates hypertension and end-organ inflammation

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Abstract

The lymphocyte adaptor protein LNK (also known as SH2B3) is primarily expressed in hematopoietic and endothelial cells, where it functions as a negative regulator of cytokine signaling and cell proliferation. Single-nucleotide polymorphisms in the gene encoding LNK are associated with autoimmune and cardiovascular disorders; however, it is not known how LNK contributes to hypertension. Here, we determined that loss of LNK exacerbates angiotensin II–induced (Ang II–induced) hypertension and the associated renal and vascular dysfunction. At baseline, kidneys from Lnk^–/– mice exhibited greater levels of inflammation, oxidative stress, and glomerular injury compared with WT animals, and these parameters were further exacerbated by Ang II infusion. Aortas from Lnk^–/– mice exhibited enhanced inflammation, reduced nitric oxide levels, and impaired endothelial-dependent relaxation. Bone marrow transplantation studies demonstrated that loss of LNK in hematopoietic cells is primarily responsible for the observed renal and vascular inflammation and predisposition to hypertension. Ang II infusion increased IFN-γ–producing CD8⁺ T cells in the spleen and kidneys of Lnk^–/– mice compared with WT mice. Moreover, IFN-γ deficiency resulted in blunted hypertension in response to Ang II infusion. Together, these results suggest that LNK is a potential therapeutic target for hypertension and its associated renal and vascular sequela.

Authors

Mohamed A. Saleh, William G. McMaster, Jing Wu, Allison E. Norlander, Samuel A. Funt, Salim R. Thabet, Annet Kirabo, Liang Xiao, Wei Chen, Hana A. Itani, Danielle Michell, Tianxiao Huan, Yahua Zhang, Satoshi Takaki, Jens Titze, Daniel Levy, David G. Harrison, Meena S. Madhur

Sirtuin 3–dependent mitochondrial dynamic improvements protect against acute kidney injury

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Abstract

Acute kidney injury (AKI) is a public health concern with an annual mortality rate that exceeds those of breast and prostate cancer, heart failure, and diabetes combined. Oxidative stress and mitochondrial damage are drivers of AKI-associated pathology; however, the pathways that mediate these events are poorly defined. Here, using a murine cisplatin-induced AKI model, we determined that both oxidative stress and mitochondrial damage are associated with reduced levels of renal sirtuin 3 (SIRT3). Treatment with the AMPK agonist AICAR or the antioxidant agent acetyl-l-carnitine (ALCAR) restored SIRT3 expression and activity, improved renal function, and decreased tubular injury in WT animals, but had no effect in Sirt3^–/– mice. Moreover, Sirt3-deficient mice given cisplatin experienced more severe AKI than WT animals and died, and neither AICAR nor ALCAR treatment prevented death in Sirt3^–/– AKI mice. In cultured human tubular cells, cisplatin reduced SIRT3, resulting in mitochondrial fragmentation, while restoration of SIRT3 with AICAR and ALCAR improved cisplatin-induced mitochondrial dysfunction. Together, our results indicate that SIRT3 is protective against AKI and suggest that enhancing SIRT3 to improve mitochondrial dynamics has potential as a strategy for improving outcomes of renal injury.

Authors

Marina Morigi, Luca Perico, Cinzia Rota, Lorena Longaretti, Sara Conti, Daniela Rottoli, Rubina Novelli, Giuseppe Remuzzi, Ariela Benigni

Intronic locus determines SHROOM3 expression and potentiates renal allograft fibrosis

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Abstract

Fibrosis underlies the loss of renal function in patients with chronic kidney disease (CKD) and in kidney transplant recipients with chronic allograft nephropathy (CAN). Here, we studied the effect of an intronic SNP in SHROOM3, which has previously been linked to CKD, on the development of CAN in a prospective cohort of renal allograft recipients. The presence of the rs17319721 allele at the SHROOM3 locus in the donor correlated with increased SHROOM3 expression in the allograft. In vitro, we determined that the sequence containing the risk allele at rs17319721 is a transcription factor 7–like 2–dependent (TCF7L2-dependent) enhancer element that functions to increase SHROOM3 transcription. In renal tubular cells, TGF-β1 administration upregulated SHROOM3 expression in a β-catenin/TCF7L2–mediated manner, while SHROOM3 in turn facilitated canonical TGF-β1 signaling and increased α1 collagen (COL1A1) expression. Inducible and tubular cell–specific knockdown of Shroom3 markedly abrogated interstitial fibrosis in mice with unilateral ureteric obstruction. Moreover, SHROOM3 expression in allografts at 3 months after transplant and the presence of the SHROOM3 risk allele in the donor correlated with increased allograft fibrosis and with reduced estimated glomerular filtration rate at 12 months after transplant. Our findings suggest that rs17319721 functions as a cis-acting expression quantitative trait locus of SHROOM3 that facilitates TGF-β1 signaling and contributes to allograft injury.

Authors

Madhav C. Menon, Peter Y. Chuang, Zhengzhe Li, Chengguo Wei, Weijia Zhang, Yi Luan, Zhengzi Yi, Huabao Xiong, Christopher Woytovich, Ilana Greene, Jessica Overbey, Ivy Rosales, Emilia Bagiella, Rong Chen, Meng Ma, Li Li, Wei Ding, Arjang Djamali, Millagros Saminego, Philip J. O’Connell, Lorenzo Gallon, Robert Colvin, Bernd Schroppel, John Cijiang He, Barbara Murphy

mTORC2 regulates renal tubule sodium uptake by promoting ENaC activity

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Abstract

The epithelial Na⁺ channel (ENaC) is essential for Na⁺ homeostasis, and dysregulation of this channel underlies many forms of hypertension. Recent studies suggest that mTOR regulates phosphorylation and activation of serum/glucocorticoid regulated kinase 1 (SGK1), which is known to inhibit ENaC internalization and degradation; however, it is not clear whether mTOR contributes to the regulation of renal tubule ion transport. Here, we evaluated the effect of selective mTOR inhibitors on kidney tubule Na⁺ and K⁺ transport in WT and Sgk1^–/– mice, as well as in isolated collecting tubules. We found that 2 structurally distinct competitive inhibitors (PP242 and AZD8055), both of which prevent all mTOR-dependent phosphorylation, including that of SGK1, caused substantial natriuresis, but not kaliuresis, in WT mice, which indicates that mTOR preferentially influences ENaC function. PP242 also substantially inhibited Na⁺ currents in isolated perfused cortical collecting tubules. Accordingly, patch clamp studies on cortical tubule apical membranes revealed that mTOR inhibition markedly reduces ENaC activity, but does not alter activity of K⁺ inwardly rectifying channels (ROMK channels). Together, these results demonstrate that mTOR regulates kidney tubule ion handling and suggest that mTOR regulates Na⁺ homeostasis through SGK1-dependent modulation of ENaC activity.

Authors

Catherine E. Gleason, Gustavo Frindt, Chih-Jen Cheng, Michael Ng, Atif Kidwai, Florian Lang, Michel Baum, Lawrence G. Palmer, David Pearce

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