The Role Of Renal Local Renin Angiotensin System In The Progression Of Diabetic Nephropathy And Its Involved Mechanisms

PART I Blockade of Renal Renin-Angiotensin System by AT1 Antisense Objective Local renin-angiotensin system (RAS) may play an important role in the pathogenesis of various kidney diseases. However, direct evidence has not been provided so far due to methodology limitations, and the mechanisms underlying local RAS have not been fully elucidated. In the present study, RAS suppressive effect of angiotensin type 1 receptor (AT1) antisense (AS) oligodeoxynucleotide (ODN), transferred unilaterally into one kidney of rat by local gene transfer technique, was evaluated.Methods In male Wistar rats, AT1 AS-ODN was locally delivered to one kidney through unilateral injection via renal artery followed by electroporation of the injected kidney. FITC-labeled AS-ODN was used to visualize the localization of transferred ODN on frozen section of the transfected kidney. Renal AT1 suppression was determined by radioautography for receptor binding capacity, RT-PCR for mRNA expression, Western blot as well as immunohistochemistry for protein expression. The effects of AS-ODN were compared with those caused by AT1 sense ODN (Sen-ODN) or procedure alone (Sham). The gene transfer protocol was optimized in terms of voltage for electroporation and duration of transferred AS-ODN. Results FITC-labeled AS-ODN was distributed mainly in glomeruli of transfected kidney, while it was not detectable in contralateral untransfected kidney. AT1 AS-ODN transfection decreased AT1 binding by some 70%, mRNA level by 43% and protein expression by 67% compared with those in the contralateral kidney 3 days after transfection. Similarly, AT1 mRNA expression was 47% and 51% less, and protein level was 63% and 71% lower compared to those with Sen-ODN transfections and Sham, respectively. Immunohistochemistry showed that reduced AT1 in glomeruli was primarily localized in the mesangium.Conclusion Our study shows that local transfection of AT1 AS-ODN can effectively inhibit AT1 in transfected glomeruli. This method can be used to study short-term effects of renal RAS.part IIRenal Transfection of AT1 Antisense Inhibits PKCβ Activation in Experimental Diabetic Rat KidneysObjective Protein kinase Cβ(PKCβ) has been considered as the key profibrotic enzyme in diabetic kidneys. Angiotensin II activates PKCβ through its type 1 receptor (AT1). However, whether the interaction between AT1 and PKCP depends upon the systemic milieu, i.e. changes in hyperglycemia, angiotensin II level, blood pressure, is unknown. This study is to examine the renal effect of ATI on activation of PKCβ. Methods Hyperglycemia was induced by streptozotocin (55mg/kg) in male WKY rats. After three weeks, AT1 antisense oligodeoxynucleotide (ODN) or control ODN was transfected into the left kidney through renal artery injection of the DNA then followed by electroporation on the same kidney. Three days later, AT1 expression in the renal cortex, and PKCβ expression ratio of cytomembrane to cytosol in the renal cortex were assessed by Western blot. Sham-operated diabetic rats orally treated with AT1 antagonist Losartan (20mg/kg/d) were also examined.Results Losartan treated diabetic rats showed significantly decreased systolic blood pressure compared with the sham-operated diabetic animals, while the antisense ODN and control ODN did not. Western blot showed some 70.1% and 76% reduction of cortical AT1 expression in antisense and losartan treated kidneys. Compared with the sham-operated and control ODN transfected rats, AT1 antisense significantly attenuated the increased PKCβ expression ratio of cytomembrane to cytosol seen in diabetic kidney, which was duplicated by losartan treatment, the ratio decreased by 79.1 % and 74% in antisense and losartan treated kidneys.Conclusion Our results suggest that locally transfected antisense for AT1 can inhibit the activation of PKCβ in streptozotocin-induced diabetic rat kidneys.PART III Effects of AT1_a receptor on renal extracellular matrix remolding in chimeric diabetic miceObjective To explore the effects of AT1_a receptor on extracellular matrix remolding in diabetic mice.Methods Chimeric mice were made up with AT1_A-deficient (Agtrla_/_) and intact cells. AT1_A-intact cells within the kidney can be stained ubiquitously by β-galactosidase. Hyperglycemia