Up-regulation of protease-activated receptor-1 in diabetic glomerulosclerosis

Patients with diabetes are under a hypercoagulable state leading to generation of thrombin. It is not known whether thrombin plays a role in the progression of diabetic nephropathy. We analyzed gene expression of two thrombin receptors, protease-activated receptor-1 (PAR-1) and PAR-4 in the kidney of diabetic db/db mice. Mice developed hyperglycemia from 7 to 10 weeks of age and showed renal abnormalities such as mesangial expansion and urinary albumin excretion at 10 weeks of age. PAR-1 mRNA was up-regulated in isolated glomeruli in db/db mice compared with age-matched db/m littermates, but PAR-4 mRNA was not. In situ hybridization studies showed that PAR-1 mRNA was detected mainly at the glomerulus, and that intensive signals were observed in mesangial cells and podocytes. The up-regulation of PAR-1 in glomeruli in diabetic mice may play a role in the progression of glomerulosclerosis and abnormal urinary albumin excretion in diabetic nephropathy.     

Dissection of protease-activated receptor-1-dependent and -independent responses to thrombin in skeletal myoblasts

Thrombin exerts a number of effects on skeletal myoblasts in vitro. It stimulates proliferation and intracellular calcium mobilization and inhibits differentiation and apoptosis induced by serum deprivation in these cells. Many cellular responses to thrombin are mediated by protease-activated receptor-1 (PAR-1). Expression of PAR-1 is present in mononuclear myoblasts in vitro, but repressed when fusion occurs to form myotubes. In the current study, we used PAR-1-null mice to determine which of thrombin's effects on myoblasts are mediated by PAR-1. Thrombin inhibited fusion almost as effectively in cultures prepared from the muscle of PAR-1-null myoblasts as in cultures prepared from wild-type mice. Apoptosis was inhibited as effectively in PAR-1-null myoblasts as in wild-type myoblasts. These effects in PAR-1-null myoblasts were mediated by a secreted inhibitor of apoptosis and fusion, as demonstrated previously for normal rat myoblasts. Thrombin failed to induce an intracellular calcium response in PAR-1-null myoblast cultures, although these cells were able to mobilize intracellular calcium in response to activation of other receptors. PAR-1-null myoblasts also failed to proliferate in response to thrombin. These results demonstrate that thrombin's effects on myoblast apoptosis and fusion are not mediated by PAR-1 and that PAR-1 is the only thrombin receptor capable of inducing proliferation and calcium mobilization in neonatal mouse myoblasts.     

Up-regulation of protease-activated receptor-2 by bFGF in cultured human synovial fibroblasts

Protease-activated receptors (PARs) have been implicated in the development of acute and chronic inflammatory responses. We have examined the expression of mRNA for PARs and their regulation by growth factors and cytokines in synovial fibroblasts derived from patients with rheumatoid arthritis (RA). Messenger RNA for PAR-1, -2 and -3 was detected in these cells, but not that for PAR-4. Expression of mRNA for PAR-2 was up-regulated by bFGF in a concentration-dependent manner, whereas expression of mRNA for PAR-1 and PAR-3 was not affected. Levels of mRNA encoding PAR-1, PAR-2 and PAR-3 did not increase in response to IL-1beta and TNF-alpha. Expression of mRNA for PAR-2 was maximal 12 h after addition of bFGF, and maximal levels of immunoreactive PAR-2 were reached after 24 h. Furthermore, PAR-2 agonist peptide (SLIGKV-NH(2)), but not the inactive reverse peptide (VKGILS-NH(2)), induced transitory cytosolic Ca(2+) mobilization in cells, and its response was increased by pretreatment with bFGF. An important role could be played by bFGF in the regulation of functional PAR-2 expression in cultured RA synovial fibroblasts.     

