Roles of connective tissue growth factor and prostanoids in early streptozotocin-induced diabetic rat kidney: the effect of aspirin treatment.

BACKGROUND: Connective tissue growth factor (CTGF) is a cysteine-rich growth factor induced by transforming growth factor-beta (TGF-beta) and is thought to play a critical role in TGF-beta-stimulated extracellular matrix accumulation. To explore its involvement in early diabetic nephropathy, we investigated the time course of CTGF gene expression and its regulation in streptozotocin (STZ)-induced diabetic rat kidney. METHODS: Northern blot analysis for CTGF, TGF-beta, and fibronectin expression was performed in the glomeruli of STZ-induced diabetic rats from 3 days to 12 weeks after the induction of diabetes, together with histological examination. To investigate the role of prostanoids in this process, aspirin was administered in one group of diabetic rats. Furthermore, CTGF expression was analyzed in rat mesangial cells cultured under high-glucose conditions. RESULTS: Glomerular expression of CTGF and TGF-beta1 mRNA was coordinately upregulated as early as day 3, followed by fibronectin induction and mesangial matrix accumulation. Chronic aspirin treatment in diabetic rats significantly attenuated mesangial expansion, and effectively suppressed CTGF induction, as well as inhibiting the upregulation of TGF-beta1 and fibronectin expression. In cultured mesangial cells, aspirin treatment abolished high glucose-stimulated CTGF upregulation. CONCLUSIONS: These results indicate that CTGF expressed in the glomeruli is upregulated in the early stage of STZ-induced diabetic nephropathy in rats, and could be a critical mediator of the development of diabetic glomerulosclerosis. In addition, the modulatory effects of aspirin during this process suggest a role of the cyclooxygenase pathway in the progression of diabetic nephropathy.

Regulation of stanniocalcin 1 and 2 expression in the kidney by klotho gene.

The klotho gene product and stanniocalcin (STC) 1 and 2 are recently identified molecules implicated in calcium and phosphorus homeostasis. In the present study, we investigated the regulation of STC1 and STC2 gene expression in the kidney by klotho gene expression. Mice deficient in klotho expression (klotho mice) have hypercalcemia and hyperphosphatemia, and increased renal gene expression of STC1 and STC2 compared with wild-type mice. Administration of vitamin D or CaCl(2) to wild-type mice causes upregulation of STC1 but STC2 gene expression is not altered significantly. On the other hand, treatment of klotho mice with low phosphorus diet results in partial decrease in STC2 gene expression with normalization of hyperphosphatemia. These findings indicate that klotho gene expression plays a crucial role in the regulation of renal stanniocalcin gene expression in vivo, at least partly, through the control of circulating calcium and phosphate concentrations.

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.

Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis.

Connective tissue growth factor (CTGF) is one of the candidate factors mediating downstream events of transforming growth factor-beta (TGF-beta), but its role in fibrogenic properties of TGF-beta and in tubulointerstitial fibrosis has not yet been clarified. Using unilateral ureteral obstruction (UUO) in rats, we analyzed gene expression of TGF-beta1, CTGF, and fibronectin. We further investigated the effect of blockade of endogenous CTGF on TGF-beta-induced fibronectin expression in cultured rat renal fibroblasts by antisense oligodeoxynucleotide (ODN) treatment. After UUO, CTGF mRNA expression in the obstructed kidney was significantly upregulated subsequent to TGF-beta1, followed by marked induction of fibronectin mRNA. By in situ hybridization, CTGF mRNA was detected mainly in the interstitial fibrotic areas and tubular epithelial cells as well as in parietal glomerular epithelial cells in the obstructed kidney. The interstitial cells expressing CTGF mRNA were also positive for alpha-smooth muscle actin. CTGF antisense ODN transfected into cultured renal fibroblasts significantly attenuated TGF-beta-stimulated upregulation of fibronectin mRNA and protein compared with control ODN transfection, together with inhibited synthesis of type I collagen. With the use of a reporter assay, rat fibronectin promoter activity was increased by 2.5-fold with stimulation by TGF-beta1, and this increase was abolished with antisense CTGF treatment. Thus CTGF plays a crucial role in fibronectin synthesis induced by TGF-beta, suggesting that CTGF blockade could be a possible therapeutic target against tubulointerstitial fibrosis.

Overexpression of brain natriuretic peptide in mice ameliorates immune-mediated renal injury.

One of major causes of end-stage renal disease is glomerulonephritis, the treatment of which remains difficult clinically. It has already been shown that transgenic mice that overexpress brain natriuretic peptide (BNP), with a potent vasorelaxing and natriuretic property, have ameliorated glomerular injury after subtotal nephrectomy. However, the role of natriuretic peptides in immune-mediated renal injury still remains unknown. Therefore, the effects of chronic excess of BNP on anti-glomerular basement membrane nephritis induced in BNP-transgenic mice (BNP-Tg) were investigated and the mechanisms how natriuretic peptides act on mesangial cells in vitro were explored. After induction of nephritis, severe albuminuria (approximately 21-fold above baseline), tissue damage, including mesangial expansion and cell proliferation, and functional deterioration developed in nontransgenic littermates. In contrast, BNP-Tg exhibited much milder albuminuria (approximately fourfold above baseline), observed only at the initial phase, and with markedly ameliorated histologic and functional changes. Up-regulation of transforming growth factor-beta (TGF-beta) and monocyte chemoattractant protein-1 (MCP-1), as well as increased phosphorylation of extracellular signal-regulated kinase (ERK), were also significantly inhibited in the kidney of BNP-Tg. In cultured mesangial cells, natriuretic peptides counteracted the effects of angiotensin II with regard to ERK phosphorylation and fibrotic action. Because angiotensin II has been shown to play a pivotal role in the progression of nephritis through induction of TGF-beta and MCP-1 that may be ERK-dependent, the protective effects of BNP are likely to be exerted, at least partly, by antagonizing the renin-angiotensin system locally. The present study opens a possibility of a novel therapeutic potential of natriuretic peptides for treating immune-mediated renal injury.