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.

[Role of CD2-associated protein in podocyte differentiation.].

To study the cellular changes and the potential role of CD2-associated protein (CD2AP) in podocyte differentiation, conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium under permissive condition at 33 °C. After transfection with CD2AP small interfering RNA (siRNA) the cells were shifted to non-permissive condition at 37 °C. Simultaneously, untransfected cells were taken as differentiation control. The podocyte proliferation rate was determined by MTT method. The expressions of CD2AP, WT1, synaptopodin and nephrin mRNAs were examined by RT-PCR. CD2AP, WT1 and nephrin protein expressions were examined by Western blot. The distribution of CD2AP, nephrin, F-actin and tubulin in differentiated and undifferentiated podocytes was detected by laser scanning confocal microscopy. The results showed: (1) CD2AP, WT1 and nephrin were stably expressed in differentiated and undifferentiated podocytes while synaptopodin was only expressed in differentiated podocytes. (2) CD2AP and nephrin mRNA and protein expressions were up-regulated during podocyte differentiation (P<0.05). (3) CD2AP and tubulin were distributed in the cytoplasm and perinulcear region in undifferentiated podocytes, and F-actin was predominantly localized to a cortical belt and paralleled to the cell axis. Under differentiation condition, CD2AP distribution profile was presented as peripheral accumulation, tubulin took on fascicular style and F-actin extended into foot processes in podocytes. CD2AP colocalized with nephrin and F-actin in undifferentiated podocytes. (4) After transfection with CD2AP siRNA, the expression of CD2AP was partially inhibited and cell growth was arrested; Synaptopodin, the differentiation podocyte marker, was apparently down-regulated; The differentiation of podocytes was delayed. The results demonstrate that podocyte differentiation is accompanied by cytoskeleton rearrangement and cell morphology change. CD2AP might play an essential role in podocyte differentiation.

[Protein kinase C-betaI, betaII in mouse diabetic nephropathy kidney and its relation to nephroprotective actions of the angiotensin receptor blocker telmisartan].

OBJECTIVE: To investigate the localization and expression of protein kinase C (PKC)-betaI, betaII in diabetic nephropathy (DN) mouse kidney and its relation to angiotensin receptor blocker telmisartan (Micardis). METHODS: Eighteen mice were divided into three groups: normal group, DN group and Micardis-treated group (n = 6, each group). The expression of PKC-betaI, betaII, transforming growth factor- beta 1 (TGF-beta1) and vascular endothelial growth factor (VEGF) in glomeruli was measured by semiquantitative immunofluorescence histochemistry, the localization of PKC-betaI, betaII was detected by confocal immunofluorescence laser scanning microscopy and the expression of PKC-betaI, betaII in renal cortex, outer and inner medulla were evaluated by semiquantitative Western blotting. RESULTS: Compared to normal mice, the expression of PKC-betaI and betaII on apical membrane of proximal tubule epithelial cells of DN mice was significantly increased, whereas the expression of PKC-betaII on cortical and inner medullary collecting duct was decreased. Western blotting detected increasing expression of PKC-betaI in the renal cortex and outer medulla (P < 0.01), and decreasing expression of PKC-betaII in renal cortex of DN mice (P < 0.01). Enhanced expression of PKC-betaI as well as TGF-beta1 and VEGF (P < 0.01) were shown in the glomeruli of DN mice, where the expression of PKC-betaII was decreased (P < 0.05). Meanwhile, PKC-betaI exhibited a positive correlation to TGF-beta1 (r = 0.649, P = 0.030), but no correlation to VEGF (r = 0.387, P = 0.079). Micardis could partly attenuate above changes. CONCLUSION: The localization and expression of PKC-betaI, betaII are altered in DN mice, PKC-betaI, betaII may change the function of proximal tubule and PKC-betaI may contribute to glomerular hypertrophy through influencing the expression of glomerular TGF-beta1. Treatment with Micardis can partly improve the abnormal expression and distribution of PKC-betaI, betaII in kidneys of DN mice, which suggests that renin-angiotensin-system is implicated in the pathogenesis of DN by regulating the expression and activation of PKC-betaI, betaII isoforms.

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

AIM: To investigate the effects of angiotensin receptor blocker (ARB) telmisartan on the expression and distribution of protein kinase C (PKC)-alpha 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-alpha in the renal cortex, and the outer and inner medulla were assessed by immunohistochemistry and semiquantitative Western blotting. In addition, expressions of PKC-alpha, transforming growth factor-beta1 (TGF-beta1), and vascular endothelial growth factor (VEGF) in glomeruli were measured by semiquantitative immunohistochemistry. RESULTS: Diabetic and normal mice showed similar distributions of PKC-alpha in the kidneys. The expression of PKC-alpha 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-alpha 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-alpha in the renal cortex and outer medulla, but not in the inner medulla of DN mice. Enhanced expressions of PKC-alpha, TGF-beta1, and VEGF were shown in the glomeruli of DN mice, where PKC-alpha exhibited a correlation to VEGF, but no correlation to TGF-beta1. ARB telmisartan attenuated alterations of PKC-alpha as mentioned earlier in the DN mice. CONCLUSION: Our findings suggest that PKC-alpha 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-alpha.

