Allogenic fetal membrane-derived mesenchymal stem cells contribute to renal repair in experimental glomerulonephritis.

Mesenchymal stem cells (MSC) have been reported to be an attractive therapeutic cell source for the treatment of renal diseases. Recently, we reported that transplantation of allogenic fetal membrane-derived MSC (FM-MSC), which are available noninvasively in large amounts, had a therapeutic effect on a hindlimb ischemia model (Ishikane S, Ohnishi S, Yamahara K, Sada M, Harada K, Mishima K, Iwasaki K, Fujiwara M, Kitamura S, Nagaya N, Ikeda T. Stem Cells 26: 2625-2633, 2008). Here, we investigated whether allogenic FM-MSC administration could ameliorate renal injury in experimental glomerulonephritis. Lewis rats with anti-Thy1 nephritis intravenously received FM-MSC obtained from major histocompatibility complex-mismatched ACI rats (FM-MSC group) or a PBS (PBS group). Nephritic rats exhibited an increased urinary protein excretion in the PBS group, whereas the FM-MSC group rats had a significantly lower level of increase (P < 0.05 vs. PBS group). FM-MSC transplantation significantly reduced activated mesangial cell (MC) proliferation, glomerular monocyte/macrophage infiltration, mesangial matrix accumulation, as well as the glomerular expression of inflammatory or extracellular matrix-related genes including TNF-α, monocyte chemoattractant protein 1 (MCP-1), type I collagen, TGF-ß, type 1 plasminogen activator inhibitor (PAI-1) (P < 0.05 vs. PBS group). In vitro, FM-MSC-derived conditioned medium significantly attenuated the expression of TNF-α and MCP-1 in rat MC through a prostaglandin E(2)-dependent mechanism. These data suggest that transplanted FM-MSC contributed to the healing process in injured kidney tissue by producing paracrine factors. Our results indicate that allogenic FM-MSC transplantation is a potent therapeutic strategy for the treatment of acute glomerulonephritis.

Impaired recovery of blood flow after hind-limb ischemia in mice lacking guanylyl cyclase-A, a receptor for atrial and brain natriuretic peptides.

OBJECTIVE: Atrial and brain natriuretic peptides (ANP and BNP, respectively) function via guanylyl cyclase (GC)-A, resulting in diuresis, natriuresis, and blood vessel dilation. Here, we investigated the role of endogenous ANP/BNP-GC-A signaling on reparative vascular remodeling using a hind-limb ischemia model. METHODS AND RESULTS: In GC-A-deficient mice (GC-A-KO), hind-limb ischemia resulted in autoamputation or severe ulcers in 60% of mice (6/10) during the 28-day observation period. In wild-type (WT) mice, partial amputation or mild ulcers were detected in only 20% of mice (2/10). Laser Doppler perfusion imaging revealed that the recovery of blood flow in the ischemic limb was significantly inhibited in GC-A-KO mice compared with WT mice. Immunostainings with anti-PECAM-1 antibody demonstrated that, in GC-A-KO, the capillary density of the ischemic tissue was significantly diminished compared to WT. Furthermore, bone marrow transplantation showed the predominant role of GC-A on local ischemic tissue rather than on vascular progenitor cells mobilized from bone marrow during vascular remodeling. In cultured human endothelial cells, ANP treatment significantly stimulated mRNA expressions of vascular endothelial growth factor and endothelial nitric oxide synthase via Erk1/2-dependent mechanism. CONCLUSIONS: These results suggest that endogenous ANP and BNP play important roles in reparative vascular remodeling in ischemic tissue.

Expression of CCN1 (CYR61) in developing, normal, and diseased human kidney.

