Objective This study was designed to explore the effect of hyperglycemia on the expression of Toll-like receptor 4 (TLR4 ) in renal tubular epithelial cells and its significance in diabetic nephropathy. Methods In vitro cultured renal tubular epithelial cells ( NRK-52E) were divided into LG group (cultured in 5mmol / L glucose DMEM) and HC group (cultured in 25mmol / L glucose DMEM). Cells were harvested at different time points. Immunohistochemistry, Rt-PCR, Western Blot were used to detect TLR4 protein and mRNA expression, and the levels of IL-6 and TNF-α from the cell culture supernatant were determined by EL1SA assay. Results After 6 hours, there was increased expression TLR4 mRNA in HC group, which appeared to be maintained for 24 hours and began to decrease after 48 hours ( P < 0.05). TLR4 protein expression increased in HC group after 24 hours, and increased even further after 48 hours. Compared with LG groups, the difference had statistical significance ( P <0.05). In HG group, IL-6 and TNF-α expression in the supernatant from the NRK-52E culture were significantly increased ( P < 0.05) , and the expression of IL-6 and TNF-α was positive correlated with the expression of TLR4 protein ( r =0.799,0.820). Conclusion High glucose triggers an increase in expression of TLR4 in NRK-52E cells, itself leading to an increase in expression of inflammatory factors such as TNF-α and IL-6. In this way, TLR4 participates in the progress of diabetic nephropathy.

Objective To study the histone acetylation level of CD4+ T cells in peripheral blood of lupus nephritis ,to explore the role of histone acetylation in pathogenesis of lupus nephritis .Methods According to Feng X ,Bernstein ,Wagner S J and other scholars′s classification criteria for LN ,those who met the following conditions considered for the activity of LN :proteinuria >0 .5 g/d ,change or activity of urinary sediment (hematuria > 5 red blood cell /Hp ,or pyuria > 5 Hp white blood cells ,or 1 cell type/Hp) ,serum creatinine increased >1 .2 mg/L ,and the exclusion of infection ,kidney stones and other causes .Lupus nephritis pa‐tients were divided into inactive group (group I) 8 people ,active group (group A) 10 people .18 patients with LN and 8 normal con‐trols were collected peripheral blood 50 mL ,density gradient centrifugation method (Ficoll method) for separation of mononuclear cells in peripheral blood;CD4+ T cell was analyzed by immunomagnetic beads ,extracted histone acetylation level and detected H3/H4 protein by the acetylation of histone H3/H4 kits and the relationship of histone H3/H4 acetylation with diseases was analyzed . Results First ,compared with group N ,the histone H3 and H4 of CD4+ T cells in peripheral blood both in A and I of group LN pa‐tients showed low acetylation status (P<0 .01);Second ,the acetylation level of histone H4 in group A was lower than that in group I (P<0 .01) ,histione H3 acetyl the level of the group of two groups had no statistical significant (P>0 .05) .Third ,it was nega‐tively related to the acetylation level of histone H4 and 24 h urinary protein excretion(r= -0 .661 ,P<0 .05) .Conclusion Histone H3 and histone H4 of the CD4+ T cells showed low acetylation may be involved in the pathogenesis of lupus nephritis .The acetyla‐tion level of histone H4 in CD4+ T cells may be related to the activity of the LN .

