The Immunoregulating Of1,25-dihydroxychole-Calciferol On The Tubulointerstitial Fibrosis In Diabetes Mellitus

Objective:Diabetic kidney disease (DKD) is one of the important microvascular complications of diabetes mellitus and is believed to be the major causes of end stage renal disease. The injury of kidney tubules before glomerulus in DM is also thought of the primary pathologic abnormality of the DKD. The theory of the immuno-injury participate in the development of DKD is still being debated for the time being. Meanwhile, that the pathway of TLR-NF-κB activation may be the first barricade of the inherent immunity and participate in the natural defensive reaction also under wide investigation. But over-activation will result in inflammatory reaction which would promote fibrosis of the target tissues. There are few study up till now, on the relationship between the inherent immunity and the renal tubulointerstitial fibrosis in DKD. On the other hand,1,25-dihydroxycholecalciferol is a hormone, however, which is widely recognized to be involeved in the immuno-regulation. Some studies showed that by two-ways,1,25(OH)2D3had regulating effects on the TLRs in the inherent immunity. At the same time, many studies indicated that the1,25(OH)2D3had the renoprotection through the down-regulating effects on NF-KB expression.. However, there has been no any report in the literature about whether or not1,25(OH)2D3had a down-regulating effect on NF-κB expression by inhibiting the activation of the TLRs. Under such a circumstances, our investigation was launched and focused on (1) To make sure that whether or not the TLRs-MyD88-NF-κB pathway might be involved in the development of RIF in DKD. To elucidate the DKD is a immunity kidney disease in which the inherent immunity participative.(2) Whether the1,25-dihydroxycholecalciferol can ameliorate the development of DKD through down-regulating the over-activated of the TLRs.(3) To evaluate the safety of1,25-dihydroxycholecalciferol and look for an optimal dose in immunosurpressive therapy of kidney. Through this experiment, the theory for the immuno-injury in DKD could be established, while a concored theoretical basis could also be established for1,25(OH)2D3which was involved in the immune-regulating mechanism besides those effects on calcium and phosphorus metabolism. Methods:(1) Animal experiment:The DM rats models were made by intravenous injection of STZ. Then, they were randomly devided into seven groups, including control group(group C), diabetic unintervention group (group D), diabetic group intervention orally with a low dose1,25(OH)2D3(group L,0.025ug/kg/d),a middle dose1,25(OH)2D3(group M,0.15ug/kg/d), a high dose1,25(OH)2D3(group H,0.3ug/kg/d), diabetic group intervention orally with losartan potassium (group A,10.4mg/kg/d), diabetic group intervention injection with insulin(group Y,16U/kg/d). Sixteen weeks later, the urinary microalbumin, urinary NAG and blood biochemical indicator were determined. Renal pathological changes were studied by both HE and Masson staining. The tubulointerstitial injury score were assessed by optical microscope. Besides these, the expression of MCP-1、α-SMA and NF-κB in tubulointerstitial were also investigated by immunohistochemisty. The expression of MCP-1、α-SMA. NF-κB、TLR4and MyD88were detected by Real-time quantitive polymerase chain reaction and Western blot. The expression of both TLR4and MyD88in tubulointerstitial was studied by immunofluorescence.(2) Cellular experiment:The renal tubular epithelial cells of normal rats were incubated in vitro. NRK-52E cells were exposed to three concentrations of glucose (including5.5mmol/L,25mmol/L,50mmol/L respectively) and harvested at the0.2,4,6,8.24hour. The expression of TLR4and MyD88were examined by Western blot and Real-time PCR. In order to determine the glucose concentration and the time point for the harvesting based upon the expression peak of the TLR4and MyD88, the experiment treated with1,25(OH)2D3was subsequently carried out. The cells were divided into six groups according to the experimental conditions, with DMEM as the medium:Group normal glucose Control (5.5mmol/L, LG group); Group high glucose (25mmol/L, HG group); Groups1,25(OH)2D3, cultured with high concentration of glucose (25mmol/L)DMEM and with various concentrations of1,25(OH)2D3(10-9mol/L,10-8mol/L,10-7mol/L)(HG+10-9, HG+10-8, HG+10-7) Group ARB, cultured with high glucose DMEM+Losartan potassium (HG+ARB). The expression of TLR4and MyD88mRNA were studied by Real-time PCR at hour6and the expression of TLR4and MyD88protein were examined by Western blot and cellular immunofluorescence at hour24. Results:1、The results of animal experiment:(1)In the rats of diabetic group without intervention, blood glucose, urinary microalbumin, urinary NAG, blood urea nitrogen, serum creatinine, kidney weight/body weight, tubulointerstitial injury score, the expression of MCP-1and α-SMA in renal tissue was significantly increased compared with the normal control group. The urinary microalbumin, urinary NAG, blood urea nitrogen, serum creatinine, tubulointerstitial injury score and the expression of MCP-1and α-SMA in renal tissue of the diabetic group intervented orally with a high dose1,25(OH)2D3, diabetic group intervented by the injection of insulin and diabetic group intervented orally with losartan potassium were significantly lower than the diabetic wihtout any intervention. These expression in the diabetic group intervented orally by both a low dose and a middle dose of1,25(OH)2D3showed no difference with the diabetic without intervention.(2) In the rats of diabetic without intervention, the expression of TLR4, MyD88and NF-κB in the renal tissue significantly increased compared with normal control group. The expression of TLR4, MyD88and NF-κB in the renal tissue of the diabetic group intervented orally by a high dose of1,25(OH)2D3, diabetic group intervented injection with insulin and diabetic group intervented orally with losartan potassium were significantly lower than the diabetic group without any intervention. The expression of TLR4, MyD88, NF-κB were positively correlated with urinary microalbumin blood urea nitrogen, serum creatinine, tubulointerstitial injury score while the expression of MCP-1and a-SMA in renal tissue was nearly same.(3) Among each groups, the serum calcium, phosphate and the liver functions showed no difference.2、The results of the cellular experiment:(1) The peak times of the TLR4、MyD88mRNA expression and the protein expression were hour6and hour24in the culture with high glucose of25mmol/L and showed significantly difference in comparison with other glucose at various glucose concentrations and time points.(2) The expression of TLR4and MyD88in the group treated with the1,25(OH)2D3of1×10-/mol/L significantly decreased compared with the high glucose group. There had no difference compared with the high glucose group in the groups that the1,25(OH)2D3concentration were1×10-9mol/L and1×10-8mol/L.Conclusions:1、The kidney tubulointerstitial disease is the important component in the development of the DKD. The inflammatory reaction induced by the over-activation of the TLR4-NF-κB pathway, one of the inherent immunity, was involved in the development of the DKD.2、The over-activation of the TLR4-MyD88-NF-KB pathway in the kidney of STZ induced diabetic rats could be suppressed by1,25(OH)2D3, down-regulating the expression of the MCP-1and a-SMA. So the process of the renal tubulointerstitial fibrosis could be delayed by1,25(OH)2D3through alleviating the inflammatory reaction, repressing the generation of EMT and reducing the leakage of the urine protein consequently.3、The same results were also derived as well in vitro experiment. The expression of TLR4and MyD88was increased in the culture with25mol/L concentration glucose and was also suppressed by the1,25(OH)2D3. The over-activation of the inherent immunity could be suppressed by1,25(OH)2D3.4、In our experiment, relatively speaking, the intervention dose of1,25(OH)2D3we designed is currently believed to be safe while the hypercalciuria induced by1,25(OH)2D3needs to be further investigated.