Claudin-16 (Cldn16) is selectively expressed at tight junctions (TJs) of renal epithelial cells of the thick ascending limb of Henle’s loop, where it plays a central role in the reabsorption of divalent cations. Over 20 different mutations in the CLDN16 gene have been identified in patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), a disease of excessive renal Mg2+ and Ca2+ excretion. Here we show that disease-causing mutations can lead to the intracellular retention of Cldn16 or affect its capacity to facilitate paracellular Mg2+ transport. Nine of the 21 Cldn16 mutants we characterized were retained in the endoplasmic reticulum, where they underwent proteasomal degradation. Three mutants accumulated in the Golgi complex. Two mutants were efficiently delivered to lysosomes, one via clathrin-mediated endocytosis following transport to the cell surface and the other without appearing on the plasma membrane. The remaining 7 mutants localized to TJs, and 4 were found to be defective in paracellular Mg2+ transport. We demonstrate that pharmacological chaperones rescued surface expression of several retained Cldn16 mutants. We conclude that FHHNC can result from mutations in Cldn16 that affect intracellular trafficking or paracellular Mg2+ permeability. Knowledge of the molecular defects associated with disease-causing Cldn16 mutations may open new venues for therapeutic intervention.
P. Jaya Kausalya, Salah Amasheh, Dorothee Günzel, Henrik Wurps, Dominik Müller, Michael Fromm, Walter Hunziker
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel plays vital roles in fluid transport in many epithelia. While CFTR is expressed along the entire nephron, its function in renal tubule epithelial cells remains unclear, as no specific renal phenotype has been identified in cystic fibrosis. CFTR has been proposed as a regulator of the 30 pS, ATP-sensitive renal K channel (Kir1.1, also known as renal outer medullar K [ROMK]) that is critical for K secretion by cells of the thick ascending limb (TAL) and distal nephron segments responsive to aldosterone. We report here that both ATP and glibenclamide sensitivities of the 30 pS K channel in TAL cells were absent in mice lacking CFTR and in mice homozygous for the ΔF508 mutation. Curcumin treatment in ΔF508-CFTR mice partially reversed the defect in ATP sensitivity. We demonstrate that the effect of CFTR on ATP sensitivity was abrogated by increasing PKA activity. We propose that CFTR regulates the renal K secretory channel by providing a PKA-regulated functional switch that determines the distribution of open and ATP-inhibited K channels in apical membranes. We discuss the potential physiological role of this functional switch in renal K handling during water diuresis and the relevance to renal K homeostasis in cystic fibrosis.
Ming Lu, Qiang Leng, Marie E. Egan, Michael J. Caplan, Emile L. Boulpaep, Gerhard H. Giebisch, Steven C. Hebert
Congenital malformations of the urinary tract are a major cause of renal failure in children and young adults. They are often caused by physical obstruction or by functional impairment of the peristaltic machinery of the ureter. The underlying molecular and cellular defects are, however, poorly understood. Here we present the phenotypic characterization of a new mouse model for congenital ureter malformation that revealed the molecular pathway important for the formation of the functional mesenchymal coating of the ureter. The gene encoding the T-box transcription factor Tbx18 was expressed in undifferentiated mesenchymal cells surrounding the distal ureter stalk. In Tbx18–/– mice, prospective ureteral mesenchymal cells largely dislocalized to the surface of the kidneys. The remaining ureteral mesenchymal cells showed reduced proliferation and failed to differentiate into smooth muscles, but instead became fibrous and ligamentous tissue. Absence of ureteral smooth muscles resulted in a short hydroureter and hydronephrosis at birth. Our analysis also showed that the ureteral mesenchyme derives from a distinct cell population that is separated early in kidney development from that of other mesenchymal cells of the renal system.
Rannar Airik, Markus Bussen, Manvendra K. Singh, Marianne Petry, Andreas Kispert
To investigate the function of Cx43 during hypertension, we studied the mouse line Cx43KI32 (KI32), in which the coding region of Cx32 replaces that of Cx43. Within the kidneys of homozygous KI32 mice, Cx32 was expressed in cortical and medullary tubules, as well as in some extra- and intraglomerular vessels, i.e., at sites where Cx32 and Cx43 are found in WT mice. Under such conditions, renin expression was much reduced compared with that observed in the kidneys of WT and heterozygous KI32 littermates. After exposure to a high-salt diet, all mice retained a normal blood pressure. However, whereas the levels of renin were significantly reduced in the kidneys of WT and heterozygous KI32 mice, reaching levels comparable to those observed in homozygous littermates, they were not further affected in the latter animals. Four weeks after the clipping of a renal artery (the 2-kidney, 1-clip [2K1C] model), 2K1C WT and heterozygous mice showed an increase in blood pressure and in the circulating levels of renin, whereas 2K1C homozygous littermates remained normotensive and showed unchanged plasma renin activity. Hypertensive, but not normotensive, mice also developed cardiac hypertrophy. The data indicate that replacement of Cx43 by Cx32 is associated with decreased expression and secretion of renin, thus preventing the renin-dependent hypertension that is normally induced in the 2K1C model.
