Renal Transforming Growth Factor-beta Research Articles

Foxp3-Positive Regulatory T Cells Contribute to Antifibrotic Effects in Renal Fibrosis via an Interleukin-18 Receptor Signaling Pathway.

Renal interstitial fibrosis is a common lesion in the process of various progressive renal diseases. Interleukin (IL)-18 is a proinflammatory cytokine that plays an important role in the induction of Th1 responses and is associated with renal interstitial fibrosis, but the mechanism of fibrosis remains unclear. Here we used IL-18 receptor alpha knockout (IL-18Rα KO) mice to investigate the role of an IL-18Rα signaling pathway in renal fibrosis in a murine model of unilateral ureteral obstruction. IL-18 Rα KO mice showed decreased renal interstitial fibrosis and increased infiltration of CD4+ T cells and Foxp3+ regulatory T cells (Tregs) compared to wildtype (WT) mice. The expression of renal transforming growth factor beta 1 (TGF-β1, which is considered an important cytokine in renal interstitial fibrosis) was not significantly different between WT and IL-18Rα KO mice. The adoptive transfer of CD4+ T cells from the splenocytes of IL-18Rα KO mice to WT mice reduced renal interstitial fibrosis and increased the number of Foxp3+ Tregs in WT mice. These results demonstrated that Foxp3+ Tregs have a protective effect in renal interstitial fibrosis via an IL-18R signaling pathway.

Protective role of zinc oxide nanoparticles in kidney injury induced by cisplatin in rats

Cisplatin used as chemotherapy for various cancers may leads to accumulation of platinum within the kidney and disturb its function. Zinc oxide nanoparticles (ZnO-NPs) are of low toxicity nanomaterials and have many medical fields so this study aims to indicate ZnO-NPs effect in kidney injury induced by cisplatin. Adult male rats were pre-injected with one dose of ZnO-NPs (5 mg/kg IP) and after 2 h from injection, the rats were injected with also only one dose of cisplatin (6 mg/kg IP) and two additional groups were served as controls treated with either ZnO-NPs or cisplatin only, respectively, and normal control was involved and euthanization occurred after 7 and 12 days. Cisplatin-induced nephropathy increased kidney function parameters; serum creatinine, blood urea nitrogen and microalbuminuria. Conversely, these parameters were down regulated after ZnO-NPs treatment. ZnO-NPs reversed the decrease of renal superoxide dismutase, catalase and glutathione reductase and the increase of renal malondialdehyde induced by cisplatin. In addition, the annexin V demonstrated that the proportion of viable cells was significantly elevated and the proportion of apoptotic and necrotic cells significantly reduced. Also, the level of renal transforming growth factor beta 1 decreased in group pre-treated with ZnO-NPs. The Nuclear factor-E2-related factor, heme oxygenase-1 and endothelial nitric oxide synthase expression genes were up regulated while Bcl-2-associated X protein expression was down regulated in kidney tissue via ZnO-NPs. Histopathological and immunohistochemical observations were context with these findings. In conclusion, ZnO-NPs treatment revealed renoprotective effect against cisplatin drug, probably via its antioxidant, anti-inflammatory and antiapoptotic properties.

Complement C5a inhibition moderates lipid metabolism and reduces tubulointerstitial fibrosis in diabetic nephropathy.

Complement C5 mediates pro-inflammatory responses in many immune-related renal diseases. Given that the C5a level is elevated in diabetes, we investigated whether activation of C5a/C5aR signalling plays a pathogenic role in diabetic nephropathy (DN) and the therapeutic potential of C5a inhibition for renal fibrosis. Human renal biopsies from patients with DN and control subjects were used for immunohistochemical staining of complement C5 components. Renal function and tubulointerstitial injury were compared between db/m mice, vehicle-treated mice and C5a inhibitor-treated db/db mice. A cell culture model of tubule epithelial cells (HK-2) was used to demonstrate the effect of C5a on the renal fibrotic pathway. Increased levels of C5a, but not of its receptor C5aR, were detected in renal tubules from patients with DN. The intensity of C5a staining was positively correlated with the progression of the disease. In db/db mice, administration of a novel C5a inhibitor, NOX-D21, reduced the serum triglyceride level and attenuated the upregulation of diacylglycerolacyltransferase-1 and sterol-regulatory element binding protein-1 expression and lipid accumulation in diabetic kidney. NOX-D21-treated diabetic mice also had reduced serum blood urea nitrogen and creatinine levels with less glomerular and tubulointerstitial damage. Renal transforming growth factor beta 1 (TGF-β1), fibronectin and collagen type I expressions were reduced by NOX-D21. In HK-2 cells, C5a stimulated TGF-β production through the activation of the PI3K/Akt signalling pathway. Blockade of C5a signalling by NOX-D21 moderates altered lipid metabolism in diabetes and improved tubulointerstitial fibrosis by reduction of lipid accumulation and TGF-β-driven fibrosis in diabetic kidney.