[Urine protein from patients with lupus nephritis stimulates transdifferentiation and high expression of collagen in renal tubular cells].

OBJECTIVE: To investigate the effects of urine protein on the renal tubular-interstitial fibrosis in the patients with lupus nephritis (LN). METHODS: Protein was isolated and purified from the urine of six patients with primary LN, 1 male and 5 females, aged 27.4, and incubated with renal tubular cells of the line HK-2 for 0, 1, 2, 12, 24, or 48 h respectively. The mRNA expressions of transforming growth factor beta1(TGF-beta1), collagen I (COL I), and alpha-smooth muscle actin (alpha-SMA) in the HK-2 cells were detected by RT-PCR, and the. protein expressions of TGF-beta1, COL I, and alpha-SMA were detected with Western blotting and indirect immunofluorescence. RESULTS: The urine protein from the LN patients dose-and time-dependently increased the mRNA and protein expressions of TGF-beta1, COL I, and alpha-SMA in the HK-2 cells. The TGF-beta1 mRNA level 48 h after incubation was 0.39 +/- 0.03, significantly higher than that at the beginning of incubation (0.27 +/- 0.02, P < 0.01), and the TGF-beta1 protein level 48 h after incubation was 0.37 +/- 0.03, 1.7 times that at the beginning of incubation (0.27 +/- 0.04, P < 0.01). The COL I mRNA level 48 h after incubation was 0.38 +/- 0.02, significantly higher than the baseline level (0.22 +/- 0.03, P < 0.01); and the COL I protein level 48 h after incubation was 0.44 +/- 0.03, significantly higher than the baseline level (0.19 +/- 0.02, P < 0.01). The alpha-SMA mRNA level 48 h after incubation was 0.66 +/- 0.04, significantly higher than the baseline level (0.44 +/- 0.03, P < 0.01), and the alpha-SMA protein level 48 h after incubation was 0.43 +/- 0.02, significantly higher than the baseline level (0.24 +/- 0.03, P < 0.01). CONCLUSION: Urine protein may play an important role in the renal tubular-interstitial fibrosis by inducing the production of extracellular matrix and phenotype change in HK-2 cells.

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

AIM: 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 alpha (TNF-alpha), 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-alpha 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-alpha and expression of TNF-alpha 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-alpha 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.

[Role of connective tissue growth factor in human renal tubular epithelial cell transdifferentiation in vitro].

OBJECTIVE: To observe the effect of connective tissue growth factor (CTGF) on the transdifferentiation of human renal tubular epithelial cells and to explore the influence of CTGF antisense oligodeoxynucleotide (ASODN) transfection on the transdifferentiation process induced by transforming growth factor-beta1 (TGF-beta1). METHODS: Human renal tubular epithelial cells of the strain HKC were cultured and divided into 3 groups: (1) negative control group, (2) low dose CTGF group, treated with recombinant human CTGF (rhCTGF) with the terminal concentration of 2.5 microg/L, and (3) high dose CTGF group, treated with rhCTGF with the terminal concentration of 5.0 microg/L). To evaluate the contribution of CTGF to the transdifferentiation induced by TGF-beta1, Another HKC cells were divided into 4 groups: (1) untreated control group (Group C), (2) Group T, stimulated by TGF-beta1 (10.0 microg/L), (3) Group S, stimulated by sense ODN transfection + TGF-beta1 (10.0 microg/L), and (4) Group A, stimulated by antisense ODN transfection + TGF-beta1 (10.0 microg/L). RT-PCR was used to detect the mRNA expression of alpha-smooth muscle actin (alpha-SMA) and collagen type IV (col IV) mRNA. Indirect immunofluorescence assay and flow cytometry were used to assess the level of intracellular alpha-SMA protein. ELISA was used to determine the concentration of col IV in the media. RESULTS: The normal HKC cells were round and the HKC cells stimulated with rhCTGF became elongated. Upon the stimulation of different concentrations of rhCTGF, the expression of alpha-SMA mRNA increased markedly (both P < 0.01), while the mRNA expression of collagen type IV gene was down-regulated significantly (both P < 0.01). The percentage of alpha-SMA positive cells was significantly higher in the stimulated groups than that in negative control with significant difference among any 2 groups (38.9%, 65.5% vs. 2.4% respectively, all P < 0.01). Under this condition, collagen type IV secreted into the culture medium was lowered markedly upon the induction of CTGF (P < 0.01). RT-PCR analysis showed that the CTGF gene expression was upregulated by TGF-beta1 stimulation and peaked in 3 hours, and the alpha-SMA expression was upregulated by TGF-beta1 stimulation, however, peaked in 6 hours. The CTGF mRNA expression of the HKC cells transfected with CTGF ASODN that was stimulated by TGF-beta1 10 microg/L was significantly suppressed (P < 0.01) and the alpha-SMA mRNA expression induced by TGF-beta1 10 microg/L was significantly inhibited by CTGF ASODN transfection (P < 0.01). Indirect immunofluorescence assay showed that normal HKC cells did not express alpha-SMA, 48 hours after stimulation of TGF-beta1 10 microg/L the HKC cells showed expression of alpha-SMA in the cytoplasm, and the intracytoplasmic alpha-SMA expression was significantly down-regulated by the transfection of CTGF ASODN (P < 0.01). CONCLUSION: CTGF can promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblast (MyoF) in vitro, and CTGF blockade results in a dramatic inhibition of TGF-beta-induced transdifferentiation of renal tubular cells. So CTGF may be a crucial factor in promoting tubular-epithelial myofibroblast transdifferentiation.

