Regulation Of Expression Of Renin/prorenin Receptor In Rat Mesangial Cells And Streptozotocin-induced Diabetic Rats Kidney

Since renin was discovered in 1898, researches of the renin-angiotensin system (RAS) become more and more emphasized and many new components and mechanisms have been observed. Lots of experiments testified that RAS played primary role in the pathophysiology of cardiovascular, nervous and renal diseases, such as diabetic nephropathy. RAS includes renin, agiontensinogen, angiotensinâ… (Angâ… ), angiotensin converting enzyme (ACE), angiotensinâ…¡(Angâ…¡) and its receptors, etc. Angâ…¡binds with its receptors at the plasma membrane, and phosphorylation of the receptor activates downstream signaling and induces intracellular responses. So far the frontiers of RAS has been focused on Angâ…¡and its receptors (AT₁R and AT₂R) and signaling pathway. Most of the scientists have the idea that the upstream components of RAS have no receptors. In recent years, data from immunofluorescence, molecular biology and animal studies suggest that local angiotensin generating systems exist, which may operate independently of their systemic counterpart. Such local tissue-specific renin-angiotensin systems have been found in several organs including heart, kidney, brain, vascular, adrenal gland, testes and lung. The discovery of local RAS made a great breakthrough in the study of the RAS.As the upstream of RAS cascade, renin, the rate-limiting enzyme, would be a better treatment target because the downsteam of RAS becomes more and more complicated. The physiological plasma concentration of renin is in the picomolar range, but it is admitted that in tissues, especially in interstitial fluids, the renin concentration may be 100-fold greater. This induces a hypothesis that there are some binding proteins or receptors which can bind to renin or prorenin, elevating the renin concentration to a high level that causes certain biological effects. Recently, scientists found that renin not only cleaves angiotensinogen to generate angiotensinâ… (Angâ… ), but also has its own binding protein(s) or receptor(s). Two such receptors have been identified so far: the mannose-6-phosphate receptor and renin/prorenin receptor (RnR). The first binding protein only leads to internalization, and plays no role in either extracellular or intracellar effects. In 2002, renin/prorenin receptor was cloned and the receptor messenger RNA is detected in heart, brain, kidney, placenta and liver. (Pro)renin binding to this receptor induced angiotensin-independent effect including phosphorylation and activation of mitogen-actived protein kinase extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) activation. Immunofluorescence studies on normal human kidney and heart showed that RnR is localized in the mesangium of glomeruli and in the sub-endothelium of coronary and kidney arteries. RnR is a 350-amino-acid protein with a single transmembrane domain, and some reports believe that the receptor is located intracellularly. In 2004 and 2005, Ichihara et al reported that prorenin probably involved in the diabetic nephropathy by a nonproteolytic mechanism and a synthesized "handle region peptide" (HRP) could competitively bind to RnR as a decoy peptide and thus prevented the development of diabetic nephropathy and the progression of cardiac fibrosis in animal models. Recently, Huang et al reported that in mesangial cells renin increased transforming growth factor-β1 (TGF-β1) , fibronectin, collagen I and plasminogen activator inhibitor-type 1 (PAI-1) through RnR-mediated mechanism, independent of angiotensinâ…¡(Angâ…¡. Schefe et al indicated that a transcription factor promyelocytic zinc finger protein (PLZF) was an upstream regulator of RnR and that renin stimulation of rat H9c2 cardiomyoblasts induced an increase of cell number and a decrease of apoptosis. All of these studies suggest that RnR plays an important role in physiological and pathophysiological situations, especially in cardiovascular and renal diseases.The application of angiotensin converting enzyme inhibitor (ACEI) and angiotensin receptor blockers (ARBs) has been one of the greatest therapeutic breakthrough in the treatment of renal fibrosis in the past two decades. Numerous experimental studies by using pharmacological or genetic means to manipulate Angâ…¡action as well as clinical studies have shown that Angâ…¡blockade reduces renal injury and fibrosis. Human and animal data suggest that, even at high doses, these drugs only slow down progression to end-stage organ failure, but not halted. The limited effectiveness of these drugs may be due, in part, to the elevated renin levels they induce and the Angâ…¡-independent or RnR-dependent profibrotic actions of renin. Therefore, the study of the biological effects of RnR in kidney may further elucidate the mechanisms of diabetic nephropathy and provides new supports to the clinical research. Renin inhibitors, such as aliskiren, which is currently in phase III clinical trials, reduce plasma renin activity