Attenuation of renal damage in type I diabetic rats by umbelliferone - a coumarin derivative.

It is well known that diabetes is one of the non-communicable disease affecting a large population worldwide. When diabetes remains untreated or uncontrolled, it leads to further serious complications, affecting vital organs like eyes, kidney, heart, etc. The present study was designed to evaluate effects of umbelliferone, a phytochemical, in treatment of diabetic nephropathy. Experimental model used was streptozotocin (55mg/kg, ip) induced diabetic nephropathy in male Sprague Dawley rats. After 28days of streptozotocin administration, diabetic animals were treated with umbelliferone at two dose levels, 20 and 40mg/kg for next 28days. The results of the study showed that umbelliferone treatment significantly decreased the elevated plasma creatinine and blood urea nitrogen level while significantly increased the total protein and albumin level in diabetic animals. Creatinine clearance was improved in umbelliferone treated animals. Renal oxidative stress was decreased in umbelliferone treated animals significantly. Histopathological study of the kidney was carried out by specific stains like Hematoxylin-Eosin, Periodic Acid Schiff and Masson Trichrome stain. The sections of the kidney showed that umbelliferone treatment decreased the glomerular damage, mesangial matrix expansion as well as the renal fibrosis. Determination of renal transforming growth factor beta one (TGF-β1) expression by immunohistochemical analysis, western blotting and circulating TGF-β1 by ELISA assay showed that umbelliferone decreased the renal tissue and circulating TGF-β1 level. Umbelliferone treatment can significantly reduce the diabetes induced renal damage and can improve the pathological conditions related to the diabetic nephropathy by down regulation of TGF-β.

Gallic acid ameliorates renal functions by inhibiting the activation of p38 MAPK in experimentally induced type 2 diabetic rats and cultured rat proximal tubular epithelial cells

Diabetic nephropathy (DN) is one of the leading causes of morbidity and mortality in diabetic patients that accounts for about 40% of deaths in type 2 diabetes. p38 mitogen activated protein kinase (p38 MAPK), a serine–threonine kinase, plays an important role in tissue inflammation and is known to be activated under conditions of oxidative stress and hyperglycemia. The role of p38 MAPK has been demonstrated in DN, and its inhibition has been suggested as an alternative approach in the treatment of DN. In the present study, we investigated the nephroprotective effects of an anti-inflammatory phenolic compound, gallic acid (GA, 3,4,5-trihydroxybenzoic acid), in high fat diet/streptozotocin (HFD/STZ) induce type 2 diabetic wistar albino rats. GA (25 mg/kgbw and 50 mg/kgbw, p.o.) treatment for 16 weeks post induction of diabetes led to a significant reduction in the levels of blood glucose, HbA1c, serum creatinine, blood urea nitrogen and proteinuria as well as a significant reduction in the levels of creatinine clearance. GA significantly inhibited the renal p38 MAPK and nuclear factor kappa B (N-κB) activation as well as significantly reduced the levels of renal transforming growth factor beta (TGF-β) and fibronectin. Treatment with GA resulted in a significant reduction in the serum levels of proinflammatory cytokines viz. interleukin 1 beta (IL-1β), IL-6 and tumor necrosis factor alpha (TNF-α). Moreover, GA significantly lowered renal pathology and attenuated renal oxidative stress. In cultured rat NRK 52E proximal tubular epithelial cells, GA treatment inhibited high glucose induced activation of p38 MAPK and NF-κB as well as suppressed proinflammatory cytokine synthesis. The results of the present study provide in vivo and in vitro evidences that the p38 MAPK pathway plays an important role in the pathogenesis of DN, and GA attenuates the p38 MAPK-mediated renal dysfunction in HFD/STZ induced type 2 diabetic rats.

