Nephrology
Abstract
Alport syndrome is a rare hereditary renal disorder with no etiologic therapy. We found that osteopontin (OPN) is highly expressed in the renal tubules of the Alport mouse and plays a causative pathological role. OPN genetic deletion ameliorated albuminuria, hypertension, tubulointerstitial proliferation, renal apoptosis, and hearing and visual deficits in the Alport mouse. In Alport renal tubules we found extensive cholesterol accumulation and increased protein expression of dynamin-3 (DNM3) and LDL receptor (LDLR) in addition to dysmorphic mitochondria with defective bioenergetics. Increased pathological cholesterol influx was confirmed by a remarkably increased uptake of injected DiI-LDL cholesterol by Alport renal tubules, and by the improved lifespan of the Alport mice when crossed with the Ldlr–/– mice with defective cholesterol influx. Moreover, OPN-deficient Alport mice demonstrated significant reduction of DNM3 and LDLR expression. In human renal epithelial cells, overexpressing DNM3 resulted in elevated LDLR protein expression and defective mitochondrial respiration. Our results suggest a potentially new pathway in Alport pathology where tubular OPN causes DNM3- and LDLR-mediated enhanced cholesterol influx and impaired mitochondrial respiration.
Authors
Wen Ding, Keyvan Yousefi, Stefania Goncalves, Bradley J. Goldstein, Alfonso L. Sabater, Amy Kloosterboer, Portia Ritter, Guerline Lambert, Armando J. Mendez, Lina A. Shehadeh
Abstract
Autosomal dominant polycystic kidney disease (ADPKD) can be caused by mutations in the PKD1 or PKD2 genes. The PKD1 gene product is a Wnt cell-surface receptor. We previously showed that a lack of the PKD2 gene product, PC2, increases β-catenin signaling in mouse embryonic fibroblasts, kidney renal epithelia, and isolated renal collecting duct cells. However, it remains unclear whether β-catenin signaling plays a role in polycystic kidney disease phenotypes or if a Wnt inhibitor can halt cyst formation in ADPKD disease models. Here, using genetic and pharmacologic approaches, we demonstrated that the elevated β-catenin signaling caused by PC2 deficiency contributes significantly to disease phenotypes in a mouse ortholog of human ADPKD. Pharmacologically inhibiting β-catenin stability or the production of mature Wnt protein, or genetically reducing the expression of Ctnnb1 (which encodes β-catenin), suppressed the formation of renal cysts, improved renal function, and extended survival in ADPKD mice. Our study clearly demonstrates the importance of β-catenin signaling in disease phenotypes associated with Pkd2 mutation. It also describes the effects of two Wnt inhibitors, XAV939 and LGK974, on various Wnt signaling targets as a potential therapeutic modality for ADPKD, for which there is currently no effective therapy.
Authors
Ao Li, Yuchen Xu, Song Fan, Jialin Meng, Xufeng Shen, Qian Xiao, Yuan Li, Li Zhang, Xiansheng Zhang, Guanqing Wu, Chaozhao Liang, Dianqing Wu
Abstract
Progressive chronic kidney diseases (CKDs) are on the rise worldwide. However, the sequence of events resulting in CKD progression remain poorly understood. Animal models of CKD exploring these issues are confounded by systemic toxicities or surgical interventions to acutely induce kidney injury. Here we report the generation of a CKD mouse model through the inducible podocyte-specific ablation of an essential endogenous molecule, the chromatin structure regulator CCCTC-binding factor (CTCF), which leads to rapid podocyte loss (iCTCFpod–/–). As a consequence, iCTCFpod–/– mice develop severe progressive albuminuria, hyperlipidemia, hypoalbuminemia, and impairment of renal function, and die within 8–10 weeks. CKD progression in iCTCFpod–/– mice leads to high serum phosphate and elevations in fibroblast growth factor 23 (FGF23) and parathyroid hormone that rapidly cause bone mineralization defects, increased bone resorption, and bone loss. Dissection of the timeline leading to glomerular pathology in this CKD model led to the surprising observation that podocyte ablation and the resulting glomerular filter destruction is sufficient to drive progressive CKD and osteodystrophy in the absence of interstitial fibrosis. This work introduces an animal model with significant advantages for the study of CKD progression, and it highlights the need for podocyte-protective strategies for future kidney therapeutics.
