The morphology of healthy podocyte foot processes is necessary for maintaining the characteristics of the kidney filtration barrier. In most forms of glomerular disease, abnormal filter barrier function results when podocytes undergo foot process spreading and retraction by remodeling their cytoskeletal architecture and intercellular junctions during a process known as effacement. The cell adhesion protein nephrin is necessary for establishing the morphology of the kidney podocyte in development by transducing from the specialized podocyte intercellular junction phosphorylation-mediated signals that regulate cytoskeletal dynamics. The present studies extend our understanding of nephrin function by showing that nephrin activation in cultured podocytes induced actin dynamics necessary for lamellipodial protrusion. This process required a PI3K-, Cas-, and Crk1/2-dependent signaling mechanism distinct from the previously described nephrin-Nck1/2 pathway necessary for assembly and polymerization of actin filaments. Our present findings also support the hypothesis that mechanisms governing lamellipodial protrusion in culture are similar to those used in vivo during foot process effacement in a subset of glomerular diseases. In mice, podocyte-specific deletion of Crk1/2 prevented foot process effacement in one model of podocyte injury and attenuated foot process effacement and associated proteinuria in a delayed fashion in a second model. In humans, focal adhesion kinase and Cas phosphorylation — markers of focal adhesion complex–mediated Crk-dependent signaling — was induced in minimal change disease and membranous nephropathy, but not focal segmental glomerulosclerosis. Together, these observations suggest that activation of a Cas-Crk1/2–dependent complex is necessary for foot process effacement observed in distinct subsets of human glomerular diseases.
Britta George, Rakesh Verma, Abdulsalam A. Soofi, Puneet Garg, Jidong Zhang, Tae-Ju Park, Laura Giardino, Larisa Ryzhova, Duncan B. Johnstone, Hetty Wong, Deepak Nihalani, David J. Salant, Steven K. Hanks, Tom Curran, Maria Pia Rastaldi, Lawrence B. Holzman
Hemolytic uremic syndrome (HUS) is a potentially life-threatening condition. It often occurs after gastrointestinal infection with E. coli O157:H7, which produces Shiga toxins (Stx) that cause hemolytic anemia, thrombocytopenia, and renal injury. Stx-mediated changes in endothelial phenotype have been linked to the pathogenesis of HUS. Here we report our studies investigating Stx-induced changes in gene expression and their contribution to the pathogenesis of HUS. Stx function by inactivating host ribosomes but can also alter gene expression at concentrations that minimally affect global protein synthesis. Gene expression profiling of human microvascular endothelium treated with Stx implicated a role for activation of CXCR4 and CXCR7 by their shared cognate chemokine ligand (stromal cell–derived factor-1 [SDF-1]) in Stx-mediated pathophysiology. The changes in gene expression required a catalytically active Stx A subunit and were mediated by enhanced transcription and mRNA stability. Stx also enhanced the association of CXCR4, CXCR7, and SDF1 mRNAs with ribosomes. In a mouse model of Stx-mediated pathology, we noted changes in plasma and tissue content of CXCR4, CXCR7, and SDF-1 after Stx exposure. Furthermore, inhibition of the CXCR4/SDF-1 interaction decreased endothelial activation and organ injury and improved animal survival. Finally, in children infected with E. coli O157:H7, plasma SDF-1 levels were elevated in individuals who progressed to HUS. Collectively, these data implicate the CXCR4/CXCR7/SDF-1 pathway in Stx-mediated pathogenesis and suggest novel therapeutic strategies for prevention and/or treatment of complications associated with E. coli O157:H7 infection.