Systemic delivery of IL-10 by an AAV vector prevents vascular remodeling and end-organ damage in stroke-prone spontaneously hypertensive rat

Interleukin-10 (IL-10) ameliorates various T-helper type 1 cell-mediated chronic inflammatory diseases. Although the therapeutic benefits of IL-10 include antiatherosclerotic effects, pathophysiological effects of IL-10 on vascular remodeling in hypertension have not yet been elucidated. These studies were designed to determine whether sustained IL-10 expression, mediated by an adeno-associated virus (AAV) vector, prevents vascular remodeling and target-organ damage in the stroke-prone spontaneously hypertensive rat (SHR-SP)-an animal model of malignant hypertension. A single intramuscular injection of an AAV1 vector encoding rat IL-10 introduced long-term IL-10 expression. These IL-10-transduced rats had decreased stroke episodes and proteinuria, resulting in improved survival. Histological examination revealed a reduced level of deleterious vascular remodeling of resistance vessels in the brain and kidney of these rats. Immunohistochemical analysis indicated that IL-10 inhibited the enhanced renal transforming growth factor-beta expression and perivascular infiltration of monocytes/macrophages and nuclear factor-kappaB-positive cells normally observed in the SHR-SP. Four weeks after IL-10 vector injection, systolic blood pressure significantly decreased and this effect persisted for several months. Overall, AAV vector-mediated systemic IL-10 expression prevented vascular remodeling and inflammatory lesions of target organs in the SHR-SP. This approach provides significant insights into the prevention strategy of disease onset with unknown genetic predisposition or intractable polygenic disorders.

Protective Effect of Gui Qi Mixture on the Progression of Diabetic Nephropathy in Rats

Huang Qi (root of Astragalus membranaceus) and Dang Gui ( Angelica sinensis), two of the most widely used herbs in traditional Chinese medicine, have been proven to be effective in the treatment of diabetes mellitus (DM) although the underlying molecular mechanisms are not fully elucidated. This study was designed to investigate the protective effect of Dang Gui and Huang Qi mixture (GQM) on the development of diabetic nephropathy in rats with streptozotocin (STZ)-induced DM and the possible underlying molecular mechanism. The diabetic animal model was made by a single intraperitoneal injection of STZ and then treated with GQM or benazepril. Blood glucose, triglyceride (TG), cholesterol (CHO), high density lipoprotein (HDL), serum creatinine (Scr), creatinine clearance rate (Ccr), blood urea nitrogen (BUN), urine beta (2)-microglobin (beta (2)-MG), kidney/body weight (K/B) ratio, glomerular area (GA), renal transforming growth factor-beta (1) (TGF-beta (1)) mRNA expression and blood and renal angiotensin II (AngII) expression were determined 8 weeks after the treatment. The blood glucose, CHO and TG levels, BUN, SCr, Ccr. K/B ratio, GA, the excretion of beta (2)-MG, renal TGF-beta (1) mRNA expression and blood and renal AngII expression were significantly increased while the HDL level was decreased 8 week after STZ injection. The changes in blood glucose, TG, CHO and HDL were reversed by GQM, not by benazepril, whereas the changes in other variables were reversed by both GQM and benazepril. Our results suggest that GQM alleviates the disorder in blood glucose and lipids, protects against the progression of renal nephropathy in diabetic rats, probably by inhibiting the expression of AngII and TGF-beta (1) mRNA.

Retinoic acid treatment protects MRL/lpr lupus mice from the development of glomerular disease

Retinoic acid (tRA) is an active metabolite of vitamin A with potent anti-inflammatory properties. We analyzed the effects of tRA on the development of lupus nephritis in MRL/lpr mice. MRL/lpr mice received chow supplemented with vehicle or tRA (daily 10 mg/kg) from 8 to 14 weeks until their sacrifice. MRL/wt mice served as an additional control. tRA-treated MRL/lpr mice showed reduced lymphoadenopathy and splenomegaly as compared to vehicle-treated controls. Treatment reduced proteinuria to almost basal levels. Plasma IgG and anti-DNA antibodies increased comparably in both vehicle and tRA-treated mice. Vehicle-treated mice showed characteristic renal lesions. In contrast tRA-treated mice showed almost normal glomerular histology with a pronounced reduction in endocapillary cell proliferation. T-cell and macrophage infiltrates were reduced after tRA treatment within glomeruli and interstitium as compared to vehicle-treated animals. In spite of this, immune complex and complement deposition were comparable in both groups. Adoptively transferred T cells from vehicle-treated to tRA-treated MRL/lpr mice did not induce renal lesions or proteinuria. These beneficial effects of tRA treatment were associated with reduced renal expression of chemokines and inflammatory cytokines. Surprisingly, renal transforming growth factor-beta (TGF-beta) mRNA levels of tRA-treated mice were elevated, possibly indicating that TGF-beta acts as an anti-inflammatory signal in this lupus model. tRA treatment reduces lymphoproliferation and glomerulonephritis in MRL/lpr mice. This occurs in spite of unaltered anti-DNA titers and glomerular immune complex deposition, and cannot be overcome by T-cell transfer from nephritic MRL/lpr mice.

