While macro- and microscopic kidney development appear to proceed normally in mice that lack Foxi1, electron microscopy reveals an altered ultrastructure of cells lining the distal nephron. Northern blot analyses, cRNA in situ hybridizations, and immunohistochemistry demonstrate a complete loss of expression of several anion transporters, proton pumps, and anion exchange proteins expressed by intercalated cells of the collecting ducts, many of which have been implicated in hereditary forms of distal renal tubular acidosis (dRTA). In Foxi1-null mutants the normal epithelium with its two major cell types — principal and intercalated cells — has been replaced by a single cell type positive for both principal and intercalated cell markers. To test the functional consequences of these alterations, Foxi1–/– mice were compared with WT littermates in their response to an acidic load. This revealed an inability to acidify the urine as well as a lowered systemic buffer capacity and overt acidosis in null mutants. Thus, Foxi1–/– mice seem to develop dRTA due to altered cellular composition of the distal nephron epithelium, thereby denying this epithelium the proper gene expression pattern needed for maintaining adequate acid-base homeostasis.
Sandra Rodrigo Blomqvist, Hilmar Vidarsson, Sharyn Fitzgerald, Bengt R. Johansson, Anna Ollerstam, Russell Brown, A. Erik G. Persson, Göran Bergström, Sven Enerbäck
Kidney podocytes and their slit diaphragms form the final barrier to urinary protein loss. This explains why podocyte injury is typically associated with nephrotic syndrome. The present study uncovered an unanticipated novel role for costimulatory molecule B7-1 in podocytes as an inducible modifier of glomerular permselectivity. B7-1 in podocytes was found in genetic, drug-induced, immune-mediated, and bacterial toxin–induced experimental kidney diseases with nephrotic syndrome. The clinical significance of our results is underscored by the observation that podocyte expression of B7-1 correlated with the severity of human lupus nephritis. In vivo, exposure to low-dose LPS rapidly upregulates B7-1 in podocytes of WT and SCID mice, leading to nephrotic-range proteinuria. Mice lacking B7-1 are protected from LPS-induced nephrotic syndrome, suggesting a link between podocyte B7-1 expression and proteinuria. LPS signaling through toll-like receptor-4 reorganized the podocyte actin cytoskeleton in vitro, and activation of B7-1 in cultured podocytes led to reorganization of vital slit diaphragm proteins. In summary, upregulation of B7-1 in podocytes may contribute to the pathogenesis of proteinuria by disrupting the glomerular filter and provides a novel molecular target to tackle proteinuric kidney diseases. Our findings suggest a novel function for B7-1 in danger signaling by nonimmune cells.
Jochen Reiser, Gero von Gersdorff, Martin Loos, Jun Oh, Katsuhiko Asanuma, Laura Giardino, Maria Pia Rastaldi, Novella Calvaresi, Haruko Watanabe, Karin Schwarz, Christian Faul, Matthias Kretzler, Anne Davidson, Hikaru Sugimoto, Raghu Kalluri, Arlene H. Sharpe, Jordan A. Kreidberg, Peter Mundel
Hepatocyte nuclear factor–1β (HNF-1β) is a Pit-1, Oct-1/2, UNC-86 (POU)/homeodomain-containing transcription factor that regulates tissue-specific gene expression in the liver, kidney, and other organs. Humans with autosomal dominant mutations of HNF-1β develop maturity-onset diabetes of the young type 5 (MODY5) and congenital cystic abnormalities of the kidney. Autosomal recessive polycystic kidney disease (ARPKD) is an inherited cystic disorder that produces renal failure in infants and children and is caused by mutations of PKHD1. The proximal promoter of the mouse Pkhd1 gene contains an evolutionarily conserved HNF-1–binding site that is located near a region of deoxyribonuclease hypersensitivity. HNF-1β and the structurally related HNF-1α bind specifically to the Pkhd1 promoter and stimulate gene transcription. Mutations of the HNF-1 site or expression of a dominant-negative HNF-1β mutant inhibit Pkhd1 promoter activity in transfected cells. Transgenic mice expressing a dominant-negative HNF-1β mutant under the control of a kidney-specific promoter develop renal cysts, similarly to humans with MODY5. Pkhd1 transcripts are absent in the cells lining the cysts but are present in morphologically normal surrounding tubules. These studies identify a link between two cystic disease genes, HNF1β (MODY5) and PKHD1 (ARPKD). HNF-1β directly regulates the transcription of Pkhd1, and inhibition of PKHD1 gene expression may contribute to the formation of renal cysts in humans with MODY5.