Metabonomic analysis of the therapeutic effect of exendin-4 for the treatment of tBHP-induced injury in mouse glomerulus mesangial cells

Although previous studies have reported the protective effect of glucagon-like peptide-1 (GLP-1) in diabetes nephropathy, the molecular mechanism such as nephroprotection remains elusive. In this study, we explored the molecular mechanism of exendin-4 as an GLP-1 receptor agonist for the treatment of tert-butyl hydroperoxide (t-BHP)-induced injury in mouse glomerulus mesangial cells (SV40 MES 13 cells) via an NMR-based metabonomic analysis. We found that exendin-4 protected mesangial cells from t-BHP-mediated toxicity, decreased the percentage of t-BHP-treated cells undergoing apoptosis, and restored glucose consumption in the t-BHP-treated group. A supervised partial least-squares discriminant analysis (PLS-DA) revealed that the metabolic profiles could be distinguished between the control, t-BHP-treated, and exendin-4-pretreated groups. Our findings indicate that exendin-4 pretreatment can cause distinct changes in energy, glycerol phospholipid, and amino acid metabolism. Our study provides novel insight into the metabolic mechanism of exendin-4-mediated nephroprotective effects.     

Induction of monocyte chemoattractant protein-1 release from A549 cells by agonists of protease-activated receptor-1 and -2

Hypersecretion of cytokines and serine proteases has been observed in asthma. However, the influence of proteases and protease-activated receptors (PARs) on monocyte chemoattractant protein-1 (MCP-1) release from airway epithelial cells remains largely unknown. In the present study, A549 cells were challenged with agonists of PARs, and levels of MCP-1 released in the supernatant and mRNA expression were examined by ELISA and real time polymerase chain reaction (PCR), respectively. The results show that thrombin, tryptase, elastase and trypsin induced an up to 6.5-, 1.8-, 1.6-, and 3.1-fold increase in MCP-1 release from A549 cells, respectively, following a 16-h incubation period. The protease-induced secretion of MCP-1 can be abolished by specific protease inhibitors. Agonist peptides of PAR-1 and PAR-2 stimulate MCP-1 secretion up to 15- and 12.7-fold, respectively. Real-time PCR showed that MCP-1 mRNA is up-regulated by the serine proteases tested and by agonist peptides of PAR-1 and PAR-2. In conclusion, serine proteases can stimulate MCP-1 release from A549 cells possibly through a PARs-related mechanism, suggesting that they are likely to contribute to MCP-1-related airway inflammatory disorders in man.     

Association between endothelin-1 and collagen deposition in db/db diabetic mouse kidneys

Endothelin-1 has been implicated in diabetic kidney injury, but there are few firm data establishing the temporal and spatial expression of kidney endothelin-1 in diabetes. We performed an immunohistochemical and histopathological analysis to determine endothelin-1 peptide expression in the kidneys of diabetic db/db mice and non-diabetic db/m controls. Diabetic mice were studied at 8 weeks, before histological damage is evident, and again at 16 weeks, when significant glomerular injury has occurred. Urinary endothelin-1 was 6.2- and 3.6-fold higher in 8- and 16-week diabetic mice compared to age-matched controls (P<0.01 db/db vs. db/m). Compared to non-diabetic kidneys, immunoreactive endothelin-1 was first elevated 2.5-fold (P=0.02) in the tubulointerstitial compartment at 8-week and remained high (3.8-fold, P<0.01) at 16 weeks. In contrast, glomerular endothelin-1 was elevated 3.2-fold (P=0.03) only in 16-week diabetic mice. Glomerular and tubulointerstitial endothelin-1 were unchanged in 8- and 16-week non-diabetic mice. Elevated endothelin-1 in diabetic mice associated temporally and spatially with collagen deposition, especially in the tubulointerstitial compartment. The localization of kidney endothelin-1 is consistent with a role for this peptide in renal fibrogenesis. These results also highlight the potential role of ET-1 in the pathogenesis of early tubulointerstitial changes in diabetes.     