Authors
Marta Christov, Abbe R. Clark, Braden Corbin, Samy Hakroush, Eugene P. Rhee, Hiroaki Saito, Dan Brooks, Eric Hesse, Mary Bouxsein, Niels Galjart, Ji Yong Jung, Peter Mundel, Harald Jüppner, Astrid Weins, Anna Greka
Abstract
Renal fibrosis is a common pathogenic response to injury in chronic kidney disease (CKD). The receptor-interacting protein kinase-3 (RIPK3), a regulator of necroptosis, has been implicated in disease pathogenesis. In mice subjected to unilateral ureteral obstruction–induced (UUO-induced) or adenine diet–induced (AD-induced) renal fibrosis, models of progressive kidney fibrosis, we demonstrate increased kidney expression of RIPK3. Mice genetically deficient in RIPK3 displayed decreased kidney fibrosis and improved kidney function relative to WT mice when challenged with UUO or AD. In contrast, mice genetically deficient in mixed-lineage kinase domain-like protein (MLKL), a downstream RIPK3 target, were not protected from UUO-induced kidney fibrosis. We demonstrate a pathway by which RIPK3 promotes fibrogenesis through the AKT-dependent activation of ATP citrate lyase (ACL). Genetic or chemical inhibition of RIPK3 suppressed the phosphorylation of AKT and ACL in response to TGF-β1 in fibroblasts. Inhibition of AKT or ACL suppressed TGF-β1–dependent extracellular matrix production and myofibroblast differentiation in fibroblasts. Pharmacological inhibition of ACL suppressed UUO-induced kidney fibrosis. RIPK3 expression was highly regulated in human CKD kidney. In conclusion, we identify a pathway by which RIPK3 promotes kidney fibrosis independently of MLKL-dependent necroptosis as a promising therapeutic target in CKD.
Authors
Mitsuru Imamura, Jong-Seok Moon, Kuei-Pin Chung, Kiichi Nakahira, Thangamani Muthukumar, Roman Shingarev, Stefan W. Ryter, Augustine M.K. Choi, Mary E. Choi
Abstract
BACKGROUND. In type 1 diabetes (T1D), adjuvant treatment with inhibitors of the renin-angiotensin-aldosterone system (RAAS), which dilate the efferent arteriole, is associated with prevention of progressive albuminuria and renal dysfunction. Uncertainty still exists as to why some individuals with long-standing T1D develop diabetic kidney disease (DKD) while others do not (DKD resistors). We hypothesized that those with DKD would be distinguished from DKD resistors by the presence of RAAS activation. METHODS. Renal and systemic hemodynamic function was measured before and after exogenous RAAS stimulation by intravenous infusion of angiotensin II (ANGII) in 75 patients with prolonged T1D durations and in equal numbers of nondiabetic controls. The primary outcome was change in renal vascular resistance (RVR) in response to RAAS stimulation, a measure of endogenous RAAS activation. RESULTS. Those with DKD had less change in RVR following exogenous RAAS stimulation compared with DKD resistors or controls (19%, 29%, 31%, P = 0.008, DKD vs. DKD resistors), reflecting exaggerated endogenous renal RAAS activation. All T1D participants had similar changes in renal efferent arteroilar resistance (9% vs. 13%, P = 0.37) irrespective of DKD status, which reflected less change versus controls (20%, P = 0.03). In contrast, those with DKD exhibited comparatively less change in afferent arteriolar vascular resistance compared with DKD resistors or controls (33%, 48%, 48%, P = 0.031, DKD vs. DKD resistors), indicating higher endogenous RAAS activity. CONCLUSION. In long-standing T1D, the intrarenal RAAS is exaggerated in DKD, which unexpectedly predominates at the afferent rather than the efferent arteriole, stimulating vasoconstriction. FUNDING. JDRF operating grant 17-2013-312.
