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Type 2 diabetes mellitus is a leading health issue worldwide. Among cases of diabetes mellitus nephropathy (DN), the major complication of type 2 diabetes mellitus, the nephrotic phenotype is often intractable to clinical intervention and demonstrates the rapid decline of renal function to end-stage renal disease. We recently identified the gene for glypican-5 (GPC5), a cell-surface heparan sulfate proteoglycan, as conferring susceptibility for acquired nephrotic syndrome and additionally identified an association through a genome-wide association study between a variant in GPC5 and DN of type 2 diabetes mellitus. In vivo and in vitro data showed a progressive increase of GPC5 in type 2 DN along with severity; the excess was derived from glomerular mesangial cells. In this study, diabetic kidney showed that accumulation of fibroblast growth factor (Fgf)2 strikingly induced progressive proteinuria that was avoided in Gpc5 knockdown mice. The efficacy of Gpc5 inhibition was exerted through expression of the Fgf receptors 3 and 4 provoked in the diabetic kidney attributively. Extraglomerular Fgf2 was pathogenic in DN, and the deterrence of Gpc5 effectively inhibited the glomerular accumulation of Fgf2, the subsequent increase of mesangial extracellular matrix, and the podocytes' small GTPase activity. These findings elucidate the pivotal role of GPC5, identified as a susceptible gene in the genome-wide association study, in hyperglycemia-induced glomerulopathy.
Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
Lipoprotein glomerulopathy (LPG) is a renal disease often accompanied by dyslipidemia and increased serum apoE levels. apoESendai (Arg145Pro), a rare mutant based on the apoE3 sequence carrying an apoE2 charge, causes LPG in humans and transgenic mice, but its effects on the artery wall are unknown. Macrophage expression of apoESendai may also directly influence renal and arterial homeostasis. We investigated the effects of macrophage-expressed apoESendai in apoE(-/-) mice with or without LDL receptor (LDLR). Murine bone marrow transduced to express apoE2, apoE3, or apoESendai was transplanted into lethally irradiated mice. Macrophage apoESendai expression reduced aortic lesion size and inflammation by 32 and 28%, respectively, compared with apoE2 in apoE(-/-) recipients. No differences in lesion size or inflammation were found between apoESendai and apoE3 in apoE(-/-) recipients. Macrophage apoESendai expression also reduced aortic lesion size by 18% and inflammation by 29% compared with apoE2 in apoE(-/-)/LDLR(-/-) recipients. Glomerular lesions compatible with LPG with increased mesangial matrix, extracellular lipid accumulation, and focal mesangiolysis were only observed in apoE(-/-)/LDLR(-/-) mice expressing apoESendai. Thus, macrophage expression of apoESendai protects against atherosclerosis while causing lipoprotein glomerulopathy. This is the first demonstration of an apoprotein variant having opposing effects on vascular and renal homeostasis.
Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.
Superoxide dismutase (SOD) is a major defender against excessive superoxide generated under hyperglycemia. We have recently reported that renal SOD1 (cytosolic CuZn-SOD) and SOD3 (extracellular CuZn-SOD) isoenzymes are remarkably down-regulated in KK/Ta-Ins2(Akita) diabetic mice, which exhibit progressive diabetic nephropathy (DN), but not in DN-resistant C57BL/6- Ins2(Akita) (C57BL/6-Akita) diabetic mice. To determine the role of SOD1 and SOD3 in DN, we generated C57BL/6-Akita diabetic mice with deficiency of SOD1 and/or SOD3 and investigated their renal phenotype at the age of 20 weeks. Increased glomerular superoxide levels were observed in SOD1(-/-)SOD3(+/+) and SOD1(-/-)SOD3(-/-) C57BL/6-Akita mice but not in SOD1(+/+)SOD3(-/-) C57BL/6-Akita mice. The SOD1(-/-)SOD3(+/+) and SOD1(-/-)SOD3(-/-) C57BL/6-Akita mice exhibited higher glomerular filtration rate, increased urinary albumin levels, and advanced mesangial expansion as compared with SOD1(+/+)SOD3(+/+) C57BL/6-Akita mice, yet the severity of DN did not differ between the SOD1(-/-)SOD3(+/+) and SOD1(-/-)SOD3(-/-) C57BL/6-Akita groups. Increased renal mRNA expression of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF), reduced glomerular nitric oxide (NO), and increased renal prostaglandin E2 (PGE2) production were noted in the SOD1(-/-)SOD3(+/+) and SOD1(-/-)SOD3(-/-) C57BL/6-Akita mice. This finding indicates that such renal changes in fibrogenic cytokines, NO, and PGE2, possibly caused by superoxide excess, would contribute to the development of overt albuminuria by promoting mesangial expansion, endothelial dysfunction, and glomerular hyperfiltration. The present results demonstrate that deficiency of SOD1, but not SOD3, increases renal superoxide in the setting of diabetes and causes overt renal injury in nephropathy-resistant diabetic mice, and that SOD3 deficiency does not provide additive effects on the severity of DN in SOD1-deficient C57BL/6-Akita mice.