Jacques-Antoine Haefliger, Nathalie Krattinger, David Martin, Thierry Pedrazzini, Alessandro Capponi, Britta Döring, Achim Plum, Anne Charollais, Klaus Willecke, Paolo Meda
Ischemia/reperfusion (I/R) of several organs results in complement activation, but the kidney is unique in that activation after I/R occurs only via the alternative pathway. We hypothesized that selective activation of this pathway after renal I/R could occur either because of a loss of complement inhibition or from increased local synthesis of complement factors. We examined the relationship between renal complement activation after I/R and the levels and localization of intrinsic membrane complement inhibitors. We found that loss of polarity of complement receptor 1–related protein y (Crry) in the tubular epithelium preceded activation of the alternative pathway along the basolateral aspect of the tubular cells. Heterozygous gene-targeted mice that expressed lower amounts of Crry were more sensitive to ischemic injury. Furthermore, inhibition of Crry expressed by proximal tubular epithelial cells in vitro resulted in alternative pathway–mediated injury to the cells. Thus, altered expression of a complement inhibitor within the tubular epithelium appears to be a critical factor permitting activation of the alternative pathway of complement after I/R. Increased C3 mRNA and decreased factor H mRNA were also detected in the outer medulla after I/R, suggesting that altered synthesis of these factors might further contribute to complement activation in this location.
Joshua M. Thurman, Danica Ljubanović, Pamela A. Royer, Damian M. Kraus, Hector Molina, Nicholas P. Barry, Gregory Proctor, Moshe Levi, V. Michael Holers
Dialysis dependency is one of the leading causes of morbidity and mortality in the world, and once end-stage renal disease develops, it cannot be reversed by currently available therapy. Although administration of large doses of bone morphogenetic protein–7 (BMP-7) has been shown to repair established renal injury and improve renal function, the pathophysiological role of endogenous BMP-7 and regulatory mechanism of its activities remain elusive. Here we show that the product of uterine sensitization-associated gene–1 (USAG1), a novel BMP antagonist abundantly expressed in the kidney, is the central negative regulator of BMP function in the kidney and that mice lacking USAG-1 (USAG1–/– mice) are resistant to renal injury. USAG1–/– mice exhibited prolonged survival and preserved renal function in acute and chronic renal injury models. Renal BMP signaling, assessed by phosphorylation of Smad proteins, was significantly enhanced in USAG1–/– mice with renal injury, indicating that the preservation of renal function is attributable to enhancement of endogenous BMP signaling. Furthermore, the administration of neutralizing antibody against BMP-7 abolished renoprotection in USAG1–/– mice, indicating that USAG-1 plays a critical role in the modulation of renoprotective action of BMP and that inhibition of USAG-1 is a promising means of development of novel treatment for renal diseases.
Motoko Yanagita, Tomohiko Okuda, Shuichiro Endo, Mari Tanaka, Katsu Takahashi, Fumihiro Sugiyama, Satoshi Kunita, Satoru Takahashi, Atsushi Fukatsu, Masashi Yanagisawa, Toru Kita, Takeshi Sakurai
Previous research on proteins that inhibit kidney stone formation has identified a relatively small number of well-characterized inhibitors. Identification of additional stone inhibitors would increase understanding of the pathogenesis and pathophysiology of nephrolithiasis. We have combined conventional biochemical methods with recent advances in mass spectrometry (MS) to identify a novel calcium oxalate (CaOx) crystal growth inhibitor in normal human urine. Anionic proteins were isolated by DEAE adsorption and separated by HiLoad 16/60 Superdex 75 gel filtration. A fraction with potent inhibitory activity against CaOx crystal growth was isolated and purified by anion exchange chromatography. The protein in 2 subfractions that retained inhibitory activity was identified by matrix-assisted laser desorption/ionization–time-of-flight MS and electrospray ionization–quadrupole–time-of-flight tandem MS as human trefoil factor 1 (TFF1). Western blot analysis confirmed the mass spectrometric protein identification. Functional studies of urinary TFF1 demonstrated that its inhibitory potency was similar to that of nephrocalcin. The inhibitory activity of urinary TFF1 was dose dependent and was inhibited by TFF1 antisera. Anti–C-terminal antibody was particularly effective, consistent with our proposed model in which the 4 C-terminal glutamic residues of TFF1 interact with calcium ions to prevent CaOx crystal growth. Concentrations and relative amounts of TFF1 in the urine of patients with idiopathic CaOx kidney stone were significantly less (2.5-fold for the concentrations and 5- to 22-fold for the relative amounts) than those found in controls. These data indicate that TFF1 is a novel potent CaOx crystal growth inhibitor with a potential pathophysiological role in nephrolithiasis.
