| Obstructive nephropathy is a common disease of urology. The contralateral kidney can still maintain the basic physiological events in the very early stage, due to the compensatory mechanisms. But as the diseases progressed, it will fall into chronic renal failure. The mechanism about obstructive nephropathy has been extensive and intensive researched, which is contrary to the contralateral kidney. According to the mechanism in renal interstitial fibrosis, cell proliferation and EMT play a very important role in the deposit of extracellular matrix. Especially cell proliferation appears even in the very early stage, and goes through the whole process. Our hypothesis is that obstructive nephropathy will induce the activation of aldosterone, the secretion of cytokines, which will influence the contralateral renal via blood circulation. And increased local expression of cytokines via NF-κB, which in turn leads to cell proliferation in the contralateral kidney, and fall into renal failure.In this experiment, we used UUO model to simulate the pathology of obstructive nephropathy in early injure. And the effect of Eplerenone, a mineralocorticoid receptor blocker and Chinese herbs were observed, whether can inhibit cell proliferation via aldosterone stimulation, and provide theoretical and experimental basis for traditional Chinese medicine(TCM).PartⅠTheory of “Preventive Treatment” in Traditional Chinese Medicine and the application in obstructive nephropathy“Preventive Treatment” is an important part of traditional Chinese medicine, including“prevention deals with latent diseases”,“prevent an asymptomatic disease from progressing to symptomatic disease”,“prevent disability” and “ preventing recurrence”. This theory covers from the occurrence of the disease, development, outcome to prognosis.Renal diseases such as edema, urinary infection, end-stage renal disease are interrelated each other. To prevent renal disease,it also needs“Preventive Treatment”, especially“prevent progressing”,“prevent disability”and“preventing recurrence”.In this article, we briefly introduced the theory and history of“Preventive Treatment” in TCM, described the characteristics of obstructive nephropathy, and the relationship between them.The obstruction only effect the local renal at the early stage, as the disease progressed, it will finally influence the contralateral renal. The contralateral renal damage is called“sick the same organ”in TCM. The mechanism is obstruct renal will influence contralateral kidney as the progressed. According to this theory and characteristics of obstructive nephropathy, we use“Shenluotong”decoction to treatment UUO rats, and provide theoretical basis for TCM.PartⅡEffect of Shenluotong Decoction on pathology in the contralateral kidney of UUO ratsObstructive: To establish the obstructive nephropathy rat model and observe the general situation of rats, changes in the contralateral kidney pathology and the indices about fibrosis, then verify the protection in contralateral kidney with shenluotong and mineralocorticoid receptor blockers.Methods: 48 Wistar rats weight 200±10g were used in this study. After 7 days adaptive feeding, rats are randomly divided into 4 groups: Sham group, UUO group, UUO plus Eplerenone and UUO plus Shenluotong group. The rats except Sham group had the UUO operation and treated with Eplerenone of 100mg/Kg/d in diet and Sheluotong 26g/(kg/d) in drinking water respectively. At the tenth day, serum, contralateral kidney and obstructive nephropathy were harvested for further study. The data were counted by SPSS13.0 software. The data were expressed as mean ± standard deviation. All results are considered significant at P<0.05 and P<0.01.Results:1 The general situation in rats The vital signs of Sham group rats were in good condition: daily diet and water drinking were stable, body weight was gradually growth, hair was shiny, and sensitive to external stimulation. After UUO surgery, rats in UUO group were decreased in diet and water, and the body weight also growed slowly. Ten days after surgery, obstructive renal/body weight in UUO group increased significantly compare to the Sham group(P<0.05). And the condition was same to the contralateral renal(P<0.05), but there was no obvious difference between these three UUO surgery groups.2 The changes of Pathomorphology in renal tissue Pathomorphology changes are determined by HE and Masson staining. HE staining showed that there was no change on both kidneys in the Sham group, basic structure was normal, tubular epithelial cells arranged regularly, no edema and few inflammation cells in interstitial. In UUO group, the obstructive kidney was significantly enlarged, denatured, necrotic or shed, renal tubular epithelial cells were observed; renal interstitial space was widened; large numbers of inflammation cells infiltrated in renal interstitial. And in contralateral kidney, there were also many inflammation cells infiltrated in renal interstitial, which was