Copyright © 2012 Elsevier Inc. All rights reserved.
Bone morphogenetic protein7 (BMP7) attenuates renal tubular and interstitial damage in a variety of experimental models. The function of BMP in the glomerulus is, however, not well understood. In the present study, we generated transgenic mice carrying cDNA for noggin, an endogenous inhibitor of BMPs, driven by the podocyte-specific promoter nephrin. Transgenic founder mice could be divided into two groups based on gross histological analyses at 2 months of age. One group was characterized by the presence of cystic glomeruli with collapsed capillary tufts and a decrease in mesangial cell number, representing a developmental defect during glomerular morphogenesis ("cystic" Tg mice). In contrast, the kidneys appeared to be normal in the other group ("non-cystic" Tg mice). In both groups, however, massive mesangial expansion developed at 10 months of age. The lesion was characterized by the accumulation of fibronectin, but not type I collagen, type IV collagen or laminin. This phenotype is similar to the fibronectin nephropathy. These results suggest that endogenous BMP can have an important role in regulating glomerular structural homeostasis.
Severity of fibrosis after injury is determined by the nature of the injury and host genetic susceptibility. Metabolism of collagen, the major component of fibrotic lesions, is, in part, regulated by integrins. Using a model of glomerular injury by adriamycin, which induces reactive oxygen species (ROS) production, we demonstrated that integrin alpha1-null mice develop more severe glomerulosclerosis than wild-type mice. Moreover, primary alpha1-null mesangial cells produce more ROS both at baseline and after adriamycin treatment. Increased ROS synthesis leads to decreased cell proliferation and increased glomerular collagen IV accumulation that is reversed by antioxidants both in vivo and in vitro. Thus, we have identified integrin alpha1beta1 as a modulator of glomerulosclerosis. In addition, we showed a novel pathway where integrin alpha1beta1 modulates ROS production, which in turn controls collagen turnover and ultimately fibrosis. Because integrin alpha1beta1 is expressed in many cell types this may represent a generalized mechanism of controlling matrix accumulation, which has implications for numerous diseases characterized by fibrosis.
