Anguo Deng

Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro.

Connective tissue growth factor (CTGF) has been reported to play an important role in mediating the profibrotic effects of transforming growth factor‐β (TGF‐β) in various renal diseases. To elucidate the role of CTGF in renal tubular epithelial‐myofibroblast transdifferentiation, we examined the expression of α‐smooth muscle actin (α‐SMA), vimentin, tenascin‐C, and collagen IV expression upon the stimulation of CTGF in cultured human proximal tubular epithelial cell line (HKC), and further investigated the effects of endogenous CTGF blockade on the transdifferentiation process induced by TGF‐β. It is revealed that upon the stimulation of recombinant human CTGF (rhCTGF, 2.5 or 5.0 μg/L), the expression of α‐SMA and tenascin‐C mRNA increased significantly (p < 0.01), while collagen IV gene expression decreased significantly (p < 0.01), all in a dose‐dependent manner. The percentage of α‐SMA‐positive cells was significantly larger in the rhCTGF‐stimulated groups than that in negative control (38.9%, 65.5% vs. 2.4%, respectively, p < 0.01) as confirmed by flow cytometry. Both cytoplasmic and secretory tenascin‐C expression was upregulated by the stimulation of rhCTGF (p < 0.01). Under this condition, collagen IV secreted into the culture media was lowered markedly (p < 0.01). On RT‐PCR analysis, TGF‐β1 upregulated CTGF gene expression, preceding that of α‐SMA. The α‐SMA mRNA expression induced by TGF‐β1 was significantly inhibited by CTGF antisense oligodeoxynucleotide (ODN) transfection (p < 0.01). With prolonged incubation time, CTGF antisense ODN also inhibited intracellular α‐SMA protein synthesis, as demonstrated by indirect immuno‐fluorescence. So it is concluded that CTGF could promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblasts in vitro, both directly and as a downstream mediator of TGF‐β, and CTGF blockade would be a possible therapeutic target against tubulointerstitial fibrosis.

Effect of telmisartan on expression of protein kinase C-α in kidneys of diabetic mice

AbstractAim:To investigate the effects of angiotensin receptor blocker (ARB) telmisartan on the expression and distribution of protein kinase C (PKC)-α in the kidneys of diabetic mice.Methods:Diabetic mice were induced with streptozotocin and a group of them were randomly selected for treatment with telmisartan. After 6 weeks, the expression and localization of PKC-α in the renal cortex, and the outer and inner medulla were assessed by immunohistochemistry and semiquantitative Western blotting. In addition, expressions of PKC-α, transforming growth factor-β1 (TGF-β1), and vascular endothelial growth factor (VEGF) in glomeruli were measured by semiquantitative immunohistochemistry.Results:Diabetic and normal mice showed similar distributions of PKC-α in the kidneys. The expression of PKC-α was found in glomeruli, epithelial cells of proximal tubules, and medullary-collecting duct, while not in the medullary and cortical thick ascending limb, and was different in the epithelial cells of proximal tubules of diabetic nephropathy (DN) mice, PKC-α was mostly translocated from the basement membrane to the apical membrane, whereas it was largely translocated from the apical membrane to the basement membrane in epithelial cells of the inner medullary-collecting duct. Western blotting detected increased expression of PKC-α in the renal cortex and outer medulla, but not in the inner medulla of DN mice. Enhanced expressions of PKC-α, TGF-β1, and VEGF were shown in the glomeruli of DN mice, where PKC-α exhibited a correlation to VEGF, but no correlation to TGF-β1. ARB telmisartan attenuated alterations of PKC-α as mentioned earlier in the DN mice.Conclusion:Our findings suggest that PKC-α may play a role in the pathogenesis of DN, and that the nephroprotective effects of ARB telmisartan may be partly associated with its influence on PKC-α.

Changes in angiopoietin expression in glomeruli involved in glomerulosclerosis in rats with daunorubicin-induced nephrosis

AbstractAim:To study the potential pathological role of endogenous angiopoietins in daunorubicin-induced progressive glomerulosclerosis in rats.Methods:Seventy male Wistar rats were allocated randomly into a daunorubicin group (DRB; n=40) or a control group (n=30). The rats in the DRB group were injected with DRB (15 mg/kg), in their tails. Subsequently, at intervals of 1, 2, 4, 6, 8, and 12 weeks, 5 male Wistar rats in each group were chosen randomly for 24 h urinary protein quantitative measurements (24 h UPQM), and determination of plasma tumor necrosis factor α (TNF-α), angiopoietin-1 (Ang1), and angiopoietin-2 (Ang2) levels. Kidney sections were examined by electron microscopy, Periodic Acid Schiff (PAS) staining, immunohistochemical staining and in situ hybridization histochemistry.Results:As glomerulosclerosis progressed in the DRB group, expression of Ang1 mRNA and protein in glomeruli decreased and expression of TNF-α protein, Ang2 mRNA and protein in glomeruli increased. Expression of Ang1 mRNA and protein in glomeruli were negatively correlated with 24 h UPQM, Fn protein expression, and mean area of extracellular matrix (MAECM). In comparison, expression of Ang2 mRNA and protein in glomeruli were positively correlated with 24 h UPQM, Fn protein expression and MAECM; furthermore, there was a positive correlation between plasma Ang2 and 24 h UPQM. Plasma TNF-α and expression of TNF-α in glomeruli were positively correlated with expression of Ang2 mRNA and protein in glomeruli. There was a negative correlation between Ang1 protein expression and Ang2 protein expression in glomeruli.Conclusion:During DRB-induced glomerulosclerosis, podocyte injury led to a shift in the balance of Ang1 and Ang2 in glomeruli. Increased TNF-α in plasma and glomeruli may upregulate Ang2 expression in glomeruli. Elevated Ang2 in both plasma and glomeruli may mediate protein permeability through the glomerular filtration barrier. Moreover, local expression of Ang2 may facilitate the progress of glomerulosclerosis by upregulating a component expression of extracellular matrix.