was induced by peritoneal injection of streptozotocin on male chimeric mice. 12 weeks later, kidneys were harvested and frozen quickly in dry ice-acetone. LacZ staining and immunohistochemistry were done on serial frozen sections. Extracellular matrix index were measured by PAS staining, and expression of TGFβ1, PAI1, AGE, nitrotyrosine were semi-quantitated by immunohistochemical method in lacz-positive(AT1_A-intact cells) and lacz-negative (AT1_A-deficient) glomeruli respectively.Results The expression of ECM and TGFβ1, PAI1, AGE, nitrotyrosine in both types of glomeruli were increased significantly in diabetic mice compared with control mice (p<0.05) . Interestingly, we found that the expression of ECM and TGFβ1, PAI1, AGE, nitrotyrosine in AT1_A-deficient glomeruli is higher than AT1_A-intact glomeruli in normal chimeric mice (p<0.05), but the increasing extent in AT1_A -deficient glomeruli is lower than that of AT1_A -intact glomeruli (p<0.01). Conclusions The deficiency of AT1_a results in compensative upregulation of TGFβ1, PAI1,AGE, nitrotyrosine in glomeruli. The results also suggest that local RAS plays some role in extracellular matrix remolding by manipulate the expression of TGFβ1,PAI1,AGE, nitrotyrosine in the kidney of diabetic mice , but there should exist other pathways to explore the whole scenario.PART IV Changes of RAS expression in AT1a gene knockout glomeruli and its effect on extracellular matrix remolding under diabetic condition Objective: To explore the changes of RAS expression in AT1a gene knockout glomeruli and its effect on extracellular matrix remolding under diabetic condition. Methods: Hyperglycemia was induced by peritoneal injection of streptozotocin in male AT1a gene knockout and wildtype mice. 12 weeks later, kidneys were harvested and frozen quickly in dry ice-acetone. Glomeruli were collected by laser capture microdissection and RNA was extracted from these glomeruli. mRNA levels of AT1a,AT1b, AT2, angiotensin, ACE, renin, and CYP11B2 were assessed by Realtime PCR; Extracellular matrix index were measured by PAS staining, and expression of TGFβ1, PAI1, MCP1 and renin were semi-quantitated by immunohistochemical method in AT1a gene knockout and wildtype mice respectively. Results: Contrast to wildtype mice glomeruli, mRNA expression of AT1b, angiotensin, renin, CYP11B2 were upregulated significantly in diabetic AT1a knockout glomeruli (p<0.05) , ACE expression were no difference between two gene type glomeruli, AT2 expression were not detected in AT1a knockout glomeruli and downregulated in diabetic wildtype glomeruli; The expression of ECM and TGFβ1, PAI1, MCP1 and renin in both kinds glomeruli were increased significantly in diabetic mice compared with control mice (p<0.05) , and were higher in AT1a knockout glomeruli than widtype glomeruli(p<0.05).Conclusion: This study has demonstrated that AT1a gene knockout cannot reduce extracellular matrix remolding in diabetic mice, upregulation of AT1b, CYP11B2 and renin may play major role in the process.PART V The effect of AT1a gene knockout on podocyte and its associate protein under diabetic conditionObjective: To explore the effect of AT1a gene knockout on podocyte and its associate protein under diabetic conditionMethods: Hyperglycemia was induced by peritoneal injection of streptozotocin in male AT1a gene knockout and wildtype mice. 12 weeks later, kidneys were harvested and frozen quickly in dry ice-acetone. Glomeruli were collected by laser capture microdissection and RNA was extracted from these glomeruli, mRNA levels of nephrin and α-actinin 4 were assessed by Real- time PCR; podocyte number were measured by WT1 immunohistochemistry in AT1a gene knockout and wildtype mice respectively; distribution of nephrin and α-actinin 4 were observed by immunofluorescence method.Results: Compared with wildtype mice, mRNA and protein expression of nephrin were decreased and distribution has changed significantly in diabetic AT1a knockout glomeruli (p<0.05) ; mRNA and protein expression of α-actinin 4 were decreased in diabetic mice (p<0.05), but there are no difference between two gene type glomeruli; The number of podocytes has no change under diabetic condition in both gene type mice.Conclusion: AT1a gene knockout downrgulte nephrin expression in diabetic mice, have no effects on α-actinin 4 expression and podocyte number.