Thomas Hiesberger, Yun Bai, Xinli Shao, Brian T. McNally, Angus M. Sinclair, Xin Tian, Stefan Somlo, Peter Igarashi
Yingjian Li, Junwei Yang, Chunsun Dai, Chuanyue Wu, Youhua Liu
Tubulointerstitial fibrosis is the final common result of a variety of progressive injuries leading to chronic renal failure. Transforming growth factor-β (TGF-β) is reportedly upregulated in response to injurious stimuli such as unilateral ureteral obstruction (UUO), causing renal fibrosis associated with epithelial-mesenchymal transition (EMT) of the renal tubules and synthesis of extracellular matrix. We now show that mice lacking Smad3 (Smad3ex8/ex8), a key signaling intermediate downstream of the TGF-β receptors, are protected against tubulointerstitial fibrosis following UUO as evidenced by blocking of EMT and abrogation of monocyte influx and collagen accumulation. Culture of primary renal tubular epithelial cells from wild-type or Smad3-null mice confirms that the Smad3 pathway is essential for TGF-β1–induced EMT and autoinduction of TGF-β1. Moreover, mechanical stretch of the cultured epithelial cells, mimicking renal tubular distention due to accumulation of urine after UUO, induces EMT following Smad3-mediated upregulation of TGF-β1. Exogenous bone marrow monocytes accelerate EMT of the cultured epithelial cells and renal tubules in the obstructed kidney after UUO dependent on Smad3 signaling. Together the data demonstrate that the Smad3 pathway is central to the pathogenesis of interstitial fibrosis and suggest that inhibitors of this pathway may have clinical application in the treatment of obstructive nephropathy.
Misako Sato, Yasuteru Muragaki, Shizuya Saika, Anita B. Roberts, Akira Ooshima
The AP-1 transcription factor, composed of Jun and Fos proteins, plays a crucial role in the fine tuning of cell proliferation. We showed previously that AP-1 complexes are activated during the proliferative response that parallels the development of renal lesions after nephron reduction, but little is known about the specific role of individual Jun/Fos components in the deterioration process. Here we used JunD knockout (JunD–/–) mice and an experimental model of chronic renal injury (75% nephron reduction) to explore the role of JunD. Nephron reduction resulted in an initial compensatory growth phase that did not require JunD. JunD, however, was essential to inhibit a second wave of cell proliferation and to halt the development of severe glomerular sclerosis, tubular dilation, and interstitial fibrosis. We show that the effects of junD inactivation are not cell autonomous and involve upregulation of the paracrine mitogen, TGF-α. Expression of a transgene (REM) encoding a dominant negative isoform of the EGFR, the receptor for TGF-α, prevented the second wave of cell proliferation and the development of renal lesions in bitransgenic JunD–/–/REM mice. We propose that JunD is part of a regulatory network that controls proliferation to prevent pathological progression in chronic renal diseases.
Evangéline Pillebout, Jonathan B. Weitzman, Martine Burtin, Carla Martino, Pierre Federici, Moshe Yaniv, Gérard Friedlander, Fabiola Terzi
Tumors associated with osteomalacia elaborate the novel factor(s), phosphatonin(s), which causes phosphaturia and hypophosphatemia by cAMP-independent pathways. We show that secreted frizzled-related protein-4 (sFRP-4), a protein highly expressed in such tumors, is a circulating phosphaturic factor that antagonizes renal Wnt-signaling. In cultured opossum renal epithelial cells, sFRP-4 specifically inhibited sodium-dependent phosphate transport. Infusions of sFRP-4 in normal rats over 2 hours specifically increased renal fractional excretion of inorganic phosphate (FEPi) from 14% ± 2% to 34% ± 5% (mean ± SEM, P < 0.01). Urinary cAMP and calcium excretion were unchanged. In thyro-parathyroidectomized rats, sFRP-4 increased FEPi from 0.7% ± 0.2% to 3.8% ± 1.2% (P < 0.05), demonstrating that sFRP-4 inhibits renal inorganic phosphate reabsorption by PTH-independent mechanisms. Administration of sFRP-4 to intact rats over 8 hours increased FEPi, decreased serum phosphate (1.95 ± 0.1 to 1.53 ± 0.09 mmol/l, P < 0.05) but did not alter serum 1α, 25-dihydroxyvitamin D, renal 25-hydroxyvitamin D 1α-hydroxylase cytochrome P450, and sodium-phosphate cotransporter mRNA concentrations. Infusion of sFRP-4 antagonizes Wnt action as demonstrated by reduced renal β-catenin and increased phosphorylated β-catenin concentrations. The sFRP-4 is detectable in normal human serum and in the serum of a patient with tumor-induced osteomalacia. Thus, sFRP-4 displays phosphatonin-like properties, because it is a circulating protein that promotes phosphaturia and hypophosphatemia and blunts compensatory increases in 1α, 25-dihydroxyvitamin D.