Dissociation of renal TGF-beta and hypertrophy in female rats with diabetes mellitus.

Prepubertal onset of diabetes mellitus (DM) in male rats delays diabetic renal hypertrophy and suppresses renal transforming growth factor-beta (TGF-beta) compared with onset in adults. Because there are sex differences in normal and pathological renal growth, we performed similar experiments in female rats and examined the effects of prior ovariectomy. As in male rats, adult onset of DM increased renal weight approximately 35%, total renal TGF-beta approximately 35%, and mRNA for TGF-beta inducible gene H3 (betaIG-H3) approximately 200%. TGF-beta levels did not increase with DM in prepubertal animals, but renal weight increased approximately 40%, similar to the enlargement seen in adults. In nondiabetic rats, ovariectomy suppressed renal TGF-beta levels by 25-50% in both age groups, but betaIG-H3 was stable in younger animals and increased by approximately 200% in older animals after ovariectomy. Ovariectomy increased kidney weight approximately 10% in both age groups. DM further increased kidney weight by an additional 40% after ovariectomy with an approximately 150% increase in betaIG-H3, even though TGF-beta levels were not significantly increased. Prepubertal (approximately 99% lower), diabetic (approximately 50% lower), and ovariectomized rats (approximately 90% lower) all tended toward lower estradiol levels than intact adults, although not all differences were statistically significant. Both prepubertal onset and ovariectomy suppress TGF-beta in the kidneys of female rats with DM compared with adult-onset animals, but these states have no effect on renal enlargement. Production of the extracellular matrix component betaIG-H3 is dissociated from TGF-beta under these conditions. These observations may help explain some of the sex differences demonstrated in progressive kidney diseases, including DM.

Effect of aldosterone on renal transforming growth factor-beta.

Aldosterone participates in the pathophysiology of several models of progressive chronic renal disease. Because of the causal connection between transforming growth factor-beta(1) (TGF-beta) and scarring in many such models, we hypothesized that aldosterone could evoke TGF-beta in the kidney. Aldosterone infusion for 3 days in otherwise normal rats caused a more than twofold increase in TGF-beta excretion without changes in systolic pressure or evidence of kidney damage. Concurrent treatment with amiloride did not alter this effect, indicating that aldosterone's stimulation of TGF-beta was independent of its regulation of sodium or potassium transport. However, concurrent treatment with spironolactone did block the increase in TGF-beta, indicating that the effect depends on the mineralocorticoid receptor. Renal mRNA for serum glucocorticoid kinase rose, but no change in TGF-beta message occurred, suggesting posttranscriptional enhancement of renal TGF-beta. In summary, aldosterone provokes renal TGF-beta, and this action may contribute to aldosterone's fibrotic propensity.

Suppression of cyclosporine a nephrotoxicity in vivo by transforming growth factor beta receptor-immunoglobulin G chimeric protein.

Transforming growth factor (TGF)beta is implicated in the pathogenesis of cyclosporine A (CsA) nephrotoxicity. We examined the efficacy of TGF beta receptor (R)II/immunoglobulin (Ig)G Fc, a soluble chimeric protein consisting of the extracellular domain of human TGF beta RII and IgG1 Fc, on CsA nephrotoxicity in mice. Subcutaneous injection of CsA (25 mg/kg/d) was given daily to mice maintained on a low-sodium diet. On days 1 and 7, an expression vector carrying cDNA for either TGF beta RII/IgG Fc or beta-galactosidase was transfected into the skeletal muscles by electroporation. At 2 or 3 weeks of CsA administration, plasma and renal TGF beta 1 levels, and tubulointerstitial injury and fibrosis were evaluated. After 2 weeks of CsA administration, plasma and renal TGF beta 1 levels increased to the maximum and then declined toward the baseline levels. Renal TGF beta 1 mRNA remained elevated until 3 weeks. Tubulointerstitial alterations became appreciable in 2 weeks and intensified by 3 weeks. At 2 weeks, the TGF beta RII/IgG Fc intervention abolished the increase in plasma TGF beta 1, attenuated the increase in renal TGF beta 1 by 50%, and markedly suppressed the histologic alterations. At 3 weeks, the histologic alterations remained markedly suppressed by the intervention, with no appreciable effects on the renal TGF beta 1 mRNA and protein. The introduction of TGF beta RII/IgG Fc by gene transfer effectively abrogated CsA-induced tubulointerstitial alterations. Suppression of tubulointerstitial changes was evident at 3 weeks when renal TGF beta 1 mRNA and protein were comparable to those with CsA alone, indicating that early anti-TGF beta intervention is effective in suppressing the progression of CsA nephrotoxicity despite persistent increases in renal TGF beta 1 expression.