CCN1 (cysteine-rich protein 61; Cyr61) is an extracellular matrix-associated signaling molecule that functions in cell migration, adhesion, and differentiation. We previously reported that CCN1 is induced at podocytes in rat anti-Thy-1 glomerulonephritis, a well-known model of reversible glomerular injury, but its expression and significance in the human kidney remain totally unknown (Sawai K, Mori K, Mukoyama M, Sugawara A, Suganami T, Koshikawa M, Yahata K, Makino H, Nagae T, Fujinaga Y, Yokoi H, Yoshioka T, Yoshimoto A, Tanaka I, Nakao K. J Am Soc Nephrol 14: 1154-1163, 2003). Here we report that, in the human kidney, CCN1 expression was confined to podocytes in normal adult and embryonic glomeruli from the capillary loop stage. Podocyte CCN1 expression was decreased in IgA nephropathy, diabetic nephropathy, and membranous nephropathy, whereas it remained unchanged in minimal change disease and focal segmental glomerulosclerosis. Downregulation of CCN1 was significantly greater in diseased kidneys with severe mesangial expansion. CCN1 protein was also localized in the thick ascending limb of Henle's loop, distal and proximal tubules, and collecting ducts, which was not altered in diseased kidneys. In vitro, recombinant CCN1 protein enhanced endothelial cell adhesion, whereas it prominently inhibited mesangial cell adhesion. CCN1 also completely suppressed mesangial cell migration, suggesting its role as a mesangial-repellent factor. In cultured podocytes, CCN1 markedly induced the expression of cyclin-dependent kinase inhibitor p27(Kip1) as well as synaptopodin in a dose-dependent manner and suppressed podocyte migration. These data indicate that CCN1 is expressed in podocytes, can act on glomerular cells to modulate glomerular remodeling, and is downregulated in diseased kidneys, suggesting that impairment of CCN1 expression in podocytes may contribute to the progression of glomerular disease with mesangial expansion.

Altered gene expression related to glomerulogenesis and podocyte structure in early diabetic nephropathy of db/db mice and its restoration by pioglitazone.

Glomerular injury plays a pivotal role in the development of diabetic nephropathy. To elucidate molecular mechanisms underlying diabetic glomerulopathy, we compared glomerular gene expression profiles of db/db mice with those of db/m control mice at a normoalbuminuric stage characterized by hyperglycemia and at an early stage of diabetic nephropathy with elevated albuminuria, using cDNA microarray. In db/db mice at the normoalbuminuric stage, hypoxia-inducible factor-1alpha (HIF-1alpha), ephrin B2, glomerular epithelial protein 1, and Pod-1, which play key roles in glomerulogenesis, were already upregulated in parallel with an alteration of genes related to glucose metabolism, lipid metabolism, and oxidative stress. Podocyte structure-related genes, actinin 4alpha and dystroglycan 1 (DG1), were also significantly upregulated at an early stage. The alteration in the expression of these genes was confirmed by quantitative RT-PCR. Through pioglitazone treatment, gene expression of ephrin B2, Pod-1, actinin 4alpha, and DG1, as well as that of oxidative stress and lipid metabolism, was restored concomitant with attenuation of albuminuria. In addition, HIF-1alpha protein expression was partially attenuated by pioglitazone. These results suggest that not only metabolic alteration and oxidative stress, but also the alteration of gene expression related to glomerulogenesis and podocyte structure, may be involved in the pathogenesis of early diabetic glomerulopathy in type 2 diabetes.

Redistribution of connexin43 expression in glomerular podocytes predicts poor renal prognosis in patients with type 2 diabetes and overt nephropathy.

BACKGROUND: Significance of podocyte injury in the progression of diabetic nephropathy is not well-understood. In this study, we examined whether alteration of gap junction protein connexin43 (Cx43) expression in podocytes is associated with the progression of overt diabetic nephropathy. METHODS: We recruited 29 type 2 diabetic patients with overt nephropathy who underwent renal biopsy. Nephrectomized kidney samples obtained from seven subjects with localized neoplasm and biopsy specimens from five patients diagnosed as minor glomerular abnormalities were used as controls. Cx43 staining on paraffin-embedded kidney sections were studied by immunohistochemistry. RESULTS: In controls, Cx43 was expressed at podocytes in a linear pattern along the glomerular basement membrane. In contrast, downregulation and loss of uniformly linear staining of Cx43 (Cx43 heterogeneity) in podocytes were observed in diabetic nephropathy. Cx43 intensity correlated with current renal function (R = 0.647, P < 0.005), whereas the magnitude of Cx43 heterogeneity correlated well with the degree of future decline in renal function (R = -0.705, P < 0.001). CONCLUSIONS: Alteration of Cx43 expression in podocytes was closely associated with the progression of overt diabetic nephropathy. These results indicate that change in Cx43 expression at podocytes represents a progressive stage in overt diabetic nephropathy and that it may be a convenient way to predict future decline in renal function.