Theresa Berndt, Theodore A. Craig, Ann E. Bowe, John Vassiliadis, David Reczek, Richard Finnegan, Suzanne M. Jan De Beur, Susan C. Schiavi, Rajiv Kumar
Under pathologic conditions, renal tubular epithelial cells can undergo epithelial to mesenchymal transition (EMT), a phenotypic conversion that is believed to play a critical role in renal interstitial fibrogenesis. However, the underlying mechanism that governs this process remains largely unknown. Here we demonstrate that integrin-linked kinase (ILK) plays an important role in mediating tubular EMT induced by TGF-β1. TGF-β1 induced ILK expression in renal tubular epithelial cells in a time- and dose-dependent manner, which was dependent on intracellular Smad signaling. Forced expression of ILK in human kidney proximal tubular epithelial cells suppressed E-cadherin expression and induced fibronectin expression and its extracellular assembly. ILK also induced MMP-2 expression and promoted cell migration and invasion in Matrigel. Conversely, ectopic expression of a dominant-negative, kinase-dead form of ILK largely abrogated TGF-β1–initiated tubular cell phenotypic conversion. In vivo, ILK was markedly induced in renal tubular epithelia in mouse models of chronic renal diseases, and such induction was spatially and temporally correlated with tubular EMT. Moreover, inhibition of ILK expression by HGF was associated with blockade of tubular EMT and attenuation of renal fibrosis. These findings suggest that ILK is a critical mediator for tubular EMT and likely plays a crucial role in the pathogenesis of chronic renal fibrosis.
Yingjian Li, Junwei Yang, Chunsun Dai, Chuanyue Wu, Youhua Liu
Aldosterone controls the final sodium reabsorption and potassium secretion in the kidney by regulating the activity of the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN). ASDN consists of the last portion of the distal convoluted tubule (late DCT), the connecting tubule (CNT), and the collecting duct (CD) (i.e., the cortical CD [CCD] and the medullary CD [MCD]). It has been proposed that the control of sodium transport in the CCD is essential for achieving sodium and potassium balance. We have tested this hypothesis by inactivating the α subunit of ENaC in the CD but leaving ENaC expression in the late DCT and CNT intact. Under salt restriction or under aldosterone infusion, whole-cell voltage clamp of principal cells of CCD showed no detectable ENaC activity, whereas large amiloride-sensitive currents were observed in control littermates. The animals survive well and are able to maintain sodium and potassium balance, even when challenged by salt restriction, water deprivation, or potassium loading. We conclude that the expression of ENaC in the CD is not a prerequisite for achieving sodium and potassium balance in mice. This stresses the importance of more proximal nephron segments (late DCT/CNT) to achieve sodium and potassium balance.
Isabelle Rubera, Johannes Loffing, Lawrence G. Palmer, Gustavo Frindt, Nicole Fowler-Jaeger, Daniel Sauter, Tom Carroll, Andrew McMahon, Edith Hummler, Bernard C. Rossier
In fibrotic renal disease, elevated TGF-β and angiotensin II lead to increased plasminogen activator inhibitor type 1 (PAI-1). PAI-1 appears to reduce glomerular mesangial matrix turnover by inhibiting plasminogen activators, thereby decreasing plasmin generation and plasmin-mediated matrix degradation. We hypothesized that therapy with a mutant human PAI-1 (PAI-1R) that binds to matrix vitronectin but does not inhibit plasminogen activators, would enhance plasmin generation, increase matrix turnover, and decrease matrix accumulation in experimental glomerulonephritis. Three experimental groups included normal, untreated disease control, and PAI-1R–treated nephritic rats. Plasmin generation by isolated day 3 glomeruli was dramatically decreased by 69%, a decrease that was reversed 43% (P < 0.02) by in vivo PAI-1R treatment. At day 6, animals treated with PAI-1R showed significant reductions in proteinuria (48%, P < 0.02), glomerular staining for periodic acid–Schiff positive material (33%, P < 0.02), collagen I (28%, P < 0.01), collagen III (34%, P < 0.01), fibronectin (48%, P < 0.01), and laminin (41%, P < 0.01), and in collagen I (P < 0.01) and fibronectin mRNA levels (P < 0.02). Treatment did not alter overexpression of TGF-β1 and PAI-1 mRNAs, although TGF-β1 protein was significantly reduced. These observations strongly support our hypothesis that PAI-1R reduces glomerulosclerosis by competing with endogenous PAI-1, restoring plasmin generation, inhibiting inflammatory cell infiltration, decreasing local TGF-β1 concentration, and reducing matrix accumulation.
Yufeng Huang, Masashi Haraguchi, Daniel A. Lawrence, Wayne A. Border, Ling Yu, Nancy A. Noble