The effect of ramipril on albumin excretion in diabetes and hypertension: the role of increased lysosomal activity and decreased transforming growth factor-beta expression.

Albumin excretion is modulated post-filtration by lysosomal processing that produces a spectrum of albumin-derived material in urine, much of which is not detected by conventional immunoassays. This study aimed to determine the efficacy of ramipril treatment (+ RAM) after 24 weeks on total albumin excretion (intact plus albumin-derived peptides) in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats with (d) and without (c) diabetes. Intact albumin excretion was analysed by radioimmunoassay and total albumin excretion was analysed by measuring radioactivity derived from circulating [ C]albumin. Renal lysosomal activity was determined by urinary [ H]dextran sulphate desulphation. Renal transforming growth factor-beta 1 (TGF-beta 1), TGF-beta inducible gene-h3 (beta ig-h3) and angiotensinogen mRNA production were analysed by real time reverse transcriptase-polymerase chain reaction. Hypertension (SHR-c and SHR-d) resulted in a significant increase in intact albumin excretion, which was significantly reduced by ramipril treatment (P < 0.05 for SHR-c + RAM and 0.001 for SHR-d + RAM compared to non-treated). This was accompanied by a significant decrease in blood pressure (P < 0.001 for SHR-c + RAM and SHR-d + RAM), renal beta ig-h3 mRNA production (P < 0.05 for SHR-c + RAM and SHR-d + RAM), and an increase in lysosomal activity. Diabetes (WKY-d and SHR-d) primarily caused a significant increase in total albumin excretion, predominantly in the form of albumin-derived fragments in the WKY-d group and intact albumin in the SHR-d group. Ramipril treatment reduced total albumin excretion in the WKY-d + RAM group (P < 0.001). Ramipril prevents increases in both intact albumin and total albumin excretion in hypertensive and diabetic states, respectively.

Albuminuria in hypertension is linked to altered lysosomal activity and TGF-beta1 expression.

Increased intraglomerular pressure is considered a major factor for increased albumin excretion in hypertension. However, other factors should also be considered because recent studies in both humans and rats have demonstrated that proteins undergoing filtration and renal passage are extensively modified by renal cell lysosomal processing; >95% of albumin is degraded to peptides that are not detected by routine immunochemical assays. Changes in postglomerular lysosomal processing may therefore be responsible for the increased intact albumin excreted in hypertension-related kidney disease. We hypothesize that transforming growth factor-beta, which is known to decrease lysosomal activity, may be upregulated in hypertension and may play a role in increased intact albumin excretion. The aims of this study were to determine the effect that hypertension has on (1) renal cell lysosomal processing of albumin and dextran sulfate, (2) glomerular permeability, and (3) renal transforming growth factor-beta(1) expression. Spontaneously hypertensive rats and Wistar-Kyoto rats were used at 8, 16, and 24 weeks. We demonstrate that albuminuria in hypertension is linked to an inhibition of lysosomal processing as determined by (1) size exclusion chromatography analysis of urinary [(14)C]albumin structural integrity and (2) ion exchange analysis of urinary [(3)H]dextran sulfate. This inhibition gives rise to an increased proportion of radioimmunoassay detectable (intact) albumin and intact dextran sulfate independent of changes in glomerular capillary wall permeability as determined by the fractional clearance of [(3)H]Ficolls of various radii. These changes may be correlated with increased renal transforming growth factor-beta(1) expression.