Tania N. Petruzziello-Pellegrini, Darren A. Yuen, Andrea V. Page, Sajedabanu Patel, Anna M. Soltyk, Charles C. Matouk, Dennis K. Wong, Paul J. Turgeon, Jason E. Fish, J.J. David Ho, Brent M. Steer, Vahid Khajoee, Jayesh Tigdi, Warren L. Lee, David G. Motto, Andrew Advani, Richard E. Gilbert, S. Ananth Karumanchi, Lisa A. Robinson, Phillip I. Tarr, W. Conrad Liles, James L. Brunton, Philip A. Marsden
Podocytes of the kidney adhere tightly to the underlying glomerular basement membrane (GBM) in order to maintain a functional filtration barrier. The clinical importance of podocyte binding to the GBM via an integrin-laminin-actin axis has been illustrated in models with altered function of α3β1 integrin, integrin-linked kinase, laminin-521, and α-actinin 4. Here we expanded on the podocyte-GBM binding model by showing that the main podocyte adhesion receptor, integrin α3β1, interacts with the tetraspanin CD151 in situ in humans. Deletion of Cd151 in mouse glomerular epithelial cells led to reduced adhesive strength to laminin by redistributing α3β1 at the cell-matrix interface. Moreover, in vivo podocyte-specific deletion of Cd151 led to glomerular nephropathy. Although global Cd151-null B6 mice were not susceptible to renal disease, as has been shown previously, increasing blood and transcapillary filtration pressure induced nephropathy in these mice. Importantly, blocking the angiotensin-converting enzyme in renal disease–susceptible global Cd151-null FVB mice prolonged their median life span. Together, these results establish CD151 as a crucial modifier of integrin-mediated adhesion of podocytes to the GBM and show that blood pressure is an important factor in the initiation and progression of Cd151 knockout–induced nephropathy.
Norman Sachs, Nike Claessen, Jan Aten, Maaike Kreft, Gwendoline J.D. Teske, Anneke Koeman, Coert J. Zuurbier, Hans Janssen, Arnoud Sonnenberg
Chronic kidney disease (CKD) is a public health epidemic that increases risk of death due to cardiovascular disease. Left ventricular hypertrophy (LVH) is an important mechanism of cardiovascular disease in individuals with CKD. Elevated levels of FGF23 have been linked to greater risks of LVH and mortality in patients with CKD, but whether these risks represent causal effects of FGF23 is unknown. Here, we report that elevated FGF23 levels are independently associated with LVH in a large, racially diverse CKD cohort. FGF23 caused pathological hypertrophy of isolated rat cardiomyocytes via FGF receptor–dependent activation of the calcineurin-NFAT signaling pathway, but this effect was independent of klotho, the coreceptor for FGF23 in the kidney and parathyroid glands. Intramyocardial or intravenous injection of FGF23 in wild-type mice resulted in LVH, and klotho-deficient mice demonstrated elevated FGF23 levels and LVH. In an established animal model of CKD, treatment with an FGF–receptor blocker attenuated LVH, although no change in blood pressure was observed. These results unveil a klotho-independent, causal role for FGF23 in the pathogenesis of LVH and suggest that chronically elevated FGF23 levels contribute directly to high rates of LVH and mortality in individuals with CKD.
Christian Faul, Ansel P. Amaral, Behzad Oskouei, Ming-Chang Hu, Alexis Sloan, Tamara Isakova, Orlando M. Gutiérrez, Robier Aguillon-Prada, Joy Lincoln, Joshua M. Hare, Peter Mundel, Azorides Morales, Julia Scialla, Michael Fischer, Elsayed Z. Soliman, Jing Chen, Alan S. Go, Sylvia E. Rosas, Lisa Nessel, Raymond R. Townsend, Harold I. Feldman, Martin St. John Sutton, Akinlolu Ojo, Crystal Gadegbeku, Giovana Seno Di Marco, Stefan Reuter, Dominik Kentrup, Klaus Tiemann, Marcus Brand, Joseph A. Hill, Orson W. Moe, Makoto Kuro-o, John W. Kusek, Martin G. Keane, Myles Wolf
The specialized epithelial cell of the kidney, the podocyte, has a complex actin-based cytoskeleton. Dynamic regulation of this cytoskeleton is required for efficient barrier function of the kidney. Podocytes are a useful cell type to study the control of the actin cytoskeleton in vivo, because disruption of components of the cytoskeleton results in podocyte damage, cell loss, and a prototypic injury response called focal segmental glomerulosclerosis (FSGS). Searching for actin regulatory proteins that are expressed in podocytes, we identified a RhoA-activated Rac1 GTPase-activating protein (Rac1-GAP), Arhgap24, that was upregulated in podocytes as they differentiated, both in vitro and in vivo. Increased levels of active Rac1 and Cdc42 were measured in Arhgap24 knockdown experiments, which influenced podocyte cell shape and membrane dynamics. Consistent with a role for Arhgap24 in normal podocyte functioning in vivo, sequencing of the ARHGAP24 gene in patients with FSGS identified a mutation that impaired its Rac1-GAP activity and was associated with disease in a family with FSGS. Thus, Arhgap24 contributes to the careful balancing of RhoA and Rac1 signaling in podocytes, the disruption of which may lead to kidney disease.