Authors
Julie A. Lovshin, Geneviève Boulet, Yuliya Lytvyn, Leif E. Lovblom, Petter Bjornstad, Mohammed A. Farooqi, Vesta Lai, Leslie Cham, Josephine Tse, Andrej Orszag, Daniel Scarr, Alanna Weisman, Hillary A. Keenan, Michael H. Brent, Narinder Paul, Vera Bril, Bruce A. Perkins, David Z.I. Cherney
Abstract
Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) cause the disease familial hyperkalemic hypertension (FHHt). In the kidney, mutant CUL3 (CUL3-Δ9) increases abundance of With-No-Lysine [K] Kinase 4 (WNK4), with excessive activation of the downstream Sterile 20 (STE20)/SPS-1–related proline/alanine-rich kinase (SPAK) increasing phosphorylation of the Na+-Cl– cotransporter (NCC). CUL3-Δ9 promotes its own degradation via autoubiquitination, leading to the hypothesis that Cul3 haploinsufficiency causes FHHt. To directly test this, we generated Cul3 heterozygous mice (CUL3-Het), and Cul3 heterozygotes also expressing CUL3-Δ9 (CUL3-Het/Δ9), using an inducible renal epithelial–specific system. Endogenous CUL3 was reduced to 50% in both models, and consistent with autoubiquitination, CUL3-Δ9 protein was undetectable in CUL3-Het/Δ9 kidneys unless primary renal epithelia cells were cultured. Abundances of WNK4 and phosphorylated NCC did not differ between control and CUL3-Het mice, but they were elevated in CUL3-Het/Δ9 mice, which also displayed higher plasma [K+] and blood pressure. Abundance of phosphorylated Na+-K+-2Cl– cotransporter (NKCC2) was also increased, which may contribute to the severity of CUL3-Δ9–mediated FHHt. WNK4 and SPAK localized to puncta in NCC-positive segments but not in NKCC2-positive segments, suggesting differential effects of CUL3-Δ9. These results indicate that Cul3 haploinsufficiency does not cause FHHt, but dominant effects of CUL3-Δ9 are required.
Authors
Mohammed Z. Ferdaus, Lauren N. Miller, Larry N. Agbor, Turgay Saritas, Jeffrey D. Singer, Curt D. Sigmund, James A. McCormick
Abstract
C5a receptor 1 (C5aR1) is a G protein–coupled receptor for C5a and also an N-linked glycosylated protein. In addition to myeloid cells, C5aR1 is expressed on epithelial cells. In this study, we examined the role of C5aR1 in bacterial adhesion/colonization of renal tubular epithelium and addressed the underlying mechanisms of this role. We show that acute kidney infection was significantly reduced in mice with genetic deletion or through pharmacologic inhibition of C5aR1 following bladder inoculation with uropathogenic E. coli (UPEC). This was associated with reduced expression of terminal α-mannosyl residues (Man; a ligand for type 1 fimbriae of E. coli) on the luminal surface of renal tubular epithelium and reduction of early UPEC colonization in these mice. Confocal microscopy demonstrated that UPEC bind to Man on the luminal surface of renal tubular epithelium. In vitro analyses showed that C5a stimulation enhances Man expression in renal tubular epithelial cells and subsequent bacterial adhesion, which, at least in part, is dependent on TNF-α driven by C5aR1-mediated intracellular signaling. Our findings demonstrate a previously unknown pathogenic role for C5aR1 in acute pyelonephritis, proposing a potentially novel mechanism by which C5a/C5aR1 signaling mediates upregulation of carbohydrate ligands on renal tubules to facilitate UPEC adhesion.
Authors
Ke Li, Kun-Yi Wu, Weiju Wu, Na Wang, Ting Zhang, Naheed Choudhry, Yun Song, Conrad A. Farrar, Liang Ma, Lin-lin Wei, Zhao-Yang Duan, Xia Dong, En-Qi Liu, Zong-Fang Li, Steven H. Sacks, Wuding Zhou
Abstract
ER stress has emerged as a signaling platform underlying the pathogenesis of various kidney diseases. Thus, there is an urgent need to develop ER stress biomarkers in the incipient stages of ER stress–mediated kidney disease, when a kidney biopsy is not yet clinically indicated, for early therapeutic intervention. Cysteine-rich with EGF-like domains 2 (CRELD2) is a newly identified protein that is induced and secreted under ER stress. For the first time to our knowledge, we demonstrate that CRELD2 can serve as a sensitive urinary biomarker for detecting ER stress in podocytes or renal tubular cells in murine models of podocyte ER stress–induced nephrotic syndrome and tunicamycin- or ischemia-reperfusion–induced acute kidney injury (AKI), respectively. Most importantly, urinary CRELD2 elevation occurs in patients with autosomal dominant tubulointerstitial kidney disease caused by UMOD mutations, a prototypical tubular ER stress disease. In addition, in pediatric patients undergoing cardiac surgery, detectable urine levels of CRELD2 within postoperative 6 hours strongly associate with severe AKI after surgery. In conclusion, our study has identified CRELD2 as a potentially novel urinary ER stress biomarker with potential utility in early diagnosis, risk stratification, treatment response monitoring, and directing of ER-targeted therapies in selected patient subgroups in the emerging era of precision nephrology.