TRANILAST AMELIORATES RENAL TUBULAR DAMAGE IN UNILATERAL URETERAL OBSTRUCTION

We determined whether tranilast, the anti-allergic agent N-(3, 4-dimethoxyciannamoyl)-anthranilic acid, would diminish renal transforming growth factor-beta (TGF-beta) levels in unilateral ureteral obstruction and concomitantly affect renal tubular apoptosis and proliferation in that condition. Tranilast (150 mg./kg.) was administered to rats 1 day before unilateral ureteral obstruction and each day thereafter. Kidneys were harvested day 14 after unilateral ureteral obstruction. Tissue TGF-beta was measured by bioassay using mink lung epithelial cells. Renal tubular proliferation and apoptosis were detected by immunostaining proliferating cell nuclear antigen and the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay, respectively. Fibrosis was assessed by measuring collagen deposition with trichrome stained slides. TGF-beta bioassay showed that obstructed kidneys in controls contained significantly higher mean TGF-beta plus or minus standard deviation than unobstructed kidneys in controls (73.7 +/- 13.6 versus 14.1 +/- 5.5 pg./mg. tissue) and tranilast significantly decreased tissue TGF-beta in obstructed kidneys (15.9 +/- 4.8 pg./mg. tissue). The terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay demonstrated that obstructed kidneys in controls had significantly more mean tubular apoptosis than the unobstructed counterparts (36.6 +/- 6.7 versus 5.8 +/- 5.5 nuclei per high power field) and tranilast significantly decreased mean renal tubular apoptosis in obstructed kidneys (16.2 +/- 1.7 nuclei per high power field). In addition, immunostaining proliferating cell nuclear antigen showed that obstructed kidneys in controls had significantly more mean renal tubular proliferation than unobstructed kidneys (20.7 +/- 3.4 versus 6.2 +/- 2.1 per high power field) and tranilast significantly increased proliferating renal tubules in obstructed and unobstructed kidneys (26.5 +/- 8.3 and 14.5 +/- 3.4 per high power field, respectively). Control obstructed kidneys exhibited significantly more fibrosis, which was also blunted by tranilast. Tranilast significantly decreases tissue TGF-beta, resulting in a reduction in tubular apoptosis and an increase in tubular proliferation. This finding suggests that tranilast is a promising agent for preventing renal tubular damage in unilateral ureteral obstruction.

Increased Glomerular and Tubular Expression of Transforming Growth Factor-β1, Its Type II Receptor, and Activation of the Smad Signaling Pathway in the db/db Mouse

Activation of the renal transforming growth factor-beta (TGF-beta) system likely mediates the excess production of extracellular matrix in the diabetic kidney. To establish the role of the TGF-beta system in type 2 diabetic nephropathy, we examined the intrarenal localization and expression of the TGF-beta1 isoform, the TGF-beta type II receptor, and the Smad signaling pathway in the 16-week-old db/db mouse, a genetic model of type 2 diabetes that exhibits mesangial matrix expansion, glomerular basement membrane thickening, and renal insufficiency that closely resemble the human disease. Compared with its nondiabetic db/m littermate, the db/db mouse showed significantly increased TGF-beta1 mRNA expression by in situ hybridization in both glomerular and tubular compartments. Likewise, TGF-beta1 protein, by immunohistochemical staining, was increased in both renal compartments, but the fractional expression of TGF-beta1 protein was less than that of the mRNA in the glomerulus. In situ hybridization and immunohistochemical staining for the TGF-beta type II receptor revealed concordant and significant increases of both mRNA and protein in the glomerular and tubular compartments of diabetic animals. Finally, immunohistochemistry showed preferential accumulation of Smad3 in the nuclei of glomerular and tubular cells in diabetes. The complementary technique of Southwestern histochemistry using a labeled Smad-binding element demonstrated increased binding of nuclear proteins to Smad-binding element, indicating active signaling downstream of the TGF-beta stimulus. We therefore propose that the TGF-beta system is up-regulated at the ligand, receptor, and signaling levels throughout the renal cortex in this animal model of type 2 diabetes. Our findings suggest that the profibrotic effects of TGF-beta may underlie the progression to glomerulosclerosis and tubulointerstitial fibrosis that characterize diabetic nephropathy.

Angiotensin converting enzyme inhibitor suppresses glomerular transforming growth factor β receptor expression in experimental diabetes in rats

Activation of the renal transforming growth factor beta (TGF-beta) axis has been suggested to play a part in the development of diabetic nephropathy by a direct stimulatory effect of hyperglycaemia or through the activation of the renin-angiotensin system. Our aim was to evaluate the involvement of the renin-angiotensin system by examining the effects of ACE-inhibition on intrarenal changes in all three TGF-beta isoforms and receptors in experimental diabetes in vivo. Immunocytochemistry, western blotting and ribonuclease protection assays were carried out for each TGF-beta isoform and receptor on kidney from non-diabetic and streptozotocin-diabetic rats after treatment with the ACE inhibitor, enalapril, for 30 days. Enalapril partially prevented the renal hypertrophy and fully prevented the increase in urinary albumin excretion rate in diabetic animals. The glomerular TGF-beta Type II Receptor mRNA and protein concentrations increased over 30 days in untreated diabetic animals compared with non-diabetic controls, while enalapril-treated diabetic animals showed a normalisation of TGF-beta Type II Receptor mRNA and protein. The ACE-inhibition had pronounced inhibitory effects on the increased expression of the glomerular TGF-beta Type II Receptor in the diabetic kidney required for intracellular signalling through this growth factor axis. This suggests a new mechanism of action of the ACE-inhibition in regulating the development of diabetic nephropathy.