Shreeram Akilesh, Hani Suleiman, Haiyang Yu, M. Christine Stander, Peter Lavin, Rasheed Gbadegesin, Corinne Antignac, Martin Pollak, Jeffrey B. Kopp, Michelle P. Winn, Andrey S. Shaw
In chronic kidney disease, fibroblast dysfunction causes renal fibrosis and renal anemia. Renal fibrosis is mediated by the accumulation of myofibroblasts, whereas renal anemia is mediated by the reduced production of fibroblast-derived erythropoietin, a hormone that stimulates erythropoiesis. Despite their importance in chronic kidney disease, the origin and regulatory mechanism of fibroblasts remain unclear. Here, we have demonstrated that the majority of erythropoietin-producing fibroblasts in the healthy kidney originate from myelin protein zero–Cre (P0-Cre) lineage-labeled extrarenal cells, which enter the embryonic kidney at E13.5. In the diseased kidney, P0-Cre lineage-labeled fibroblasts, but not fibroblasts derived from injured tubular epithelial cells through epithelial-mesenchymal transition, transdifferentiated into myofibroblasts and predominantly contributed to fibrosis, with concomitant loss of erythropoietin production. We further demonstrated that attenuated erythropoietin production in transdifferentiated myofibroblasts was restored by the administration of neuroprotective agents, such as dexamethasone and neurotrophins. Moreover, the in vivo administration of tamoxifen, a selective estrogen receptor modulator, restored attenuated erythropoietin production as well as fibrosis in a mouse model of kidney fibrosis. These findings reveal the pathophysiological roles of P0-Cre lineage-labeled fibroblasts in the kidney and clarify the link between renal fibrosis and renal anemia.
Nariaki Asada, Masayuki Takase, Jin Nakamura, Akiko Oguchi, Misako Asada, Norio Suzuki, Ken-ichi Yamamura, Narihito Nagoshi, Shinsuke Shibata, Tata Nageswara Rao, Hans Joerg Fehling, Atsushi Fukatsu, Naoko Minegishi, Toru Kita, Takeshi Kimura, Hideyuki Okano, Masayuki Yamamoto, Motoko Yanagita
Kidney podocytes are highly differentiated epithelial cells that form interdigitating foot processes with bridging slit diaphragms (SDs) that regulate renal ultrafiltration. Podocyte injury results in proteinuric kidney disease, and genetic deletion of SD-associated CD2-associated protein (CD2AP) leads to progressive renal failure in mice and humans. Here, we have shown that CD2AP regulates the TGF-β1–dependent translocation of dendrin from the SD to the nucleus. Nuclear dendrin acted as a transcription factor to promote expression of cytosolic cathepsin L (CatL). CatL proteolyzed the regulatory GTPase dynamin and the actin-associated adapter synaptopodin, leading to a reorganization of the podocyte microfilament system and consequent proteinuria. CD2AP itself was proteolyzed by CatL, promoting sustained expression of the protease during podocyte injury, and in turn increasing the apoptotic susceptibility of podocytes to TGF-β1. Our study identifies CD2AP as the gatekeeper of the podocyte TGF-β response through its regulation of CatL expression and defines a molecular mechanism underlying proteinuric kidney disease.
Suma Yaddanapudi, Mehmet M. Altintas, Andreas Kistler, Isabel Fernandez, Clemens C. Möller, Changli Wei, Vasil Peev, Jan B. Flesche, Anna-Lena Forst, Jing Li, Jaakko Patrakka, Zhijie Xiao, Florian Grahammer, Mario Schiffer, Tobias Lohmüller, Thomas Reinheckel, Changkyu Gu, Tobias B. Huber, Wenjun Ju, Markus Bitzer, Maria P. Rastaldi, Phillip Ruiz, Karl Tryggvason, Andrey Shaw, Christian Faul, Sanja Sever, Jochen Reiser
Sepsis-associated acute kidney injury (AKI) is a common and morbid condition that is distinguishable from typical ischemic renal injury by its paucity of tubular cell death. The mechanisms underlying renal dysfunction in individuals with sepsis-associated AKI are therefore less clear. Here we have shown that endotoxemia reduces oxygen delivery to the kidney, without changing tissue oxygen levels, suggesting reduced oxygen consumption by the kidney cells. Tubular mitochondria were swollen, and their function was impaired. Expression profiling showed that oxidative phosphorylation genes were selectively suppressed during sepsis-associated AKI and reactivated when global function was normalized. PPARγ coactivator–1α (PGC-1α), a major regulator of mitochondrial biogenesis and metabolism, not only followed this pattern but was proportionally suppressed with the degree of renal impairment. Furthermore, tubular cells had reduced PGC-1α expression and oxygen consumption in response to TNF-α; however, excess PGC-1α reversed the latter effect. Both global and tubule-specific PGC-1α–knockout mice had normal basal renal function but suffered persistent injury following endotoxemia. Our results demonstrate what we believe to be a novel mechanism for sepsis-associated AKI and suggest that PGC-1α induction may be necessary for recovery from this disorder, identifying a potential new target for future therapeutic studies.