Downregulation of CD2-associated protein impaired the physiological functions of podocytes.

Emerging evidences show that CD2‐associated protein (CD2AP) is involved in podocyte injury and the pathogenesis of proteinuria. However, the exact molecular mechanism by which CD2AP exerts its biological function is elusive. We knocked down CD2AP gene by target siRNA in conditionally immortalized mouse podocytes, which showed lowered cell adhesion and spreading ability (P < 0.05). At the same time, cell cycle was arrested in G2/M phase (P < 0.05), and pathologic nuclear division could easily be seen in CD2AP siRNA‐transfected podocytes. The proliferation of podocytes were also inhibited significantly by CD2AP siRNA transfection (P < 0.05). Further study revealed disordered distributions of F‐actin, as well as lowered nephrin expression and phosphorylation in podocytes. These data suggest that CD2AP may play a crucial role in maintaining the normal function of podocytes and lowered CD2AP causes podocyte injury by disrupting the cytoskeleton and disturbing the nephrin‐CD2AP signaling pathway.

High glucose promotes the CTGF expression in human mesangial cells via serum and glucocorticoid-induced kinase 1 pathway.

The role of serum and glucocorticoid-induced kinase 1 (SGK1) pathway in the connective tissue growth factor (CTGF) expression was investigated in cultured human mesangial cells (HMCs) under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with pIRES2-EGFP-S422D hSGK1 (SD) could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with pIRES2-EGFP (FP) under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with pIRES2-EGFP-K127N hSGK1 (KN) could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.SummaryThe role of serum and glucocorticoid-induced kinase 1 (SGK1) pathway in the connective tissue growth factor (CTGF) expression was investigated in cultured human mesangial cells (HMCs) under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with pIRES2-EGFP-S422D hSGK1 (SD) could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with pIRES2-EGFP (FP) under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with pIRES2-EGFP-K127N hSGK1 (KN) could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.

Role of connective tissue growth factor in extracellular matrix degradation in renal tubular epithelial cells

In order to investigate the effects of connective tissue growth factor (CTGF) antisense oligodeoxynucleotide (ODN) on plasminogen activator inhibitor-1 (PAI-1) expression in renal tubular cells induced by transforming growth factor β1 (TGF-β1) and to explore the role of CTGF in the degradation of renal extracellular matrix (ECM), a human proximal tubular epithelial cell line (HKC) was cultured in vitro. Cationic lipid-mediated CTGF antisense ODN was transfected into HKC. After HKC were stimulated with TGF-β1 (5 μg/L), the mRNA level of PAI-1 was detected by RT-PCR. Intracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 in the media was determined by Western blot. The results showed that TGF-β1 could induce tubular CTGF and PAI-1 mRNA expression. The PAI-1 mRNA expression induced by TGF-β1 was significantly inhibited by CTGF antisense ODN. CTGF antisense ODN also inhibited intracellular PAI-1 protein synthesis and lowered the levels of PAI-1 protein secreted into the media. It was concluded that CTGF might play a crucial role in the degradation of excessive ECM during tubulointerstitial fibrosis, and blocking the biological effect of CTGF may be a novel way in preventing renal fibrosis.

Apoptosis signaling pathway in a subtotal nephrectomy rat model.

To investigate the role and mechanisms of apoptosis and apoptosis signaling pathway in 5/6 nephrectomy rat model (SNX), the mRNA and protein levels of caspase-3,-8,-9 and apoptosis were detected by in situ end labeling (TUNEL), immunohistochemistry, RT-PCR, Western-blotting 1, 2, 4, 8, 12, 16, 26 and 40 weeks after 5/6 nephrectomy rat model was made respectively. The rats in the model group developed glomerular sclerosis and renal interstitial fibrosis. The number of the apoptototic cells in glomeruli, renal tubule and renal interstitium was remarkably higher in the model group than that in the control group (P<0.05, P<0.01). Changes of mRNA and protein level of caspase-3,-8,-9 had the same tendency and was up-regulated wavily in the rat model compared with the control group (P<0.05). Peaks in model appeared on the 4th and the 40th week respectively. The growth amplitude of caspase-9 was remarkably higher than that of caspase-8. It is concluded that the development of 5/6 nephrectomy rat model was correlated with the apoptosis of glomeruli, renal tubule and renal interstitium. Both of death receptor and mitochondria signaling path ways are involved in the process and the latter might play a primary role.