Somchai Chutipongtanate, Yasushi Nakagawa, Suchai Sritippayawan, Jeeraporn Pittayamateekul, Paisal Parichatikanond, Bruce R. Westley, Felicity E.B. May, Prida Malasit, Visith Thongboonkerd
The pathogenesis of cachexia in patients with uremia is unknown. We tested the hypothesis that uremia-associated cachexia is caused by leptin signaling through the hypothalamic melanocortin receptor 4 (MC4-R). We performed either subtotal nephrectomy (N) or sham operations in WT, leptin receptor–deficient (db/db), and MC4-R knockout (MC4-RKO) mice. The animals were on 17% protein diets, and none of the uremic animals were acidotic. WT-N mice produced a classic syndrome of cachexia characterized by decreased food intake, increased metabolic rate, and loss of lean body mass. Corrected leptin levels were elevated. db/db mice and MC4-RKO mice resisted the cachexic effects of uremia on weight gain, body composition, and metabolic rate. Likewise, treatment of WT mice with intracranial agouti-related peptide reversed the cachexic effects of uremia on appetite, weight gain, body composition, and metabolic rate. Gene expression of ubiquitin C and proteasome subunits C2, C3, and C9 was not changed in the uremic animals, suggesting that other pathways are involved in this model of nonacidotic uremic cachexia. The results of this study suggest that elevated circulating levels of cytokines such as leptin may be an important cause of uremia-associated cachexia via signaling through the central melanocortin system.
Wai Cheung, Pin X. Yu, Brian M. Little, Roger D. Cone, Daniel L. Marks, Robert H. Mak
TNF is essential for the development of glomerulonephritis, an immune-mediated disorder that is a major cause of renal failure worldwide. However, TNF has proinflammatory and immunosuppressive properties that may segregate at the level of the 2 TNF receptors (TNFRs), TNFR1 and TNFR2. TNFR1-deficient mice subjected to immune complex–mediated glomerulonephritis developed less proteinuria and glomerular injury, and fewer renal leukocyte infiltrates at early time points after disease induction, and this was associated with a reduced systemic immune response to nephrotoxic rabbit IgG. However, proteinuria and renal pathology were similar to those in wild-type controls at later time points, when lack of TNFR1 resulted in excessive renal T cell accumulation and an associated reduction in apoptosis of these cells. In sharp contrast, TNFR2-deficient mice were completely protected from glomerulonephritis at all time points, despite an intact systemic immune response. TNFR2 was induced on glomerular endothelial cells of nephritic kidneys, and TNFR2 expression on intrinsic cells, but not leukocytes, was essential for glomerulonephritis and glomerular complement deposition. Thus, TNFR1 promotes systemic immune responses and renal T cell death, while intrinsic cell TNFR2 plays a critical role in complement-dependent tissue injury. Therefore, therapeutic blockade specifically of TNFR2 may be a promising strategy in the treatment of immune-mediated glomerulonephritis.
Volker Vielhauer, George Stavrakis, Tanya N. Mayadas
Autosomal dominant polycystic kidney disease (ADPKD) is the most common human monogenic genetic disorder and is characterized by progressive bilateral renal cysts and the development of renal insufficiency. The cystogenesis of ADPKD is believed to be a monoclonal proliferation of PKD-deficient (PKD–/–) renal tubular epithelial cells. To define the function of Pkd1, we generated chimeric mice by aggregation of Pkd1–/– ES cells and Pkd1+/+ morulae from ROSA26 mice. As occurs in humans with ADPKD, these mice developed cysts in the kidney, liver, and pancreas. Surprisingly, the cyst epithelia of the kidney were composed of both Pkd1–/– and Pkd1+/+ renal tubular epithelial cells in the early stages of cystogenesis. Pkd1–/– cyst epithelial cells changed in shape from cuboidal to flat and replaced Pkd1+/+ cyst epithelial cells lost by JNK-mediated apoptosis in intermediate stages. In late-stage cysts, Pkd1–/– cells continued immortalized proliferation with downregulation of p53. These results provide a novel understanding of the cystogenesis of ADPKD patients. Furthermore, immortalized proliferation without induction of p53 was frequently observed in 3T3-type culture of mouse embryonic fibroblasts from Pkd1–/– mice. Thus, Pkd1 plays a role in preventing immortalized proliferation of renal tubular epithelial cells through the induction of p53 and activation of JNK.
Saori Nishio, Masahiko Hatano, Michio Nagata, Shigeo Horie, Takao Koike, Takeshi Tokuhisa, Toshio Mochizuki