The Renoprotective Effects And Mechanisms Of All-trans Retinoic Acid On Chronic Renal Failure Rats

Chronically developed renal diseases resulted by any kinds of causes present a common feature of progressing renal fibrosis and some will deteriorate to renal failure for which renal replacement will have to be prescribed [ 1 ] . The number of patients whose renal failure is caused by chronic renal diseases is increasing. So it has become one focus to prevent and delay renal function deterioration. Lots of studies have revealed various mechanisms responsible for chronic renal insufficiency, including renal heamodynamic changes, increased metabolism of glomeruli and renal tubules, increased oxygen tension, lipid metabolism abnormality, macromolucules deposition in mesangial calls, coagulation abnormality and the effects of inflammatory factors, chemotactic factors and interleukins [2] . The methods being adopted to delay renal insufficiency include low-protein diet, blood pressure control and correcting dyslipidomia, renin angiotensin system blockade ect. However the effect of these methods is less than 30% [3] , which obliges us to study new ways of treatment.All-trans retinoic acid (atRA) is the bioactive derivative of Vitamin A in human body. It has extensive effects in development of vertebrates, cell differentiation and maintaining internal environments [4] . As a group of ligands of nuclear receptors family, atRA activates retinoic acid receptor (RAR) and retinoic acid X receptor (RXR) to form dimmers RAR-RAR or RAR-RXR which modulates lots of genes expression [ 5 ]. The expression patterns of genes that modulate retinoic acid signaling system are various and the gross effect of atRA on cells and tissues is to inhibit pathologic proliferation and to induce proliferation of juvenile cells. It has been proved in vitro that it could antagonize actions of many cytokines including interleukin 1, tumor necrosis factor, transforming growth factor, platelet derived growth factor, activating protein l(AP-l) and so on, to decrease deposition of extracellular matrix. These cytokines have been proved to take important roles in promoting development of some acute nephritis and chronic renal fibrosis [ 6-8 ] . The important treatment effect of atRA is also reveled in animal models of acute nephritis. For example, treatment of atRA could decrease mesentery cells proliferation and urine protein in rats whose acute nephritis was induced by thyrogloblin-1 (Thy-1 )more than in those without atRA treatmenti 9 ]. It was also clearly proved that atRA could protectpodocytes in puromycin aminonucleoside-induced nephritic rats [10] .As mesentery cells proliferation, ECM aggregation and foot cells injury are main manifestations in early stage of glomerulosclerosis, we propose/presume/suppose atRA may possess certain treatment value for chronic renal fibrosis.In order to examine this hypothesis, 5/6 nephroectomized rats were used to study whether atRA can delay renal fibrosis of the remaining kidney tissue by observing renal function and glomerulosclerosis. The effect of atRA on mesangial cells proliferation and rennin-angiotensin system were also examined and the possible mechanisms were discussed.PARTIatRA could delay the process of glomerulosclerosis in 5/6 nephroectomized ratsObjective: Glomerulosclerosis is a common pathological event to different kidney diseases. The therapeutic strategies till now are not satisfactory. Newer interventions are needed to improve current therapeutic agents. Some evidences showed that all trans retinoic acid (atRA) could regress the mesangial cells proliferation and ameliorate podocytes damage in acute experimental glomerulonephritis, which clued on a hypothesis that atRA halts the development of glomerulosclerosis. We used 5/6 nephroectomized rats as animal model to mimic human chronic renal failure by hemodynamic mechanism.Methods: Wistar male rats were operated by 5/6 renal ablation and randomly divided into atRA treated groups (5mg/kg/d, lOmg/kg/d and 20mg/kg/d, n=40 ) and NX group (vehicle, n=40). Health rats consist of sham-operation group (n=25). Rats were sacrificed at beginning, 4 weeks, 7weeks, lOweeks and 13weeks of study, while 8 rats in atRA groups and 5 rats in sham were involved respectively. Concentrations of atRA in plasma and renal tissues were measured by Reversed Phase High Performance Liquid Chromatography (RP-HPLC). Glomerulosclerosis was evaluated by glomerulosclerosis index system. Renal function, artery blood pressure, and bone section and so on were measured at the same time.Results: The atRA concentrations either