A previous study showed that exogenous angiotensin II (AngII) induces proliferation of glomerular cells through systemic actions of AngII. In the present study, the authors examined the mode of actions of endogenous AngII in injured kidneys that were made deficient in AT1 by using in vivo transfection of antisense oligodeoxynucleotide (AS-ODN). Thy-1 nephritis was induced in rats by injection of mAb 1-22-3. Four days later, glomerular transfection was performed by unilateral whole-kidney electroporation after AT1 AS-ODN delivery through the left renal artery (n = 7). The expression of renal AT1 was assessed by autoradiography. The effect of the AS-ODN transfection was assessed 3 d later and compared with transfection with control ODN (n = 6), systemically administered pharmacologic AT1 antagonist losartan (n = 5) as well as untreated Thy-1 animals (n = 5). Fluorescence-labeled AS-ODN was found transfected in almost all glomeruli and localized primarily to the mesangium. Compared with the contralateral untransfected kidney in both normal and Thy-1 rats, AS-ODN suppressed cortical AT1 expression by some 70%. The AS-ODN transfected kidneys of Thy-1 rats had significantly lower glomerular mesangial cell proliferation (7.38 +/- 0.68 cells/glomerulus) and extracellular matrix accumulation (0.262 +/- 0.009) than kidneys transfected with control ODN (10.94 +/- 0.51 cells/glomerulus and 0.342 +/- 0.031), contralateral untransfected kidneys (9.56 +/- 1.01 cells/glomerulus and 0.371 +/- 0.011), or kidneys that were exposed to Thy-1 alone (10.45 +/- 1.06 cells/glomerulus and 0.359 +/- 0.013). There were no significant differences in systolic BP among groups. In glomeruli, immunohistochemistry detected no difference in AT2 receptor expression, number of ED1-positive macrophages or number of apoptotic cells among groups. Thus, in renal injury induced by Thy-1 nephritis, selective suppression of mesangial AT1 expression by AS-ODN significantly reduced mesangial cell proliferation and matrix. These data provide in vivo evidence that injured glomeruli are sensitive to local tissue actions of AngII, which promote proliferation and matrix accumulation within the glomerulus.
Transforming growth factor beta (TGF-beta) stimulates renal cell fibrogenesis by a poorly understood mechanism. Previously, we suggested a synergy between TGF-beta1 activated extracellular signal-regulated kinase (ERK) and Smad signaling in collagen production by human glomerular mesangial cells. In a heterologous DNA binding transcription assay, biochemical or dominant-negative ERK blockade reduced TGF-beta1 induced Smad3 activity. Total serine phosphorylation of Smad2/3, but not phosphorylation of the C-terminal SS(P)XS(P) motif, was decreased by pretreatment with the MEK/ERK inhibitors, PD98059 (10 microM) or U0126 (25 microM). This effect was not seen in the mouse mammary epithelial NMuMG cell line, indicating that ERK-dependent activation of Smad2/3 occurs only in certain cell types. TGF-beta stimulated phosphorylation of an expressed Smad3A construct, with a mutated C-terminal SS(P)XS(P) motif, was reduced by a MEK/ERK inhibitor. In contrast, MEK/ERK inhibition did not affect phosphorylation of a Smad3 construct mutated at consensus phosphorylation sites in the linker region (Smad3EPSM). Constitutively active MEK (caMEK) induced alpha2(I) collagen promoter activity, an effect blocked by co-transfected Smad3EPSM, but not Smad3A. The effects of caMEK and TGF-beta1 on collagen promoter activity were additive. These results indicate that ERK-dependent R-Smad linker region phosphorylation enhances collagen I synthesis and imply positive cross talk between the ERK and Smad pathways in human mesangial cells.
Cyclic nucleotide-dependent relaxation of vascular smooth muscle is associated with increases in the phosphorylation of the small heat shock-related protein, HSP20. To determine whether phosphorylated HSP20 directly mediates relaxation, we used gene transfection and protein transduction of HSP20 analogues. Rat mesangial cells were transfected with constructs containing wild-type HSP20-enhanced green fluorescent protein (EGFP), phosphorylation site mutated HSP20 (S16A-HSP20-EGFP), or EGFP alone. Contractile properties were determined on a silicone polymer substrata. In the presence of serum, EGFP-vector transfected control cells and S16A-HSP20 transfected cells formed wrinkles on the polymer (contracted). Activation of cyclic nucleotide signaling pathways in the EGFP-vector transfected control cells led to a time-dependent decrease in the wrinkles (relaxation). The S16A-HSP20 transfected cells were refractory to cyclic nucleotide-dependent relaxation. Cells overexpressing the wild-type HSP20 did not form wrinkles on the polymer in response to serum (refractory to contraction). Treatment of precontracted strips of intact bovine carotid artery smooth muscle with synthetic peptides containing HIV-trans-activating transcriptional activator and a phosphopeptide motif of HSP20 led to dose-dependent relaxation. These data provide evidence that phosphorylated HSP20 has a direct role in smooth muscle relaxation and that small phosphopeptide motifs of HSP20 can mimic the effects of the entire molecule.