CCN-2 is up-regulated by and mediates effects of matrix bound advanced glycated end-products in human renal mesangial cells

CCN-2, also known as connective tissue growth factor (CCN-2/CTGF) is a cysteine rich, extracellular matrix protein that acts as a pro-fibrotic cytokine in tissues in many diseases, including in diabetic nephropathy. We have published that soluble advanced glycation end products (AGEs), that are present in increased amounts in diabetes, induce CCN-2. However in vivo AGEs are known to be heavily tissue bound and whether matrix bound AGEs regulate CCN-2 has not been investigated. In this study we determined in human renal mesangial cells if CCN-2 is induced by matrix associated AGEs and if CCN-2 may then secondarily mediate effects of matrix AGEs on extracellular matrix expansion. Data generated show that CCN-2 mRNA and protein expression are induced by matrix bound AGEs, and in contrast, this was not the case for TGF-β1 mRNA regulation. Using CCN-2 adenoviral anti-sense it was found that CCN-2 mediated the up-regulation of fibronectin and the tissue inhibitor of matrix metalloproteinase, TIMP-1, that was caused by matrix bound AGEs. In conclusion, CCN-2 is induced by non-enzymatically glycated matrix and it mediates downstream fibronectin and TIMP-1 increases, thus through this mechanism potentially contributing to ECM accumulation in the renal glomerulus in diabetes.     

Differential expression of type IV collagen isoforms in rat glomerular endothelial and mesangial cells

Type IV collagen, which is encoded by six genetically distinct alpha-chains (alpha 1-alpha 6), is a major component of the kidney glomerulus. The alpha 1(IV) and alpha 2(IV) chains are present predominantly in the mesangial matrix, whereas the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains are localized almost exclusively to the glomerular basement membrane (GBM). Thickening of the GBM and expansion of the mesangial matrix are believed to contribute to the pathogenesis of diabetic nephropathy. In the present study, we evaluated the expression of alpha 1(IV), alpha 3(IV), and alpha 5(IV) chains in rat glomerular endothelial (GEndC) and mesangial cells (GMC). Under physiological concentrations of glucose (5 mM), alpha 1(IV) and alpha 5(IV) chains were detectable in GMCs, with an obvious absence of alpha 3(IV) chain. All three isoforms tested were present in GEndCs. At diabetic concentrations of glucose (25 mM), alpha 1(IV) was up-regulated in GMCs, whereas expression level of alpha 1(IV) remained unaltered in GEndCs. The alpha 3(IV) and alpha 5(IV) chains were up-regulated in GEndCs, but remained unchanged in GMCs under diabetic glucose concentrations (25 mM). Collectively, our results demonstrate that GMC might contribute to mesangial matrix expansion, mediated by alpha 1(IV) collagen, while GEndC might contribute to thickening of GBM, mediated by alpha 3(IV) collagen, in patients with diabetic nephropathy.     

Novel triazine-based pyrimidines suppress glomerular mesangial cells proliferation and matrix protein accumulation through a ROS-dependent mechanism in the diabetic milieu

Diabetic nephropathy (DN) is one of the most serious complications of diabetes worldwide. It is depicted as the leading cause of end-stage renal disease. Oxidative stress plays a key role in hyperglycemia-induced DN. The preparation and characterization of novel mono-, di-, and trisubstituted-s-triazines endowed with uracil and/or thymine are described in this paper. The synthesis of the title compounds was realized through selective nucleophilic substitution reactions of cyanuric chloride with the corresponding hydrazide nucleobases. In this study, we assessed the effects of these derivatives on the progression of diabetic nephropathy. Our results show that trisubstituted-s-triazines endowed with acylhydrazides attenuate high-glucose induced glomerular mesangial cells proliferation and matrix protein accumulation in vitro. Notably, these derivatives also display anti-oxidative properties. This suggests that the novel trisubstituted-s-triazine derivatives provide renal protection through a reactive oxygen species (ROS)-dependent mechanism. Our data provide evidence that these derivatives may serve as potential therapeutic candidates in the treatment of DN.     