Objective To determine the effect of aldosterone and its antagonist, spironolactone on epithelial-mesenchymal transition (EMT) of normal rat kidney epithelial cells (NRK-52E) in a high glucose milieu,and to explore the mechanism of renoprotection in diabetic nephropathy (DN ) in rats involving aldosterone and spironolacton. Methods NRK-52E cells were simultaneously cultured in the serum-free Dulbecco's modification of Eagle's medium Dulbecco (DMEM) for 12 hours. Then the low glucose (LG) group was cultured in LG (1000 mg/L) DMEM:The high glucose (HG) group was cultured in high glucose (4 500 mg/L) DMEM. The aldosterone (Aldo) groups were cultured in high glucose DMEM with the addition of 10,50 and 100 nmol/L aldosterone respectively. The SP group was cultured in high glucose (4 500 mg/L) DMEM plus 10~(-7)mol/L spironolactone. Immunohistochemistry, RT-PCR and Western blot were used to detect E-cadherin and α smooth muscle actin(α-SMA) mRNA expression. Results RT-PCR showed that compared with the LG Group, E-cadherin mRNA expression in the HG group was significantly lower, and α-SMA mRNA expression was significantly increased(P＜0.05). E-cadherin mRNA expression in the 50 nmol/L Aldo group and 100 nmol/L Aldo group was significantly lower than that in the HG group, while the expression of α-SMA mRNA was significantly increased in the HG group(P＜0.05), with a dose-dependent relationship with aldosterone(r=-0.70,P＜0.05;r=0.67, P＜0.05). E-cadherin mRNA in the SP group was significantly higher,while α-SMA mRNA expression was lower than that in the HG group(P＜0.01). Immunohistochemistry and Western blot showed that compared with the LG group, E-cadherin protein expression was significantly reduced, and α-SMA expression was significantly increased in the HG group(P＜0.01). In the 10 nmol/L Aldo, 50 nmol/L Aldo, and the 100 nmol/L Aldo groups, E-cadherin protein expression was significantly lower, and α-SMA protein expression was significantly higher than that in the HG group(P＜0.05), with a dose-dependent relationship with aldosterone(r=-0.83,P＜0.05;r=0.81, P＜0.05). In the SP group, E-cadherin protein expression was higher, and α-SMA protein level was lower than that in the HG group(P＜0.05). Conclusion Aldosterone can promote EMT of tubular epithelial cells in a high sugar milieu, leading to renal interstitial fibrosis in Diabetic nephropathy. Spironolactone can inhibit high glucose-induced renal tubular epithelial cells EMT, which may be an important mechanism for the inhibition of renal interstitial fibrosis.

Objective To explore the effect of aldosterone on renal epithelialmesenchymal transition in streptozocin(STZ)-induced diabetic nephropathy rats. Methods Wistar rats were intraperitoneally injected with STZ(60 mg/kg)for the preparation of diabetic model.After 4 weeks,the rats with urinary protein＞30 mg/d were regarded as successful diabetic nephropathy(n=16),and were randomly divided into diabetic nephropathy(DN group,n=8)and spironolactone group(SP group,n=8).Then eight healthy rats were selected randomly as control group(N group,n=8).SP group rats were treated with spironolactone 40 mg·kg-1·d-1,and N group and DN group rats were given equal water.After 8 weeks,rats were sacrificed to collect urine,blood plasma,kidney tissue for detection of 24 h urinary protein,creatinine and renal pathological changes.Aldosterone concentration in plasma and kidney tissue was detected by mdioimmunoassay;E-cadherin,α-SMA protein expression by immunohistochemistry,Western blotting; E-cadherin,α-SMA mRNA expression by RT-PCR. Results Compared with N group,serum creatinine, urinary protein excretion in the DN rats were significantly higher (P＜0.01,respectively), E-cadhefin protein and mRNA were significantly reduced (P＜0.01, respectively),α-SMA protein and mRNA expression was up-regulated (P＜0.01, respectively). Aldosterone level of kidney tissue in DN rats was increased obviously [(24.71±5.30) ng/g vs (16.38±2.85) ng/g, P＜0.01], which was positively correlated with urinary protein excretion, serum creatinine and α-SMA protein (r=0.737, 0.574, 0.688, P＜0.01, respectively), and negatively correlated with E-cadherin protein (r=-0.659, P＜0.O1). While no significant difference was found in serum aldosterone among three groups. Compared with DN rats, urinary protein excretion, serum creatinine were reduced (P＜0.01, respectively), E-cadherin protein and mRNA were increased (P＜0.01, respectively), α-SMA protein and mRNA expression were decreased (P ＜0.01, respectively) in SP group rats.Conclusions Local aldosterone involves in renal epithelial-mesenchymal transition in diabetic nephropathy rat. Spironolactone can block the effect of aldosterone and play a role in renal protection.