Mei Tran, Denise Tam, Amit Bardia, Manoj Bhasin, Glenn C. Rowe, Ajay Kher, Zsuzsanna K. Zsengeller, M. Reza Akhavan-Sharif, Eliyahu V. Khankin, Magali Saintgeniez, Sascha David, Deborah Burstein, S. Ananth Karumanchi, Isaac E. Stillman, Zoltan Arany, Samir M. Parikh
Renal tubulointerstitial damage is the final common pathway leading from chronic kidney disease to end-stage renal disease. Inflammation is clearly involved in tubulointerstitial injury, but it remains unclear how the inflammatory processes are initiated and regulated. Here, we have shown that in the mouse kidney, the transcription factor Krüppel-like factor–5 (KLF5) is mainly expressed in collecting duct epithelial cells and that Klf5 haploinsufficient mice (Klf5+/– mice) exhibit ameliorated renal injury in the unilateral ureteral obstruction (UUO) model of tubulointerstitial disease. Additionally, Klf5 haploinsufficiency reduced accumulation of CD11b+F4/80lo cells, which expressed proinflammatory cytokines and induced apoptosis among renal epithelial cells, phenotypes indicative of M1-type macrophages. By contrast, it increased accumulation of CD11b+F4/80hi macrophages, which expressed CD206 and CD301 and contributed to fibrosis, in part via TGF-β production — phenotypes indicative of M2-type macrophages. Interestingly, KLF5, in concert with C/EBPα, was found to induce expression of the chemotactic proteins S100A8 and S100A9, which recruited inflammatory monocytes to the kidneys and promoted their activation into M1-type macrophages. Finally, assessing the effects of bone marrow–specific Klf5 haploinsufficiency or collecting duct– or myeloid cell–specific Klf5 deletion confirmed that collecting duct expression of Klf5 is essential for inflammatory responses to UUO. Taken together, our results demonstrate that the renal collecting duct plays a pivotal role in the initiation and progression of tubulointerstitial inflammation.
Katsuhito Fujiu, Ichiro Manabe, Ryozo Nagai
Hypertension is a leading contributor to cardiovascular mortality worldwide. Despite this, its underlying mechanism(s) and the role of excess salt in cardiorenal dysfunction are unclear. Previously, we have identified cross-talk between mineralocorticoid receptor (MR), a nuclear transcription factor regulated by the steroid aldosterone, and the small GTPase Rac1, which is implicated in proteinuric kidney disease. We here show that high-salt loading activates Rac1 in the kidneys in rodent models of salt-sensitive hypertension, leading to blood pressure elevation and renal injury via an MR-dependent pathway. We found that a high-salt diet caused renal Rac1 upregulation in salt-sensitive Dahl (Dahl-S) rats and downregulation in salt-insensitive Dahl (Dahl-R) rats. Despite a reduction of serum aldosterone levels, salt-loaded Dahl-S rats showed increased MR signaling in the kidneys, and Rac1 inhibition prevented hypertension and renal damage with MR repression. We further demonstrated in aldosterone-infused rats as well as adrenalectomized Dahl-S rats with aldosterone supplementation that salt-induced Rac1 and aldosterone acted interdependently to cause MR overactivity and hypertension. Finally, we confirmed the key role of Rac1 in modulating salt susceptibility in mice lacking Rho GDP–dissociation inhibitor α. Therefore, our data identify Rac1 as a determinant of salt sensitivity and provide insights into the mechanism of salt-induced hypertension and kidney injury.
Shigeru Shibata, ShengYu Mu, Hiroo Kawarazaki, Kazuhiko Muraoka, Ken-ichi Ishizawa, Shigetaka Yoshida, Wakako Kawarazaki, Maki Takeuchi, Nobuhiro Ayuzawa, Jun Miyoshi, Yoshimi Takai, Akira Ishikawa, Tatsuo Shimosawa, Katsuyuki Ando, Miki Nagase, Toshiro Fujita