[High glucose stimulates synthesis of fibronectin in human mesangial cells via serum and glucocorticoid induced protein kinase-1 signaling pathway].

OBJECTIVE: To investigate the role of serum and glucocorticoid induced kinase-1 (SGK(1)) pathways in fibronectin (FN) synthesis in human mesangial cell (HMC) under high glucose condition and the mechanism by which SGK(1) contributes to glomerulosclerosis in diabetic nephropathy (DN). METHODS: HMCs were cultured and transfected with (P)IRES2-EGFP-(S422D) SGK(1) mutant (SD), plasmid containing SGK(1) dominant activation mutant, or blank plasmid. Non-transfected HMCs were used as control group. Then the HMCs were divided into 6 groups: transfected with SD + high glucose (SD-HG, 25 mmol/L D-glucose), transfected with FP + high glues (FP + HG), non-transfected + high glucose (NT-HG), transfeted with SD + normal glucose (SD-NG, 5.5 mmol/L D-glucose), transfected with FP + normal glues (FP + NG), and non-transfected + normal glucose (NT-NG). Eight hours after the glucose stimulation, RT-PCR was used to examine the SGK(1) mRNA expression and fibronectin (FN). Western blotting was used to detect the fibronectin (FN) protein expression. RESULTS: The SGK(1) mRNA expression of the SD + HG group was 0.709, significantly higher than those of the FP + HG and NT + HP groups (0.497 and 0.491, both P < 0.01). The SGK(1) protein expression of the SD + HG group was 1,178,497, significantly higher than those of the FP + HG and NT + HP groups (193,875 and 195,597 respectively, both P < 0.01). The FN mRNA expression of the SD + HG group was 0.749, significantly higher than those of the FP + HG and NT + HP groups (0.463 and 0.475 respectively, both P < 0.01). The FN protein expression of the SD + HG group was 659,550, significantly higher than those of the FP + HG and NT + HG groups (342,354 and 340,428 respectively, both P < 0.01). There were not significant differences in the expressions of FN mRNA and protein among different NG groups. CONCLUSION: SGK(1) may be involved in the signal transduction leading to the increase of fibronectin production in DN and therefore may play an active part in glomerulosclerosis in DN.

Role of connective growth factor in plasminogen activator inhibitor-1 and fibronectin expression induced by transforming growth factor beta1 in renal tubular cells.

BACKGROUND: Connective tissue growth factor (CTGF) contributes greatly to renal tubulointerstitial fibrosis, which is the final event leading to end-stage renal failure. This study was designed to investigate the effects of CTGF antisense oligodeoxynucleotides (ODNs) on the expressions of plasminogen activator inhibitor-1 (PAI-1) and fibronectin in renal tubular cells induced by transforming growth factor beta1 (TGF-beta1) in addition to the role of CTGF in the accumulation and degradation of renal extracellular matrix (ECM). METHODS: A human proximal tubular epithelial cell line (HKC) was cultured in vitro. Cationic lipid-mediated CTGF antisense ODNs were transfected into HKC cells. After HKC cells were stimulated with TGF-beta1 (5 microg/L), the mRNA levels of PAI-1 and fibronectin were measured by RT-PCR. Intracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 and fibronectin in the medium were determined by Western blot and ELISA, respectively. RESULTS: TGF-beta1 was found to induce tubular CTGF, PAI-1, and fibronectin mRNA expression. PAI-1 and fibronectin mRNA expression induced by TGF-beta1 was significantly inhibited by CTGF antisense ODNs. CTGF antisense ODNs also inhibited intracellular PAI-1 protein synthesis and lowered the levels of PAI-1 and fibronectin protein secreted into the medium. CONCLUSIONS: CTGF may play a crucial role in the accumulation and degradation of excessive ECM during tubulointerstitial fibrosis, and transfecting CTGF antisense ODNs may be an effective way to prevent renal fibrosis.