Discovery of a coumarin derivative as Nrf2 activator mitigating oxidative stress and fibrosis in mesangial cells under high glucose

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes mellitus. Oxidative stress and fibrosis largely contribute to the progression of DN. Recently, Nrf2 was found to be a potential target preventing DN. In the discovery of novel Nrf2 activators for the treatment of DN, we have evaluated coumarin derivatives from Wikstroemi indiaca. Molecular docking results have shown compound 4 could bind to Keap1 and activate Nrf2 significantly. Cell-based assays have revealed compound 4 activated Nrf2 and attenuated oxidative stress and fibrosis induced by high glucose in mesangial cells. Meanwhile, it was validated that disruption of the interaction between Keap1 and Nrf2 was involved in the activation of Nrf2 by compound 4 in mesangial cells under high glucose.     

Berberine inhibits aldose reductase and oxidative stress in rat mesangial cells cultured under high glucose

Diabetic nephropathy (DN), one of the most serious microvascular complications of diabetes mellitus, is a major cause of end-stage renal disease. Berberine is one of the main constituents of Coptidis rhizoma and Cortex phellodendri. In the present study, we examined effects of berberine (BBR) on renal injury in streptozotocin-induced diabetic rats, and on the changes of aldose reductase (AR) and oxidative stress in cultured rat mesangial cells exposed to high glucose. Fasting blood glucose, blood urea nitrogen, creatinine, and urine protein over 24 h were detected by using the commercially available kits. Cell proliferation, collagen synthesis, aldose reductase (AR), superoxide anion, superoxide dismutase (SOD), and malondialdehyde (MDA) were detected, respectively, by different methods. In streptozotocin-induced diabetic rats, fasting blood glucose, blood urea nitrogen, creatinine, and urine protein over 24 h were significantly decreased in rats treated with 200 mg/kg berberine for 12 weeks compared with diabetic control rats (P < 0.05). This was accompanied by a reduced AR activity and gene expression at both mRNA and protein levels. In cultured rat mesangial cells exposed to high glucose, incubation of BBR significantly decreased cell proliferation, collagen synthesis and AR activity as well as its mRNA and protein levels compared with control cells (P < 0.05). In vitro, BBR also significantly increased SOD activity and decreased superoxide anion and MDA compared with control cells (P < 0.05). These results suggested that BBR could ameliorate renal dysfunction in DN rats, which may be ascribed to inhibition of AR in mesangium, reduction of oxidative stress, and amelioration of extracellular matrix synthesis and cell proliferation. Further studies are warranted to explore the role of AR in DN and the therapeutic implications by AR inhibitors such as BBR.     

MicroRNA-21 protects from mesangial cell proliferation induced by diabetic nephropathy in db/db mice

Diabetic nephropathy (DN) is a major diabetic complication. But the initiating molecular events triggering DN are unknown. Recent researches have addressed the role of microRNAs in diabetes and its complications. In this study, we looked for microRNAs expression during early DN, and showed microRNA-21 (miR-21) expression was downregulated in response to early DN in vitro and in vivo. Over-expression of miR-21 inhibited proliferation of mesangial cells and decreased the 24-h urine albumin excretion rate in diabetic db/db mice. Moreover, we identified PTEN as a target of miR-21. We also found PI3 K and p-Akt increased in miR-21 treated mesangial cells and db/db mice. Overall, these studies for the first time provide evidence for the potential role of miR-21 in early DN.     

Cigarette smoke concentrate increases 8-epi-PGF2alpha and TGFbeta1 secretion in rat mesangial cells

Epidemiological studies have shown that cigarette smoke, an oxidant agent, is a risk factor for the development of diabetic nephropathy (DN), in which pathogenesis transforming growth factor beta(1) (TGFbeta(1)) plays a key role. In our experimental model we exposed mesangial cell cultures to cigarette smoke concentrate (CSC) to study the effect of smoking on the pathogenesis of DN. Thus, we analyzed the effect of CSC on TGFbeta(1) and lipid peroxidation (8-epi-PGF(2alpha)) in rat mesangial cells. Furthermore, since the protein kinase C (PKC) pathway appears to be a key factor for the enhanced production of TGFbeta(1), we also analyzed the effect of the selective PKCbeta inhibitor LY379196 on TGFbeta(1) response to CSC. CSC induced an increase of both TGFbeta(1) and 8-epi-PGF(2) compared to basal conditions (5 mM glucose). The CSC-induced increase in TGFbeta(1) secretion was significantly suppressed by LY379196. These data suggest that smoking could increase TGFbeta(1) production, probably due to oxidative stress and PKCbeta activation. This finding supports the concept that smoking is a risk factor for DN development.     

Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGF-beta-induced fibronectin in mesangial cells

Activation of the transforming growth factor-beta (TGF-beta) system has been implicated in the pathological changes of diabetic nephropathy such as renal hypertrophy and accumulation of extracellular matrix. Streptozotocin-induced diabetic mice were used to examine whether the Smad pathway, which transduces the TGF-beta signal, is activated in the diabetic kidney, employing Southwestern histochemistry with labeled Smad-binding element (SBE) oligonucleotides and immunoblotting of nuclear protein extracts for Smad3. Mouse mesangial cells were used to study the role of Smads in mediating the effects of high glucose and TGF-beta on fibronectin expression, using transient transfections of Smad expression vectors and TGF-beta-responsive reporter assays. By Southwestern histochemistry, the binding of nuclear proteins to labeled SBE increased in both glomeruli and tubules at 1, 3, and 6 weeks of diabetes. Likewise, immunoblotting demonstrated that nuclear accumulation of Smad3 was increased in the kidney of diabetic mice. Both increases were prevented by insulin treatment. In mesangial cells, high glucose potentiated the effect of low-dose TGF-beta1 (0.2ng/ml) on the following TGF-beta-responsive constructs: 3TP-Lux (containing AP-1 sites and PAI-1 promoter), SBE4-Luc (containing four tandem repeats of SBE sequence), and the fibronectin promoter. Additionally, Smad3 overexpression increased fibronectin promoter activity, an effect that was enhanced by high ambient glucose or treatment with TGF-beta1 (2ng/ml). The TGF-beta-stimulated activity of the fibronectin promoter was prevented by transfection with either a dominant-negative Smad3 or the inhibitory Smad7. We conclude that hyperglycemia activates the intrarenal TGF-beta/Smad signaling pathway, which then promotes mesangial matrix gene expression in diabetic nephropathy.     

Aldosterone promotes fibronectin production through a Smad2-dependent TGF-beta1 pathway in mesangial cells

Accumulating evidence demonstrates that aldosterone can cause extra-cellular matrix (ECM) accumulation, in addition to regulating sodium and potassium homeostasis. Increased extra-cellular matrix production by renal glomerular mesangial cells has been suggested to be involved in pathogenesis of glomerular sclerosis. The present studies examine whether aldosterone is also produced in renal mesangial cells, and the effect of aldosterone on ECM accumulation in these cells. In cultured renal mesangial cells, aldosterone synthase (CYP11B2), mineralocorticoid receptor (MR), and 11beta-HSD2 mRNA expressions were detected by RT-PCR. The ability of renal mesangial cells to produce aldosterone was confirmed by directly detecting aldosterone in culture medium via radioimmunoassay. Real-time RT-PCR showed that the expression of CYP11B2 mRNA in mesangial cells was significantly enhanced by AngII (P<0.001) and by potassium (P<0.05). Exposure of the cultured mesangial cells to aldosterone significantly increased fibronectin production from 12.4+/-1.9 to 74.6+/-16.8ng/ml (P<0.05). The aldosterone induced fibronectin production was abolished by aldosterone receptor antagonist spironolactone. Aldosterone also increased the TGF-beta1 reporter luciferase activity from 0.8+/-0.1 to 1.7+/-0.1 (P<0.05). Immunoblot showed TGF-beta1 protein expression was increased following aldosterone treatment. Blocking TGF-beta1 signaling pathway by knocking down Smad2 significantly blunted the aldosterone induced fibronectin production. The present studies indicate that renal mesangial cell is a target of local aldosterone action, which promotes ECM protein fibronectin production via TGF-beta1/Smad2 signaling pathway.