Effect of Micardis on the expression of renal medulla aquaporin-2 in diabetic mice.

SummaryIn current study, the effect of angiotensin receptor blocker Micardis on the localization and expression of aquaporin-2 (AQP2) was investigated in the renal medullary collecting duct of mice with diabetic nephropathy (DN). Mice were divided into three groups: normal group, DN group and Micardis-treated group. Six weeks after establishment of STZ-induced DN model in mice, the expression of AQP2 in renal medulla was detected measured by semiquantitative immunofluorescence histochemistry and Western blot techniques, and the localization of AQP2 by confocal immunofluorescence laser scanning microscopy. The results showed that the urinary osmolality was decreased in DN group as compared with normal group (2.39±0.11 vs 3.16±0.16, P<0.05). Although the localization of AQP2 on the renal medulla was unchanged, the expression of AQP2 was increased significantly in DN group as compared with normal group. Micardis could partly attenuate above changes. It was concluded that treatment with Micardis could partly rectify the abnormal expression of AQP2 in renal medulla of DN mice, which suggested that rennin-angiotensin system (RAS) is implicated in the pathogenesis of DN by regulating the expression of AQP2.In current study, the effect of angiotensin receptor blocker Micardis on the localization and expression of aquaporin-2 (AQP2) was investigated in the renal medullary collecting duct of mice with diabetic nephropathy (DN). Mice were divided into three groups: normal group, DN group and Micardis-treated group. Six weeks after establishment of STZ-induced DN model in mice, the expression of AQP2 in renal medulla was detected measured by semiquantitative immunofluorescence histochemistry and Western blot techniques, and the localization of AQP2 by confocal immunofluorescence laser scanning microscopy. The results showed that the urinary osmolality was decreased in DN group as compared with normal group (2.39±0.11 vs 3.16±0.16, P<0.05). Although the localization of AQP2 on the renal medulla was unchanged, the expression of AQP2 was increased significantly in DN group as compared with normal group. Micardis could partly attenuate above changes. It was concluded that treatment with Micardis could partly rectify the abnormal expression of AQP2 in renal medulla of DN mice, which suggested that rennin-angiotensin system (RAS) is implicated in the pathogenesis of DN by regulating the expression of AQP2.

Effects of anthopleurin-Q on the intracellular free Ca2+ concentration in cultured rat cortical neurons.

The present study was designed to investigate the mechanism underlying the intracellular free Ca(2+) concentration ([Ca(2+)]i) modulated by Anthopleurin-Q (AP-Q), a sea anemone toxin, using whole-cell patch clamp and fluorescence digital imaging techniques. Results indicated that the overall Ca(2+) concentration could be augmented in presence of AP-Q. The increase of [Ca(2+)]i induced by AP-Q was eliminated in Na(+)-free solution, Ca(2+)-free solution or in presence of TTX. However, the Ca(2+) increase induced by AP-Q could not be influenced by cyclopiazonic acid (CPA), a specific inhibitor of the endoplasmic reticulum Ca(2+)-ATPase pump. We furthermore demonstrated that voltage-gated calcium channels (VGCCs) blocker verapamil, or inhibitor of the reverse operation Na(+)-Ca(2+) exchanger NiCl2 attenuated AP-Q-induced [Ca(2+)]i elevation. Furthermore, the inactivation process of Na(+) current (I Na ) was significantly delayed with slightly change of its amplitude by AP-Q. These findings demonstrated that neuron voltage-gated Na(+) channels are also targets of AP-Q. Overall, the present results suggested that AP-Q induced calcium influx via Na(+)-dependent activation of voltage-gated sodium channels (VGSCs), VGCCs and reverse operation of the Na(+)/Ca(2+)exchanger.

The effect of ligustrazine on peritoneal transport in peritoneal dialysis

SummaryIn order to investigate the effect of ligustrazine (Lig) i.p. on peritoneal permeability in peritoneal dialysis and its side effects, creatinine was given intravenously and continuously to maintain the high plasma creatinine level. All the rabbits were divided into three groups: normal control group (group A), group B treated with 0.12 % Lig and group C treated with 0.24 % Lig. The peritoneal dialysis of all rabbits lasted 2 h. The plasma and dialysate levels of glucose, protein and creatinine were observed immediate, 30 min, 60 min, 90 min, 120 min after dialysis. Creastinine dialysate/plasma ratio (D/P), protein D/P ratio, glucose D/Do at different time points after dialysis and creatinine mass transfer area coefficient (MTAC) at 120 min were calculated. The structures of peritoneum were observed under optical microscope and electron microscope after continuously intraperitoneal injection of Lig for 14 days. The results showed that the 90-min and 120-min creatinine D/P ratios in the group C were higher than in the group A. The 120-min creatinine MATC in the group C was higher than in the group A. The rabbits treated with Lig did not show significant structure changes of peritoneum and signs of peritoneal irritation. It was suggested that Lig could increase mass transfer ability of peritoneum without significant side effects.