significantly increased than that in Sham group. Some of the tubular epithelial cells with edema had been observed. Two treatment groups, the obstructive nephropathy showed the same condition, but the degree of inflammation was much more tender than that in UUO group; the contralateral kidney also showed some inflammation cells and edema epithelial. There was no differences between these two groups. Masson staining showed the changes of fibrosis. In Sham group, there were few of fibrosis in basement membrane and renal interstitial. In UUO group, fibrosis was significantly increased in renal interstitial, the deposition of collagen had an increase tendency. And the pathology can be abolished by Eplerenone and Shenluotong.3 The expression of α-SMA by immunohistochemistryIn the sham group, α-SMA only can be observed in vessel smooth muscle cells. In UUO group, protein of α-SMA was increased, and it was observed in epithelial cells. Compared to UUO group, Eplerenone group and Shenluotong group down-regulated the level of α-SMA, and there was no differences between these two groups.Conclusions : In obstructive nephropathy animal experiment, the contralateral kidney showed compensatory hypertrophy, and renal interstitial inflammatory cell infiltration increased. The fibrosis marker α-SMA was also up-regulated and cytokine such as MCP-1、TNF- were increased.The cell proliferation obvious.Part Ⅲ Inhibition of Shenluotong Decoction on cytokines and proliferation in contralateral kidneyObstructive: To establish the obstructive nephropathy rat model and observe the situation of inflammatory infiltration and cell proliferation, then verify if inflammatory injury and cell proliferation is involved in the process of obstructive nephropathy damage in contralateral kidney and the mechanism about protection in contralateral kidney with shenluotong and mineralocorticoid receptor blockers.Methods: The methods of animal grouping, model establishment, medication and statistics were the same to PartⅡ, 10 days. The expression of MCP-1 in serum was detected by ELISA. Protein expression and location of TNF-α and PCNA in contralateral kidney were detected by immunohistochemistry and Western blot respectively.Results: 1 The expression of MCP-1 in serumCompared to the Sham group, the MCP-1 in serum was significantly increased in UUO group(P<0.01, P<0.05). Compared to the UUO group, the MCP-1 in serum was decreased in two treatment groups, especially in Eplerenone group(P<0.01, P<0.05).2 The expression of TNF-α in contralateral kidneyThe expression of TNF-α was observed in epithelial kytoplasm by immunohistochemistry. Compared to the Sham group, the expression of TNF-α was significantly increased in UUO group by Western blot. Compared with UUO group, the expression in Eplerenone group and SLT group were significantly decreased in range and strength(P<0.05).3 The expression of PCNA in contralateral kidney The positive cell about PCNA in contralateral kidney in Sham group was observed a few by immunohistochemistry. In UUO group, the positive cell was observed in epithelial cells of the distal tubule. Compare to sham group, the expression of PCNA was significantly increased but fewer than UUO group obviously. Using Western blot method to detect the expression of PCNA, compare to sham group it was up-regulated obviously in UUO group, the difference was statistically significant. Compare to UUO group, the expression of PCNA was down-regulated in both EPL group and SLT group, the difference was statistically significant(P<0.05).Conclusions : The activation of aldosterone via ERK and SGK-1 signaling pathways induced NF-k B activation, playing an important role in cell proliferation and renal failure.Part IV Inhibition of Shenluotong Decoction on ERK/NF-κB signaling pathway in contralateral kidneyObstructive: To establish the obstructive nephropathy rat model and observe the related factors of inflammatory signaling pathway-ERK/NF-κB, then verify if classic inflammatory reaction pathway is involved in the contralateral kidney damage and he mechanism about protection in contralateral kidney with shenluotong and mineralocorticoid receptor blockers.Methods: The methods of animal grouping, model establishment, medication and statistics were the same to PartⅡ, 10 days. The protein expression and location of p-ERK1/2, NF-κB in contralateral kidney were detected by immunohistochemistry and Western blot respectively.Results:1 The expression of p-ERK1/2 by immunohistochemistryThe expression of p-ERK1/2 was specially observed in nucleus. In Sham group, only a few of positive cells can be observed. In UUO group, the expression was significantly increased in epithelial cells and interstitial cells. Compared with UUO group, the expression was down-regulated in both EPL group and SLT group.2 The expression of NF-κB by immunohistochemistry In Sham group, only a few of NF-κB was expressed in kytoplasm. In UUO group, the expression was increased,and it can be observed in epithelial