in plasma or renal tissue of in atRA treated groups were much higher than those of untreated rats. That meant this medicine could be absorbed by the animal model through gavage. From7 to 10 weeks artery bloodpressures of tail in all nephroectomized rats elevated significantly. Blood pressure of rats treated by atRA became lower than those of NX group, which paralleled to the decreases of ratio of urinary albumin and urinary creatinine. The ratios of kidney weight and body weight in vehicle group were much higher than that of atRA treated rats ( P<0.05). The glomerulosclerosis scores in atRA treatment groups were lower than NX. For example, the sclerosis score at 10 weeks of study was 120.4±24.7 in 5mg/kg/d , 111.9±22.9 in lOmg/kg/d and 81.2±16.6 in 20mg/kg/d vs I51.6±32.8 in NX group, PO.05.Conclusion: 1) In this present study we proved for the very first time that atRA can delay the development of chronic glomerular sclerosis. 2) atRA could be absorbed by rats through gastrointestinal tract and accumulated in remnant kidney. PART 2The mechanism of atRA on ameliorate the remnant renal fibrosisObjective: The mechanism of chronic renal failure included many factors. In 5/6 nephroectomized animal model the glomerular hypertrophy and hyperplasia resulted by cell growth responses were associated with advanced renal fibrosis. Since mature glomerular visceral epithelial cells(podocytes) has limited proliferative capacity the enlargement of glomeruli would contribute to the podocytes damage directly. Mesangial cells proliferation was the primary reason in glomerular expansion. atRA proved by numerous researches could inhibit many cell strains including mesaigial cells. These inhibition often occurred in acute nephritis animal models mentioned at the first part of our study. We then wondered whether atRA performed such biologic activity in its remnant renal protection. In this part, we observed the morphological change of glomeruli during the process of chronic renal failure and also measured several cytokines involved in cell proliferation and kidney sclerosis, such as transforming growth factor beta 1(TGF(31), proliferative cellular nuclear antigen(PCNA) and activative protein l(AP-l).Methods: Wistar male rats were operated by 5/6 renal ablation and randomly divided into atRA treated groups (5mg/kg/d, lOmg/kg/d and 20mg/kg/d, n=40 ) and NX group ( n=40). Health rats consist of sham-operation group (n=25). Rats were sacrificed atbeginning, 4 weeks, 7weeks, lOweeks and 13weeks of study, while 8 rats in atRA groups and 5 rats in sham were involved respectively. Mesangial cell proliferation in H.E staining renal section was measured by cells calculation under light microscpe and evaluate the glomerular volume as well, the expression levels of PCNA and TGFp1 were assayed by renal immunohistochemical staining and Western blot. The mRNA expressions of two subunits of AP-1, c-jun and c-fos, were quantitated by real-time PCR.Results; First we chose 20mg/kg/d atRA treated rats as representive atRA group while observed the mesangial cell hyperplaisa and PCNA expression at different time of study. Comparing to sham the average glomerular size and cell number in 5/6 nephroectomized rats increased much than NX from the 4 week to the end. atRA treated could reduce the glomerular size expansion and cell proliferation at earlier period (at the 4th weeks of study). The expression of PCNA in atRA group decreased significantly than that of NX at the 4th and 7th weeks of study measured either by immunohistoassay or western blot. Then we measured the TGF(31 and AP-1 expression levels of 5mg/kg/d, lOmg/kg/d, 20mg/kg/d and NX at the 10th week of study. Data demonstrated that the TGF(31 levels raised much higher in all renal ablation rats than that of NX, P<0.05. TGFpi in all atRA groups decreased. 1 Omg/kg/d and 20mg/kg/d were lower than 5mg/kg/d.The mRNA expression degrees of c-jun and c-fos in subtotally nephrotomized rats increased significantly than sham. atRA could inhibit the levels of c-jun and c-fos mRNA.Conclusion: (DatRA inhibited glomerular hypertrophy and mesangial cell proliferation. ?atRA downregulated the AP-1 mRNA expression, through which reduced the TGFpi in remnant kidney of 5/6 nephrotomized rats. PART 3All-trans retinoic acid markedly inhibits over-activated of renin-angiotensin system in 5/6 nephroectomized ratsObjective: renin-angiotensin system plays an important role in chronic renal damage. It was demonstrated that atRA could downregulate several growth factors frommesangial cells, induced by angiotensin II, in vitro. Furthermore in acute nephtitis animal model