Expression Of P38 MAPK Signal Transduction Pathway And Effect Of HMG-CoA Reductase Inhibitor In Diabetic Nephropathy

Objectives:Diabetic nephropathy (DN) is one of the most common complications of diabetes, which is histologically characterized by over accumulation of extracellular matrix (ECM) proteins, such as type â…£collagen,laminin and fibronectin in glomuruli. It is very important to clarify the pathogenesis for seeking the methods to prevent and treat DN. But molecular and cellular mechanisms responsible for diabetic nephropathy are not still clear completely. Recent studies have indicated that hyperglycemia is the basic biochemical feature of diabetes and has been believed to play major role in the development of diabetics and diabetic complications. Mitogen-activated protein kinases (MAPKs) can be activated by hyperglycemia, thereby activate and further regulate the transcription of genes responsible for key cellular responses such as proliferation, differentiation and apoptosis, thus inducing and accelerating the development of DN. p38 MAPK is one of the most important mitogen-activated protein kinases, which has been posited to mediate the pathogenesis of diabetic complication. Increase of p38 MAPK activity has been observed in diabetic rats and mesangial cells cultured under high-glucose conditions. High glucose can induce matrix synthesis, at least partly by transforming growth factor-β₁ (TGF-β₁). However, TGF-β₁ not only directly induces matrix, but also induces expression of other biological-active profibrotic mediators, such as connective tissue growth factor (CTGF), which can result in collagen synthesis and renal fibrosis at last. Little is known about p38 MAPK signaling in the kidney, and about the mechanism for high glucose to induce over expression of TGF-β₁ and CTGF as well as the over accumulation of extracellular matrix proteins. Recently,experimental and clinical investigations indicated that HMG-CoA reductase inhibitor has protective effect for the renal function of diabetic rats, independent of its effect to reduce lipid, but the mechanism remains to be studied. In the current report, we tried to verify the role that p38 MAPK pathway play in the development of DN, by examining the changes of expressions and activity of the key signaling kinases and target transcription of p38 MAPK pathway, including p38 MAPK, p-p38 MAPK, CREB1, p-CREB1, p-MKK3/6 and MKP-1, in the renal tissues of diabetic rats and glomerular mesangial cells exposed to high glucose. In addition, effect of HMG-CoA reductase inhibitor on the development of DN was also studied, using diabetic rats and mesangial cells exposed to high glucose. 1 The expressions of p38 MAPK key signaling kinases in the renal tissues of diabetic rats. Methods: Uninephrectomized male Wistar rats were randomly divided into two groups: control group (CN) and diabetic group (DM). The rats of diabetic group received a single intraperitoneal injection of STZ dissolved in 0.1mol/L sodium citrate buffer (pH 4.5) at a dose of 65mg/kg. The rats of control group only received an injection of the same volume of 0.1mol/L sodium citrate. The model of diabetes was considered to be successful when the blood glucose was ≥16.7mmol/L and the glucose in urine was +++～++++ after 48 hours of the injection. Six rats from each group were respectively sacrificed at weeks 1, 2, 4, 8, 12 after STZ injection. The renal cortical tissues were obtained and used for light microscopy, western blot and reverse transcription and polymerase chain reaction (RT-PCR). Partial renal tissues were fixed in 4% formaldehyde and embedded with paraffin. Partial renal cortices were used to abstract total RNA and protein. The deposition of ECM in renal tissues and the expressions of phosphor-specific p38 MAPK, phosphor-specific CREB1, phosphor-specific MKK3/6, MKP-1, CTGF, TGF-β₁ proteins were measured by immunohistchemistry. Phosphor-specific p38 MAPK and total p38 MAPK, Phosphor-specific CREB1 and total CREB1, phosphor-specific MKK3/6, MKP-1 were measured by western blot. The mRNA expressions of p38 MAPK, CREB1, CTGF, TGF-β₁ and FN were semi-quantitatively evaluated by RT-PCR.All data (the same below) were expressed as x ±s. F and T test was separately used to analyse the difference between the two groups and within one group, which was finished with SPSS12.0 software. Results:There were obvious morphological changes of renal tissues of diabetic rats at week 12. Light microscopy showed that PAS-positive area of glomeruli was increased. Expansion of glomerular mesangium, thickening of glomerular and tubular basement membrane were observed with progression of diabetes. Immunohistochemical staining showed that positive stainings for p-p38 MAPK, p-CREB1, p-MKK3/6 and MKP-1 were observed in renal tissues of both control group and diabetic group. p-p38 MAPK, p-CREB1 and MKP-1 were observed in the nuclei of mesangial cells, endothelial cells, podocytes, and tubular epithelial