OBJECTIVE: To explore the relationship between protein kinase C (PKC) and matrix metalloproteinase (MMPs)/tissue inhibitor of metalloproteinase (TIMPs) in human mesangial cells under the high glucose medium, and to analyze the effect of PKC and MMPs/TIMPs in diabetes nephropathy (DN). METHODS: Normal human mesangial cells (NHMC) were divided into 4 groups: a control group(N, 5 mmol/L glucose), a high glucose group (H, 30 mmol/L glucose), a PKC inhibition group (P, 30 mmol/L glucose plus 10-5 mol/L chelerythrine chloride), and an mannitol group (M, 5 mmol/L glucose plus 25 mmol/L mannitol). Cell proliferation was measured by MTT at 24,48 or 72 hours. The activity of PKC was measured by ELISA and the mRNA and protein expressions of MMP2, 9 and TIMP1, 2 were examined by RT-PCR and Western blot. RESULTS: High glucose increased the activity of PKC as well as the expressions of mRNA and protein of MMP2, 9 and TIMP1, 2.The ratio of MMP-2/TIMP-2 and MMP-9/TIMP-1 was significantly decreased in the high glucose group compared with that of the control group (P<0.05). The mRNA and protein expressions of MMP2, 9 and TIMP1 were significantly increased in the PKC inhibition group compared with the control group (P<0.01). The ratio of MMP-2/TIMP-2 and MMP-9/TIMP-1 increased in the inhibition group compared with that of the high glucose group (P<0.05 or P<0.01). The activity of PKC was negatively correlated with the protein ratio of MMP-2/TIMP-2 and MMP-9/TIMP-1(rý-0.651,rý-0.702, both P<0.05). CONCLUSION High glucose can activate PKC in mesangial cells. The activity of PKC influences the expression of MMPs/TIMPs in the progressing of DN.
Cells, Cultured ; Glucose ; adverse effects ; pharmacology ; Humans ; Hyperglycemia ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mesangial Cells ; metabolism ; Protein Kinase C ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; RNA, Messenger ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism

OBJECTIVE: To examine the expressions of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in the kidney of rats with diabetic nephropathy before and after the treatment of Cordyceps sinensis, and to explore the mechanism of Cordyceps sinensis against hypoxia. METHODS: The diabetes model was produced by intraperitoneal injection of 60 mg/kg streptozotocin, then the rats whose 24 h urine protein level was above 30 mg/d were thought to have suffered diabetic nephropathy. Thirty rats were randomly divided into a diabetic nephropathy group (DN group, n=15) and a Cordyceps sinensis group (CS group, n=15), and another 15 normal rats served as a normal control group (NC group, n=15). The CS group were intragastrically administered Cordyceps sinensis extract liquid [5.0 g/(kg.d)], the other groups were intragastrically administered drinking water of equal volume. Five rats in each group were killed after 2, 4, and 6 weeks. The 24 h urine protein excretion, urine β-N-acetyl glucosaminidase (NAGase) and serum creatinine were measured; the renal pathological changes were evaluated by HE and Masson staining; the mRNA and protein expressions of HIF-1α and VEGF were dectected by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: Compared with the normal control group, the renal tubular vacuolar degeneration was obvious, and the glomerular mesangial matrix increased in the DN group. The 24 h urinary protein excretion, urine NAGase and serum creatinine also increased significantly (all P<0.05); the expressions of HIF-1α and VEGF in the renal tissue gradually increased with time, and the expression of HIF-1α was correlated with that of VEGF in the 2 groups (r=0.850, r=0.887, both P<0.05) . Compared with the DN group, the pathological changes were relieved, the 24 h urinary protein excretion, urine NAGase and serum creatinine level were decreased, and the expressions of HIF-1α and VEGF decreased in the CS group (all P<0.05), but they were still higher than those in the normal contral group (P<0.05). There was no significant difference in the mRNA and protein expression of HIF-1α between the 4th week and the 6th week after the treatment of CS (P>0.05). CONCLUSION The expressions of HIF-1α and VEGF increase in the kidney of rats with diabetic nephropathy, and the positive correlation suggests that there is chronic hypoxia in the renal tissue of diabetic nephropathy. Cordyceps sinensis may protect against chronic hypoxia injury in diabetic nephropathy by lowering the expressions of HIF-1α and VEGF.
Animals ; Cordyceps ; chemistry ; Diabetes Mellitus, Experimental ; complications ; metabolism ; Diabetic Nephropathies ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Kidney ; metabolism ; Male ; Rats ; Rats, Wistar ; Vascular Endothelial Growth Factor A ; genetics ; metabolism