it has been observed that atRA could decrease the activity of renal renin and angiotensin. At the first part of our study atRA was demonstrated as highly efficient method of limiting renal damage in the acute experimental glomerular disease. So we need to explore whether atRA could exert its effect on the renin-angiotensin system in 5/6 renal ablation model either. In the following part we measured the renin and angiotensin in plasma or kidney in different methods. Methods: We administered atRA to rats with 5/6 renal ablation by three dosage: 5mg/kg/d (n=8), lOmg/kg/d (n=8) and 20mg/kg/d (n=8) and vehicle (vehicle group, n=8) for 10 weeks. Health rats consist of sham-operation group(n=8). Renin and angiotensin II in plasma and renal tissues were measured by radioimmunoassary. mRNA expression of renin-angiotensin convert enzyme(ACE) was quantitated by real-time PCR. Angiorensin type 1 receptor(ATiR) in remnant renal cortex was measured by Western blot.Results: After 10 weeks of atRA treatment by gavarge, artery blood pressure reduced: 149±19 in 5mg/kg/d, 143±20 in lOmg/kg/d and 140±22 in 20mg/kg/d vs 192±29mmHg in NX, /><0.05. atRA inhibited the levels of rennin either in plasma or kidney (PO.05). Otherwise atRA decreased the level of angiotensin II in renal cortex (659±215 in 5mg/kg/d, 671±196 in lOmg/kg/d and 462±178 in 20mg/kg/d vs 1516±457 in NX, P<0.05), not in plasma. atRA also down-regulated ACE mRNA expressions and ATjR protein in renal cortex. Larger dose of atRA (20mg/kg/d) performed higher activity in inhibiting renin and ATiR.Conclusions: (D RAS was highly activated in 5/6 nephroectomized rats. (2) atRA could ameliorate the main elements of renin-angiotensin system. PART 4Change of all-trans retinoic acid content in chronic renal failure rats and the meaningObjective: atRA is a kind of indispensible substance in human body. The reduction in renal fibrosis induced by atRA in chronic renal failure suggests the following assumptions: (T) the intrinsic atRA amount or quality may be changed in the conditionof renal failure and extrinsic atRA is needed; (2) Vitamin A is the only precursor of atRA and its biological effects is predominantly enacted through atRA. So administration of Vitamin A might get the same effect as atRA. But the second presumption is not consistent with the clinical observation results, i.e. with the deterioration of chronic renal failure, Vitamin A content in plasma increases apparently, even up to toxic level. This contradicting phenomenon suggests the metabolism of Vitamin A and atRA in chronic renal diseases differs greatly form that in normal condition.Methods: 5/6 nephroectomized rat model was used in this trial. The 5/6 nephroectomized rats were assigned into non-intervention group (NX group, n=48) and sham group (n=30). Eight rats in NX group and 5 in sham group were killed respectively in the beginning of the trial (week 0), week 2, 4,7, 10 and 13. RP-HPLC was used to examine atRA level in blood and kidney at those time point. The specific binding protein of Vitamin A in plasma- retinol binding protein (RBP) was examined with ELISA. The expression of cytoplasma retinoic acid binding protein (CRABP) mRNA was examined with RT-PCR for assessment of the reliability of atRA content in kidney.Results: It was observed in the 5/6 renal ablation rat that the plasma RBP level increases directly with the increase of blood creatine (r=0.817). The atRA content in plasma and kidney is steady in normal conditions. It was about lOng/ml in kidney, which was apparently higher than that in plasma. In NX group the plasma atRA content presented an obvious increase in the 2nd week and it reduced to normal or below normal value. Similarly, atRA content in kidney in NX group also presented an obvious increase in the 2nd week, which was higher than that of sham group. But unlike plasma atRA, kidney atRA content did not decrease immediately and it was still higher than that in sham group in the 4th and 7th week (PO.05). Until the 10th week did it reduced to normal or below normal value. The expression of CRABP mRNA in kidney in NX group increased quickly from the 2" week and it was higher than that at sham point at all time point of 4th, 7th, 10th and 13th week (PO.05). The correlation coefficient between the expression of CRABP mRNA in kidney and the kidney atRA content was 0.805 (P = 0.055).Conclusion: ? The atRA contents in palsma and kidney of healthy rats were steady. (D atRA contents in rat plasma and kidney after 5/6 renal ablation would increase for a short time followed by decrease and they reduce to normal value in middle or late...