Authors
Yeawon Kim, Sun-Ji Park, Scott R. Manson, Carlos A.F. Molina, Kendrah Kidd, Heather Thiessen-Philbrook, Rebecca J. Perry, Helen Liapis, Stanislav Kmoch, Chirag R. Parikh, Anthony J. Bleyer, Ying Maggie Chen
Abstract
BACKGROUND. Systemic inflammation and muscle wasting are highly prevalent and coexist in patients on maintenance hemodialysis (MHD). We aimed to determine the effects of systemic inflammation on skeletal muscle protein metabolism in MHD patients. METHODS. Whole body and skeletal muscle protein turnover were assessed by stable isotope kinetic studies. We incorporated expressions of E1, E214K, E3αI, E3αII, MuRF-1, and atrogin-1 in skeletal muscle tissue from integrin β1 gene KO CKD mice models. RESULTS. Among 129 patients with mean (± SD) age 47 ± 12 years, 74% were African American, 73% were male, and 22% had diabetes mellitus. Median high-sensitivity C-reactive protein (hs-CRP) concentration was 13 (interquartile range 0.8, 33) mg/l. There were statistically significant associations between hs-CRP and forearm skeletal muscle protein synthesis, degradation, and net forearm skeletal muscle protein balance (P < 0.001 for all). The associations remained statistically significant after adjustment for clinical and demographic confounders, as well as in sensitivity analysis, excluding patients with diabetes mellitus. In attempting to identify potential mechanisms involved in this correlation, we show increased expressions of E1, E214K, E3αI, E3αII, MuRF-1, and atrogin-1 in skeletal muscle tissue obtained from an animal model of chronic kidney disease. CONCLUSION. These data suggest that systemic inflammation is a strong and independent determinant of skeletal muscle protein homeostasis in MHD patients, providing rationale for further studies using anticytokine therapies in patients with underlying systemic inflammation. FUNDING. This study was in part supported by NIH grants R01 DK45604 and 1K24 DK62849, the Clinical Translational Science Award UL1-TR000445 from the National Center for Advancing Translational Sciences, the Veterans Administration Merit Award I01 CX000414, the SatelliteHealth Normon Coplon Extramural Grant Program, and the FDA grant 000943.
Authors
Serpil M. Deger, Adriana M. Hung, Jorge L. Gamboa, Edward D. Siew, Charles D. Ellis, Cindy Booker, Feng Sha, Haiming Li, Aihua Bian, Thomas G. Stewart, Roy Zent, William E. Mitch, Naji N. Abumrad, T. Alp Ikizler
Abstract
Environmental exposures pose a significant threat to human health. However, it is often difficult to study toxicological mechanisms in human subjects due to ethical concerns. Plant-derived aristolochic acids are among the most potent nephrotoxins and carcinogens discovered to date, yet the mechanism of bioactivation in humans remains poorly understood. Microphysiological systems (organs-on-chips) provide an approach to examining the complex, species-specific toxicological effects of pharmaceutical and environmental chemicals using human cells. We microfluidically linked a kidney-on-a-chip with a liver-on-a-chip to determine the mechanisms of bioactivation and transport of aristolochic acid I (AA-I), an established nephrotoxin and human carcinogen. We demonstrate that human hepatocyte-specific metabolism of AA-I substantially increases its cytotoxicity toward human kidney proximal tubular epithelial cells, including formation of aristolactam adducts and release of kidney injury biomarkers. Hepatic biotransformation of AA-I to a nephrotoxic metabolite involves nitroreduction, followed by sulfate conjugation. Here, we identify, in a human tissue-based system, that the sulfate conjugate of the hepatic NQO1-generated aristolactam product of AA-I (AL-I-NOSO3) is the nephrotoxic form of AA-I. This conjugate can be transported out of liver via MRP membrane transporters and then actively transported into kidney tissue via one or more organic anionic membrane transporters. This integrated microphysiological system provides an ex vivo approach for investigating organ-organ interactions, whereby the metabolism of a drug or other xenobiotic by one tissue may influence its toxicity toward another, and represents an experimental approach for studying chemical toxicity related to environmental and other toxic exposures.
Authors
Shih-Yu Chang, Elijah J. Weber, Viktoriya S. Sidorenko, Alenka Chapron, Catherine K. Yeung, Chunying Gao, Qingcheng Mao, Danny Shen, Joanne Wang, Thomas A. Rosenquist, Kathleen G. Dickman, Thomas Neumann, Arthur P. Grollman, Edward J. Kelly, Jonathan Himmelfarb, David L. Eaton
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