Role of p38 mitogen-activated protein kinase activation in podocyte injury and proteinuria in experimental nephrotic syndrome.

Podocytes play an important role in maintaining normal glomerular function and structure, and podocyte injury leads to proteinuria and glomerulosclerosis. The family of mitogen-activated protein kinases (MAPK; extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase, and p38) may be implicated in the progression of various glomerulopathies, but the role of MAPK in podocyte injury remains elusive. This study examined phosphorylation of p38 MAPK in clinical glomerulopathies with podocyte injury, as well as in rat puromycin aminonucleoside (PAN) nephropathy and mouse adriamycin (ADR) nephropathy. The effect of treatment with FR167653, an inhibitor of p38 MAPK, was also investigated in rodent models. In human podocyte injury diseases, the increased phosphorylation of p38 MAPK was observed at podocytes. In PAN and ADR nephropathy, the phosphorylation of p38 MAPK and ERK was marked but transient, preceding overt proteinuria. Pretreatment with FR167653 (day -2 to day 14, subcutaneously) to PAN or ADR nephropathy completely inhibited p38 MAPK activation and attenuated ERK phosphorylation, with complete suppression of proteinuria. Electron microscopy and immunohistochemistry for nephrin and connexin43 revealed that podocyte injury was markedly ameliorated by FR167653. Furthermore, early treatment with FR167653 effectively prevented glomerulosclerosis and renal dysfunction in the chronic phase of ADR nephropathy. In cultured podocytes, PAN or oxidative stress induced the phosphorylation of p38 MAPK along with actin reorganization, and FR167653 inhibited such changes. These findings indicate that the activation of MAPK is necessary for podocyte injury, suggesting that p38 MAPK and, possibly, ERK should become a potential target for therapeutic intervention in proteinuric glomerulopathies.

Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.

Neutrophil gelatinase-associated lipocalin (Ngal), also known as siderocalin, forms a complex with iron-binding siderophores (Ngal:siderophore:Fe). This complex converts renal progenitors into epithelial tubules. In this study, we tested the hypothesis that Ngal:siderophore:Fe protects adult kidney epithelial cells or accelerates their recovery from damage. Using a mouse model of severe renal failure, ischemia-reperfusion injury, we show that a single dose of Ngal (10 microg), introduced during the initial phase of the disease, dramatically protects the kidney and mitigates azotemia. Ngal activity depends on delivery of the protein and its siderophore to the proximal tubule. Iron must also be delivered, since blockade of the siderophore with gallium inhibits the rescue from ischemia. The Ngal:siderophore:Fe complex upregulates heme oxygenase-1, a protective enzyme, preserves proximal tubule N-cadherin, and inhibits cell death. Because mouse urine contains an Ngal-dependent siderophore-like activity, endogenous Ngal might also play a protective role. Indeed, Ngal is highly accumulated in the human kidney cortical tubules and in the blood and urine after nephrotoxic and ischemic injury. We reveal what we believe to be a novel pathway of iron traffic that is activated in human and mouse renal diseases, and it provides a unique method for their treatment.

Prevention and reversal of renal injury by leptin in a new mouse model of diabetic nephropathy.