cells. p-MKK3/6 was detected mainly in cytoplasm of glomerular cells and tubular epithelial cells .Compared with the control group, the positive stainings for p-p38 MAPK and p-MKK3/6 were stronger between week 1 to 8(P<0.01). The positive expression of p-CREB1 was higher between weeks 2 to 8(P<0.01). The positive staining of MKP-1 in diabetic renal tissues was weaker, compared with control group between weeks 2 to 8(P<0.01). The positive stainings for fibronectin, collagen â…£and laminin were observed in mesangial area, capillary basement membrane and tubular interstitium. Image analysis showed that the staining intensity for fibronectin, collagen â…£and laminin markedly increased in diabetic renal tissue of 4 weeks and progressively increased with duration of diabetes(P<0.01) , compared with control group. The positive stainings of TGF-β₁ and CTGF were observed in glomerular epithelial cells and mesangial area, capillary loops, epithelial cells of Bowman's capsule and renal tubular cytoplasm. Compared with control group, the expression of TGF-β₁ and CTGF were higher after 4 weeks in diabetic kidneys and progressively heavier with duration of diabetes (P<0.01). Western blot indicated that the expressions of p-p38 MAPK and p-MKK3/6 which represent the active forms of p38 MAPK and MKK3/6 increased between 1 to 8 weeks in DM group compared with thecontrol group (P<0.01), peaked at week 4(P<0.01). The expression of p-CREB₁ was increased between 2 to 8 weeks in diabetic renal tissue. Compared with the control group, the expression of MKP-1 was decreased in diabetic rats between 2 to 8 weeks (P<0.01). RT-PCR showed that the mRNA expressions of p38MAPK, CREB₁ and MKK3/6 were the same as the activity of p38 MAPK, CREB₁ and MKK3/6. The mRNA expressions of CTGF, TGFβ₁ and fibronectin were significantly greater in diabetic group than the control group between weeks 4 to 12 (P<0.01). 2 Effects of HMG-CoA reductase inhibitor lovastatin on the expression of p38 MAPK in the renal tissues of diabetic rats. Methods: Male Wister rats were divided into three groups: normal control group, diabetic group and lovastatin treated diabetic group. The method making diabetic model was the same as part 1. After diabetic model was confirmed to be successful, the rats of lovastatin treated diabetic group were administered with lovastatin (20mgË™kg-1Ë™d-1) by gavage for 4 weeks. Blood and urine were collected and used to assay biochemical parameters. The residual kidney was removed and prepared for light microscopy, immunohistochemistry, western blot and RT-PCR. Immunohistochemical technique was used to observe the expressions of. phosphor-specific p38 MAPK, phosphor-specific CREB₁, phosphor-specific MKK3/6, MKP-1, CTGF and TGF-β₁ proteins. Phosphor-specific p38 MAPK and total p38 MAPK, Phosphor-specific CREB₁ and total CREB₁, phosphor-specific MKK3/6, MKP-1 were measured by western blot. The mRNA expressions of p38 MAPK, CREB₁, MKK3, MKK6, MKP-1, CTGF, TGF-β₁ and FN were evaluated by RT-PCR. Results: Compared with those of control rats, the glomerular enlargement, mesangial expansion and thickening of glomerular and tubular basement membrane were observed in diabetic rats, while these parameters were improved in lovastatin treated group(P< 0.01). Compared with diabetic rats, the change of renal function, including the increase of blood creatine, blood urine nitrogen and urine proteins, were ameliorated in Lovastatin treated group(P<0.01), though there was no difference of the blood glucose level and serum lipid indexes between the diabetic group and lovastatin treated group(P>0.05). Immunohistochemically, the expressions of CTGF, TGF-β₁, fibronectin, â…£collagen and laminin were lower in lovastatin treated group compared with diabetic group. The expressions of p-p38 MAPK, p-CREB₁ and p-MKK3/6 were also lower in lovastatin treated group compared with diabetic group.(P<0.01).Western blot indicated that the expressions of total p38 and CREB₁ levels were unchanged among the three groups (P>0.05), but activity of p38 MAPK, CREB₁ and MKK3/6 was higher in diabetic group than in control group, lower in lovastatin treated group than in diabetic group.(P< 0.01).The mRNA expressions of p38MAPK, CREB₁, MKK3, MKK6, CTGF and TGF-β₁ were up-regulated in diabetic group, and down-regulated in lovastatin treated group(P<0.01). 