to exert its role. Nevertheless, they increase total amount of plasma renin protein, the RnR ligand, dramatically. Therefore, it is crucial to examine whether renin inhibitors change the intrinsic activity of renin with respect to the RnR. Studies regarding the signal transduction of the RnR are of importance to evaluate the efficacy and safety of renin inhibitors.In present study, we observed whether the rat kidney mesangial cells expressed RnR and what its biological effects were. We investigated the regulation factors for RnR expression, including high glucose and Angâ…¡and its two type receptors subtype: AT₁R and AT₂R. In the kidney of streptozotocin (STZ)-induced diabetic rats and losartan treated STZ-induced diabetic rats, we observed the changes in mRNA of RnR and other components of the RAS.This paper is composed of four parts as follows:Partâ… . We used different methods to identify whether and where the receptor expressed in cultured rat kidney mesangial cells (MCs) and in (Sprague Dawley) SD rat kidney, then we studied the effects of RnR in cultured MCs. Firstly, we established a method of culturing the rat kidney glomerular MCs. The immortalized rat glomerular MCs were kindly provided by Professor Guo Muyi (Department of Pathology, Fudan University, China). The morphology of MCs was studied by light and electronic microscopy. Its molecular markers were observed by immunocytochemistry. Also, we used RT-PCR, Western blotting assessment and immunofluorescence staining to confirm the expression of RnR in rat MCs and in the rat kidney. The results obtained indicated that the MCs possessed the morphological characteristics as previously reported. The MCs grew in the form of shuttle, arborization, triangle and anomaly. These cells are positive for desmin and vimentin and negetive for cytokeratin 8．RnR mRNA and RnR protein were expressed in both MCs and rat kidney. Immunofluorescence staining analysis confirmed that the RnrR was localized in the perinuclear zone and in the mesangium of glomeruli of kidney cortex. The possibility that some RnR existed in theplasma membrane of the MCs can not be excluded. In summary, the cultured MCs possess typical characteristics of MCs previously reported. There is RnR expression in the MCs, which is consistent with the expression of RnR in the mesangium of the glomeruli.To further study the function of RnR in MCs, we analyzed the effects of HRP, a peptide antagonist to RnR, and RNA interference on the activities of the MCs.1．We detected the existence of renin activity in the culture medium by radioimmunoassay and renin mRNA expression in MCs by RT-PCR. We synthesized HRP and the HRP labeled by FITC (FITC-HRP). And observed the translocation of FITC-HRP from cell culture medium to the cytosol, and then to the nucleus. Inside the cell, the HRP was colocalized with RnR.2．HRP (10^-6 M) induced a rapid decrease of activation of ERK1/2 resulting in a rapid reduction of active ERK1/2．HRP also significantly reduced TGF-β1 mRNA expression after 24-h treatment and inhibited the proliferation of MCs after 48-h treatment. Meanwhile, HRP induced a dose-dependent increase in the matrix metalloproteinase-2 (MMP-2) activity after 72-h treatment. All these data indicate that HRP antagonizes the effects of RnR activation.3．By using the small interfering RNA technique, the effects of siRNA against RnR on the proliferation and secretion of MCs were observed. We used the sequence of StealthTM siRNA for rat RnR gene (GenBank Accession AB188298) as previously described. Confirmed that the interference efficiency of the siRNA that targeted the RnR was higher than 75%, transfection of the siRNA was shown to induce a significant reduction of TGF-β1 mRNA expression and inhibit the proliferation of MCs after 72h of transfection. The siRNA against RnR also induced an increase in the total intracellular MMP-2 protein and MMP-2 activity. Compared with treatment with LiopfectamineTM 2000 and Opti-MEM medium alone, transfection of siRNA against RnR induced a significant decrease in the typeâ…£collagen protein in the culture medicum after transfection for 72h. These results again support the view that activation of RnR promotes cell proliferation and secretion of TGF-β1 and collagenâ…£but reduces the MMP-2 activity.4．HRP suppressed the effects induced by Angâ…¡. Angâ…¡induced an increase in the level of TGF-β1 mRNA expression, stimulated the proliferation of MCs, and induced a reduction of MMP-2 activity, while HRP suppressed all these effects induced by Angâ…¡.5．HRP suppressed the effects induced by high glucose. High glucose induced an increase in the level of TGF-pi mRNA expression and this increment was suppressed by HRP1．Administration of high glucose for 72 hours induced a reduction of MMP-2 activity, and HRP also attenuated this effect.Partâ…¡. Regulation of RnR mRNA expression by Angâ…¡and high glucose in MCs.1．