cells and interstitial cells, especially in the dilated tubules. Compared with UUO group, the expression was down-regulated in both EPL group and SLT group.3 The expression of p-ERK1/2 and ERK1/2 by Western blot The expression of p-ERK1/2 and ERK1/2 were also increased obvipusly in contralateral kidney in UUO group than Sham group, and it was down-regulated in EPL group and SLT group, the difference was statistically significant.4 The expression of NF-κB by Western blot The expression of NF-κB was weak in Sham group, and was enhanced obviously in UUO group. Compared with Sham group it was statistically significant. Compared with UUO group, the expression of NF-κB was down-regulated in EPL group and SLT group, the difference was statistically significant It was also increased markedly in contralateral kidney of UUO than Sham group and down regulated by treatment.Conclusions : The contralateral kidney in obstructive nephropathy, ERK and NF-κB can be induced by inflammatory injury, and it can induce the cell proliferation in contralateral kidney, playing a role in the progress of renal interstitial fibrosis. And these can be down-regulated by Elperenone and Shenluotong. They can inhibit cell proliferation and protect the renal function and structure, slowing the progress of renal disease.Part V Inhibition of Shenluotong Decoction on aldosterone SGK-1 activationObstructive: To establish the obstructive nephropathy rat model and analysis the SGK-1 in contralateral kidney, then verify if the shenluotong and mineralocorticoid receptor blocker can protect the contralateral kidney by blocking the aldosterone-SGK-1way.Methods:The methods of animal grouping, model establishment, medication and statistics were the same to PartⅡ, 10 days. The protein expression and location of SGK-1 in contralateral kidney were detected by immunohistochemistry and Western blot respectively. The m RNA level of SGK-1 was detected by Real-time PCR.Results: 1The results of SGK-1 m RNA level in contralateral kidney by Real-time PCRThe results of SGK-1 m RNA were up-regulated in contralateral kidney in UUO group. Compared with UUO group, the expression of SGK-1 was down-regulated in EPL group and SLT group, the difference was statistically significant.2 The expression of SGK-1 by immunohistochemistry In sham group, only a few of SGK-1 was expressed in kytoplasm of epithelial cell. In UUO group, the expression was significantly increased, be observed both in cortex and medulla. The two treatment groups also had some positive cell, but not as many as UUO group.3 The expression of SGK-1 in contralateral kidney by Western blot The result of SGK-1 showed that compared to Sham group it was up-regulated in UUO group, the difference was significant(P<0.01); Compared to UUO group, the expression of SGK-1 was down-regulated in EPL group and SLT group, the difference was statistically significant(P<0.01).Conclusions:Elperenone-the blocker of aldosterone and Sheluotong have the machanism of renal protection to the contralateral kidney via inhibiting the inflammatory injury which is induced by aldosterone. They decreased the expression of NF-κB, via SGK-1 and ERK signaling pathways to inhibit cell proliferation and alleviate obstructive nephropathy contralateral kidney damage, delaying the process of fibrosis.Conclusions:1 Chinese “Preventive Treatment”include “prevention deals with latent diseases”, “prevent an asymptomatic disease from progressing to symptomatic disease”,“prevent disability”,“preventing recurrence”. This theory also contribute to the treatment of renal disease, especially in the protecting contralateral kidney in obstructive nephropathy.2 In obstructive nephropathy animal experiment, the contralateral kidney showed compensatory hypertrophy, and renal interstitial inflammatory cell infiltration increased. The fibrosis marker α-SMA was also up-regulated and cytokine such as MCP-1 、 TNF- were increased.The cell proliferation obvious.3 The activation of aldosterone via ERK and SGK-1 signaling pathways induced NF-k B activation, playing an important role in cell proliferation and renal failure.4 The contralateral kidney in obstructive nephropathy, ERK and NF-κB can be induced by inflammatory injury, and it can induce the cell proliferation in contralateral kidney, playing a role in the progress of renal interstitial fibrosis. And these can be down-regulated by Elperenone and Shenluotong. They can inhibit cell proliferation and protect the renal function and structure, slowing the progress of renal disease.5 Elperenone-the blocker of aldosterone and Sheluotong have the machanism of renal protection to the contralateral kidney via inhibiting the inflammatory injury which is induced by aldosterone. They decreased the expression of NF-κB, via SGK-1 and ERK signaling pathways to inhibit cell proliferation and alleviate obstructive nephropathy contralateral kidney damage, delaying the process of fibrosis. |
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