Antibody to transforming growth factor-β ameliorates tubular apoptosis in unilateral ureteral obstruction

Unilateral ureteral obstruction (UUO) is characterized by progressive renal atrophy, renal interstitial fibrosis, an increase in renal transforming growth factor-beta (TGF-beta), and renal tubular apoptosis. The present study was undertaken to determine the effect of a monoclonal antibody to TGF-beta (1D11) in UUO. Mechanical stretch was applied to tubular epithelial cells (NRK-52E) by a computer-assisted system. Three doses of 1D11 (either 0.5, 2, or 4 mg/rat) were administered to rats one day prior to UUO and every two days thereafter, and kidneys were harvested at day 13. Fibrosis was assessed by measuring tissue hydroxyproline and mRNA for collagen and fibronectin. Apoptosis was assessed with the terminal deoxy transferase uridine triphosphate nick end-labeling assay. TGF-beta levels were determined by bioassay. Western blot and immunostaining were used to identify proliferating cell nuclear antigen (PCNA), p53, bcl-2, and inducible nitric oxide synthase (iNOS). Stretch significantly induced apoptosis in NRK-52E cells, which was accompanied by an increased release of TGF-beta; 1D11 (10 microg/mL) totally inhibited stretch-induced apoptosis. Control obstructed kidney contained 20-fold higher TGF-beta as compared with its unobstructed kidney; 1D11 neutralized tissue TGF-beta of the obstructed kidney. Control obstructed kidney exhibited significantly more fibrosis and tubular apoptosis than its unobstructed counterpart, which was blunted by 1D11. In contrast, 1D11 significantly increased tubular proliferation. p53 immunostaining was localized to renal tubular nuclei of control obstructed kidney and was diminished by 1D11. In contrast, bcl-2 was up-regulated in the 1D11-treated obstructed kidney. Total NOS activity and iNOS activity of the obstructed kidney were increased by 1D11 treatment. The present study strongly suggests that an antibody to TGF-beta is a promising agent to prevent renal tubular fibrosis and apoptosis in UUO.

Role of hyperlipidemia in progressive renal disease: Focus on diabetic nephropathy

It has been suggested that lipids promote renal injury and that 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors confer renoprotection in certain renal diseases, including diabetic nephropathy. Sprague-Dawley rats were randomized to sham, subtotal nephrectomy (STNx) or STNx + atorvastatin groups. After 12 weeks, proteinuria, renal function, glomerular injury, renal transforming growth factor-beta (TGF-beta) gene expression and macrophage (ED1-positive cells) accumulation were assessed. In addition, the effects of HMG CoA reductase in human diabetic nephropathy were reviewed. Atorvastatin therapy was associated with a modest reduction in proteinuria and glomerulosclerosis without influencing lipid levels or renal function in STNx rats. These effects were associated with decreased renal TGF-beta 1 gene expression and less glomerular and tubulointerstitial macrophage accumulation. The renoprotective effects of HMG CoA reductase inhibitors in both insulin- and non-insulin-dependent diabetic subjects with either incipient or overt nephropathy appear to be highly variable. HMG CoA reductase inhibition appears to confer renoprotection via effects on prosclerotic cytokines such as TGF-beta and macrophage accumulation, independent of their lipid-lowering properties. The role of lipid-lowering agents in early or overt diabetic nephropathy remains to be fully ascertained.

The transforming growth factor beta system in kidney disease and repair: recent progress and future directions.