The effect of Angâ…¡on RnR mRNA expression. Angâ…¡(10^-8 M-10^-6 M) reduced the RnR mRNA and protein expression. PD123319 (AT₂R antagonist) attenuated the Angâ…¡-induced decrease in RnR mRNA expression, while losartan (AT₁R antagonist) did not influence the Angâ…¡effect. However, CGP42112A (AT₂R agonist) alone significantly reduced the RnR mRNA expression. All these data suggest that Angâ…¡suppresses the expression of RnR through the mediation of AT2R. 2．High glucose (30 mM) resulted in a reduction of the expression of RnR mRNA and protein. The level of RnR mRNA of MCs treated with high glucose plus losartan was slightly lower than that in the HG group, but the difference between the two groups was statistically insignificant. However, The level of RnR mRNA of MCs treated with high glucose plus PD123319 was significantly higher than that of the HG group and C group. A significant increase in the level of AT₂R mRNA was noted in response to high glucose treatment. There is no significant difference in AT₁R expression between HG group and control group. These results indicate that the increase of AT₂R expression by high glucose is possibly one of the mechanisms whereby high glucose suppresses the RnR expression.Partâ…¢. Expression of RnR mRNA in the kidney of STZ-induced diabetic rats and losartan-treated diabetic rats.1．Firstly, we determined the changes of RnR mRNA level in the kidney of nondiabetic SD control rats (C rats) and diabetic rats (DM rats) in different time points after STZ administration (1week, 3week, 6week, 12week and 24week). The rats were divided into 2 groups: the C rats (n=40) and DM rats (n=40) . Animals with blood glucose higher than 16．7mM were included in this study. The body weight of the DM rats was significantly lower than that of the C rats. Urinary protein excretion in diabetic rats was higher than that of C rats. In DM rats, the kidney RnR mRNA level was significantly lower than that in the C rats during the 24-week STZ treated (P<0．05) . Plasma renin activity was significantly lower in the DM rats than in the C rats (P＜0．05) . DM rats showed a significant increase in the plasma Angâ…¡and kidney Angâ…¡content compared with the C rats. The decrease in the expression of kidney RnR may be considered as a negative feedback control that may protect the kidney function from worsening.2．In another experiment, the rats were divided into three groups: C rats, DM rats andDM+losartan rats. In DM + losartan group, the rats were treated with AT₁Rantagonist (losartan), at a dose of 20 mg/kg body weight per day by gavage oncedaily (n=8) for 6 weeks (beginning 1 week after STZ administration to 7 weeksafter STZ administration); while in the DM group (n=8) , the rats were gavagedwith the same volume of water for 6 weeks. Blood glucose, body weight, kidneyweight, blood pressure, urinary volume, urinary protein excretion, serumcreatinine (Cr) were measured. Blood sample and the right kidney of each animalwere obtained after decapitation. The results obtained showed that the urinary protein excretion in DM rats significantly increased after 2 weeks of STZ administration, and that losartan almost normalized the urinary protein excretion in diabetic rats to a level only slightly higher than the control. The change of serum creatinine (Cr) was similar to the change of urinary protein excretion. RnR mRNA level in the kidney of DM rats was lower than that of the C rats, moreover, the RnR mRNA level in DM + losartan rats was lower than that of the DM rats. The kidney ATR mRNA level was lower in the DM rats than in the C rats and DM + losartan rats during the 6-week treatment period, though the difference between C rats and DM + losartan rats was statistically insignificant. Therefore, we propose our working hypothesis: the prorenin and Angâ…¡levels in the kidney increase, leading to glomerular mesangial cells proliferation and an enhancent of the expression of TGF-β1 and collagen secretion through the mediation of RnR and AT₁R, respectively, ultimately resulting in glomerulosclerosis. On the other hand, Angâ…¡reduces the expression of RnR through AT2R, which is upregulated during hyperglycemia. Therefore, the decrease of expression of kidney RnR may be considered as a negative feedback control that is of benefit for the attenuation of the nonproteolytic effects of prorenin. A clinically relevant finding in this study is that the blockade of AT₁R and an upregulation of AT₂R by losartan may further downregulate RnR, a profibrotic and proliferation factor. This may, at least, be one of the mechanisms whereby losartan exerts its renoprotective effects in diabetic nephropathy.In summary, the present study demonstrated that there is RnR expression in mesangial cells and the binding of renin/prorenin to RnR induces proliferation and fibrosis. Angâ…¡and high glucose downregulated the expression of RnR through AT₂R. The level of expression of kidney RnR in STZ-induced diabetic rats is lower than the level of the control rats. Losartan further decreased the expression of kidney RnR and elevated the expression of AT₂R in diabetic rats. This may be a new mechanism that losartan has clinically renoprotective effects in diabetic nephropathy.