3 The expression of p38 MAPK key signaling kinases and target transcription factor in mesangial cells exposed to high glucose. Methods: Glomerular mesangial cells were separated from healthy male Wistar rats. The fourth passage cells were cultured in 96-pore plate, then were washed once with serum-free RPMI 1640 and cultured with the same medium for 24 hours to synchronize the cell growth. MTT assay was used to measure the proliferation of mesangial cells at 12, 24, 48 and 72 hours, and to investigate the effect of SB203580, a p38 MAPK inhibitor, on proliferation of mesangial cells exposed to high glucose medium. Glomerular mesangial cells between the fifth and the tenth passages were randomly divided into four groups and were cultured in 24-pore plate and 25cm2 plastic culture flask: LG group(5.5mmol/L glucose) ; LG+M group (5.5mmol/L glucose+24.5mmol/L mannitol) ; HG group (30mmol/Lglucose) ;HG+SB203580 group (30mmol/L glucose +10μmol/LSB203580). Mesangial cells cultured in different conditions were harvested and used to abstract total RNA and protein at 48 hours after stimulation. The expressions of p38 MAPK, p-p38 MAPK, p-CREB₁ were measured by immunocytochemical staining and western blot. The mRNA expressions of p38 MAPK, CREB₁, MKK3, MKK6,MKP-1, CTGF and TGF-β1 were measured by RT-PCR. The contents of laminin and type IV collagen in the supernatants of the cells were detected by radioimmunoassay. Results: Compared with low glucose group, mannitol had no effect on proliferation of mesangial cells, while high concentration of glucose could promote the proliferation of mesangial cells. Immunocytochemically, the positive staining for p38 MAPK was observed in cytoplasm of the mesangial cells cultured in the medium with normal level glucose. After the cells were treated with high glucose for 48 hours, the positive staining was gradually observed in the nucleus of the cells. Positive signals for p-p38 MAPK and p-CREB₁ were also observed in the nucleus of the cells. Compared with those exposed to normal level glucose, the stainings were strengthened for p-p38 MAPK and p-CREB₁ in the mesangial cells exposed to high glucose from 48 h. Western blot indicated that the activity of p38 MAPK, Mkk3/6 and CREB₁ were higher in mesangial cells exposed to high glucose than in those exposed to normal level glucose(P<0.01), and lower in SB203580 group than in high glucose group(P<0.01). The mRNA expressions of p38 MAPK, CREB₁, MKK3, MKK6, CTGF and TGF-β1 were higher in HG group than in LG group (P<0.01), but lower in SB203580 group than in HG group (P<0.01). MKP-1 mRNA expression was lower in HG group than in LG group, and higher in SB203580 group than in HG group (P<0.01). The concentration of laminin and type IV collagen in supernatants of the cells exposed to high glucose was higher than that in low glucose group at 48 hours(P<0.05). After treament with SB203580, the contents of laminin and type IV collagen decreased compared with the high glucose group (P<0.05). 4 Effects of lovastatin on the expression of p38 MAPK key signaling kinases and target transcription in rat mesangial cells exposed to high glucose Methods: Glomerular mesangial cells between the fourth and the sixth passages were cultured in 96-pore plate. The cells were washed once with serum-free RPMI 1640, then cultured with the same medium for 24 hours to synchronize the cell growth. MTT assay was used to investigate the effect oflovastatin on proliferation of mesangial cell in high glucose medium at different time and different drug concentration. Glomerular mesangial cells between the fifth and the tenth passages were cultured in 25cm² plastic culture flask and divided into three groups: LG group(5.5mmol/L glucose);HG group(30mmol/L glucose);HG+LT group(30mmol/L glucose+1μmol/L lovastatin). Total RNA and protein were abstracted at 48 hours. The expressions of p38 MAPK, p-p38 MAPK, p-CREB₁ were measured by immunocytochemical staining and Western blot. The mRNA expressions of p38 MAPK, CREB₁, MKK3, MKK6, MKP-1, CTGF and TGF-β₁ were measured by reverse transcription and polymerase chain reaction (RT-PCR). The contents of laminin and type IV collagen in the supernatants of the GMCs were detected by radioimmunoassay. Results: Compared with low glucose group , mannitol had no effect on proliferation of mesangial cells, while high concentration of glucose could promote proliferation of the mesangial cells. 0.1umol/L lovastatin had little suppression on proliferation of the mesangial cells exposed to high glucose, and the suppression became more stronger at a dose of 1umol/L, but it showed over suppression at a dose of 10umol/L. Immunocytochemically, The positive stainings for p-p38 MAPK and p-CREB₁ were weaker in lovastatin treated group, which were observed in nucleus of the mesangial cells exposed to high glucose at 48 hours. Lovastatin also can decrease the positive staining of p38 MAPK observed in the nucleus of the cells exposed to high glucose(.P<0.01). Compared with the HG group, the activity of p38 MAPK, CREB₁, MKK3/6 was lower in lovastatin treated group. The expression of MKP-1 was decreased in HG group and increased in lovastatin treated group(P<0.01).The mRNA expressions of p38MAPK, CREB₁, MKK3, MKK6, CTGF and TGF-β₁ were up-regulated in the mesangial cells exposed to high glucose and down-regulated in the cells of lovastatin treated group(P <0.01).The concentration of laminin and type IV collagen in supernatants of the mesangial cells exposed to high glucose was higher than that in low glucose group at 48 hours. After treament with lovastatin, the contents of...