Diabetic nephropathy is the leading cause of end-stage renal disease, for which effective therapy to prevent the progression at advanced stages remains to be established. There is also a long debate whether diabetic glomerular injury is reversible or not. Lipoatrophic diabetes, a syndrome caused by paucity of adipose tissue, is characterized by severe insulin resistance, dyslipidemia, and fatty liver. Here, we show that a genetic model of lipoatrophic diabetes (A-ZIP/F-1 mice) manifests a typical renal injury observed in human diabetic nephropathy that is associated with glomerular hypertrophy, diffuse and pronounced mesangial widening, accumulation of extracellular matrix proteins, podocyte damage, and overt proteinuria. By crossing A-ZIP/F-1 mice with transgenic mice overexpressing an adipocyte-derived hormone leptin, we also reveal that leptin completely prevents the development of hyperglycemia and nephropathy in A-ZIP/F-1 mice. Furthermore, continuous leptin administration to A-ZIP/F-1 mice by minipump beginning at 40 weeks of age significantly alleviates the glomerular injury and proteinuria. These findings demonstrate the therapeutic usefulness of leptin at least for a certain type of diabetic nephropathy. The model presented here will serve as a novel tool to analyze the molecular mechanism underlying not only the progression but also the regression of diabetic nephropathy.

Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis.

Connective tissue growth factor (CTGF/CCN2) is one of the candidate factors mediating fibrogenic activity of TGF-beta. It was shown previously that the blockade of CTGF by antisense oligonucleotide (ODN) inhibits TGF-beta-induced production of fibronectin and type I collagen in cultured renal fibroblasts. The in vivo contribution of CTGF in renal interstitial fibrosis, however, remains to be clarified. With the use of a hydrodynamics-based gene transfer technique, the effects of CTGF antisense ODN are investigated in rat kidneys with unilateral ureteral obstruction (UUO). FITC-labeled ODN injection via the renal vein showed that the ODN was specifically introduced into the interstitium. At day 7 after UUO, the gene expression of CTGF, fibronectin, fibronectin ED-A, and alpha1(I) collagen in untreated or control ODN-treated obstructed kidneys was prominently upregulated. CTGF antisense ODN treatment, by contrast, markedly attenuated the induction of CTGF, fibronectin, fibronectin ED-A, and alpha1(I) collagen genes, whereas TGF-beta gene upregulation was not affected. The antisense treatment also reduced interstitial deposition of CTGF, fibronectin ED-A, and type I collagen and the interstitial fibrotic areas. The number of myofibroblasts determined by the expression of alpha-smooth muscle actin was significantly decreased as well. Proliferation of tubular and interstitial cells was not altered with the treatment. These findings indicate that CTGF expression in the interstitium plays a crucial role in the progression of interstitial fibrosis but not in the proliferation of tubular and interstitial cells during UUO. CTGF may become a potential therapeutic target against tubulointerstitial fibrosis.

Angiogenic protein Cyr61 is expressed by podocytes in anti-Thy-1 glomerulonephritis.

Dynamic recovery of glomerular structure occurs after severe glomerular damage in anti-Thy-1 glomerulonephritis (Thy-1 GN), but its mechanism remains to be investigated. To identify candidate genes possibly involved in glomerular reconstruction, screening was performed for genes that are specifically expressed by podocytes and are upregulated in glomeruli of Thy-1 GN. Among them, cysteine-rich protein 61 (Cyr61 or CCN1), a soluble angiogenic protein belonging to the CCN family, was identified. By Northern blot analysis, Cyr61 mRNA was markedly upregulated in glomeruli of Thy-1 GN from day 3 through day 7, when mesangial cell migration was most prominent. By in situ hybridization and immunohistochemistry, Cyr61 mRNA and protein were expressed by proximal straight tubules and afferent and efferent arterioles in normal rat kidneys and were intensely upregulated at podocytes in Thy-1 GN. Platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta1 (TGF-beta1), of which the gene expression in the glomeruli of Thy-1 GN was upregulated in similar time course as Cyr61, induced Cyr61 mRNA expression in cultured podocytes. Furthermore, supernatant of Cyr61-overexpressing cells inhibited PDGF-induced mesangial cell migration. In conclusion, it is shown that Cyr61 is strongly upregulated at podocytes in Thy-1 GN possibly by PDGF and TGF-beta. Cyr61 may be involved in glomerular remodeling as a factor secreted from podocytes to inhibit mesangial cell migration.