Transforming growth factor beta is a multifunctional polypeptide growth factor implicated in a variety of renal diseases. The expression of transforming growth factor beta is enhanced in renal diseases and available evidence suggests that its activity in promoting the synthesis of extracellular matrix plays a crucial role in fibrotic deposition and the decline in renal function. Transforming growth factor beta is, however, also expressed in response to renal injury and may play an important role in normal repair processes. It appears that renal diseases may result from the inappropriate regulation of transforming growth factor beta expression. The determination of the factors that mediate transforming growth factor beta activity will be of primary importance in elucidating the mechanisms leading to renal disease or repair after injury. Both in-vitro and in-vivo studies have demonstrated that proteolytic activity, thrombospondin-1, elevated glucose, angiotensin II, oxidant stress and hemodynamic forces regulate transforming growth factor beta activity through both transcriptional and post-transcriptional mechanisms. In some cases, therapies that may partly disrupt renal transforming growth factor beta activity have shown promise in slowing the progression to end-stage renal disease.

Pharmacologic control of angiotensin II ameliorates renal disease while reducing renal TGF-beta in experimental mesangioproliferative glomerulonephritis

We evaluated the effect of blocking angiotensin II (AngII) on the development of proteinuria and glomerular injury in antithymocyte serum (ATS) glomerulonephritis. Disease was induced in Sprague-Dawley rats by a single intravenous injection of rabbit ATS. Three groups of rats were considered: group 1 (n = 13), ATS rats with no therapy; group 2 (n = 13), ATS rats treated with angiotensin-converting enzyme inhibitor (40 mg/L lisinopril in the drinking water); and group 3 (n = 13), ATS rats treated with AngII receptor antagonist (50 mg/L L-158,809 in the drinking water). Treatment started 3 hours after ATS injection and lasted 4 days. An additional group of control rats (group 4, n = 13) received preimmune serum. At day 4, ATS rats developed proteinuria (46+/-5 mg/d v control 12+/-1 mg/d; P < 0.01), which was prevented by both lisinopril and L-158,809 (14+/-0.2 mg/d and 15+/-1.6 mg/d, respectively, P < 0.01 v ATS). Systolic blood pressure was comparable in ATS rats and in controls (119+/-4 mm Hg v 120+/-2 mm Hg). Systolic blood pressure values were significantly decreased after either lisinopril or L-158,809 (104+/-3 mm Hg and 101+/-5 mm Hg, respectively; P < 0.01 v ATS). Serum creatinine levels were similar in all groups. Quantitation of proliferating cells and macrophages by analysis of proliferating cell nuclear antigen-positive and ED1-positive cells/glomerular cross-section showed a marked increase in proliferating cell nuclear antigen-positive cells in glomeruli of ATS rats over controls (12.6+/-0.5 cells/glomerular cross-section v 1.9+/- 0.2 cells/glomerular cross-section; P < 0.01), which was significantly (P < 0.01) prevented by both treatments (lisinopril, 5.7+/-1.0 cells/glomerular cross-section; L-158,809, 4.8+/-1.5 cells/glomerular cross-section). The increase in ED1-positive cells (10+/-0.7 cells/glomerular cross-section v controls, 1.8+/-0.2 cells/glomerular cross-section; P < 0.01) was also significantly (P < 0.01) reduced by lisinopril and L-158,809 (4.1+/-0.7 cells/glomerular cross-sections and 2.6+/-0.6 cells/glomerular cross-section, respectively). Blocking of AngII activity prevented almost completely the formation of microaneurysms in ATS rats (percent of glomeruli with microaneurysms: ATS, 11.5%+/-3.5%; ATS + lisinopril, 0.4%+/-0.2%; ATS + L-158,809, 0.8%+/-0.8%; controls, 0%). Because AngII is a potent inducer of renal transforming growth factor-beta (TGF-beta), a cytokine involved in the regulation of cell proliferation, matrix deposition, and monocyte migration (which is overexpressed in the kidney of ATS rats), we then evaluated the effect of AngII inhibitors on renal gene expression of TGF-beta1 and on urinary TGF-beta1. TGF-beta1 mRNA levels in kidneys of ATS rats were 3.6-fold higher than those of controls and were reduced by 46% and 32% after treatment with lisinopril and L-158,809, respectively. Urinary TGF-beta1 excretion increased in ATS (37.3+/-6.0 ng/d v controls, 13.8+/-3.4 ng/d; P< 0.01) but was normalized by lisinopril and L-158,809 (7.6+/-1.9 ng/d and 6.4+/-0.4 ng/d, respectively; P < 0.01). Thus, in ATS, blocking AngII synthesis prevents proteinuria and reduces glomerular cell proliferation and inflammatory cell infiltration, possibly by reducing excessive renal TGF-beta synthesis. These findings may be relevant for future strategies in the treatment of human mesangioproliferative glomerulonephritis. (Am J Kidney Dis 1998 Mar;31(3):453-63)