Impact Of Hyperhomocysteinemia On The Kidney Of Hypertensive Rats And Effects Of Different Doses Of Folic Acid Intervention

BackgroundHypertensive nephropathy is one of the main factors leading to end-stage renal disease.Early assessment of the risk factors of renal damage and comprehensive prevention and treatment of hypertension can delay the progression of hypertensive nephropathy and have important clinical significance in preventing the occurrence of end-stage renal disease.Epidemiological studies have shown that hyperhomocysteinemia(HHcy)and low folic acid(FA)are common in Chinese patients with hypertension,and HHcy can synergistically increase the incidence of atherosclerosis,stroke and other diseases in patients with hypertension.However,there are few studies on HHcy's effect on hypertensive renal damage.Our previous clinical studies have confirmed that HHcy can increase proteinuria in patients with hypertension,but the mechanism of HHcy aggravating renal damage in hypertensive patients remains unclear.Under physiological conditions,NADPH oxidase(NOX)is the main source of ROS production.More and more studies have found that Hcy can participate in the pathophysiological process of kidney injury through NADPH oxidase.Podocyte injury is considered to be a key factor causing proteinuria and glomerular sclerosis.Whether HHcy can cause podocyte damage through NADPH oxidase in hypertension,thereby increasing proteinuria remains to be explored.Nephrine is an important structural molecule on the surface of podocytes and plays an important role in maintaining podocyte morphology and glomerular filtration function.In addition,it is also an important signal transduction molecule.When phosphorylated,its intracellular segment can interact with a variety of proteins.When bound to CD2AP,it can activate PI3K/Akt signal transduction pathway and participate in the regulation of podocyte apoptosis.Various factors,such as high glucose,angiotensin and high pressure in glomerular spheres,can lead to abnormal expression of nephrin on the surface of podocytes.However,it has not been reported whether Hcy can regulate the expression of nephrin and its signal transduction through oxidative stress,participate in podocyte apoptosis,and further worsen hypertensive nephropathy.FA is the safest and effective way to reduce homocysteine(Hcy).In the renal subgroup study of the Chinese Stroke Primary Prevention Trial,FA supplementation in patients with hypertension can alleviate chronic renal function impairment.However,the FA dose for Hcy intervention is controversial at present,and the effect and mechanism of different doses of FA intervention on renal function are still unclear.Therefore,to explore the mechanism of action of Hcy on hypertensive renal damage and reveal the efficacy and possible mechanism of different doses of FA will be conducive to delaying the occurrence of hypertensive renal damage.It also provides further theoretical basis for the application of folic acid in patients with hypertension complicated with HHcy.In order to clarify the above problems,this topic is discussed through the following two parts:PART ONEEffect of hyperhomocysteine on renal function of hypertensive rats and itsmechanismObjectiveBy establishing a hypertensive animal model with HHcy,the effect of Hcy on renal damage in hypertension was studied,and the molecular mechanism of oxidative stress involved in Hcy-induced renal damage was explored.MethodsWistar Kyoto(WKY)rats were randomized into normal control group(WKY group)and hyperhomocysteinemia group(WKY+HHcy group),and spontaneously hypertensive rats(SHRs)were randomly divided into hypertensive group(SHR group)and hypertension combined hyperhomocysteinemia group(SHR+HHcy group),with 8 rats in each group.Rats in WKY+HHcy group and SHR+HHcy group were given 2%DL-Hcy(5 ml/kg,twice daily)intraperitoneal injection for 12 weeks to construct a hyperhomocysteinemia model.Blood pressure and plasma Hcy levels were monitored.Glomerular filtration rate(GFR)and urinary albumin creatinine ratio(UACR)were measured to evaluate renal function changes,and renal PAS staining was performed to observe glomerular structural damage.Serum superoxide dismutase(SOD)and malondialdehyde(MDA)levels were measured,and the expressions of NOX2 and NOX4 in kidney were detected by qRT-PCR and western blot to evaluate the status of local oxidative stress in kidney.The mRNA and protein expression of nephrin in podocytes were detected by qRT-PCR and immunohistochemistry,respectively.Results1.The plasma Hey level of WKY and SHR rats injected intraperitoneally with DL-Hcy was significantly higher than that of the respective control group,which proved that the animal model of hypertension complicated with HHcy was successfully established.Repeated measurement analysis of variance was performed on the blood pressure of rats at week 0,week 6 and week 12.The results showed that with the increase of time,the effect of Hcy on the systolic and diastolic blood pressure of WKY rats and SHR rats was not statistically different(P>0.05).2.Both HHcy and hypertension can increase urine protein and decrease glomerular filtration rate,and the hypertension combined with HHcy group has the highest urine protein level and the lowest glomerular filtration rate,indicating that the two factors have a synergistic effect on renal damage effect.However,among the two pathogenic factors of Hcy and hypertension,hypertension is still the main risk factor for renal damage.PAS staining of the kidney showed hypertrophy of the glomerulus and slight increase of the extracellular matrix of the glomerulus in WKY+HHcy group.The glomerular capillary loops in SHR group were wrinkled and the glomerular balloon gap widened.However,the glomerular structure was most damaged in the SHR+HHcy group,the glomerular capillary loops were significantly wrinkled and thickened,and the glomerular extracellular matrix was increased.3.Both Hcy and hypertension can reduce the level of serum SOD and increase the level of MDA.However,when hypertension is combined with HHcy,the level of SOD is lower and the level of MDA is higher.Both Hcy and hypertension can lead to an imbalance of oxidation and antioxidant processes,leading to a state of chronic oxidative stress.4.The mRNA and protein expression levels of NOX2 and NOX4 in renal tissues were detected by qRT-PCR and western blot,and the results showed that Hcy up-regulated the expression levels of NOX2 and NOX4 in renal tissues,and hypertension also up-regulated the expression of NOX2 and NOX4 in renal tissue.The local oxidative stress in the kidney was strongest in the SHR+HHcy group,and the expression levels of NOX2 and NOX4 in the kidney tissues were higher than those in the WKY+HHcy group and the SHR group,respectively,suggesting that Hcy and hypertension synergistic enhance local renal oxidative stress.5.qRT-PCR and immunohistochemical results showed that Hcy down-regulated the mRNA and protein expression of nephrin on podocyte surface.The expression of nephrin on podocyte surface was the lowest in the SHR+HHcy group,which was lower than that of the SHR group and the WKY+HHcy group,respectively.With the increase of local oxidative stress in the kidney,the expression of nephrin decreased.Conclusions1.An animal model of hypertension combined with hyperhomocysteinemia was successfully constructed,and it was found that Hcy can cause early renal damage and play a synergistic role with hypertension in renal damage.2.Hcy overexpresses the GAPDH oxidases subunits NOX2 and NOX4 in renal tissues to enhance oxidative stress,and this effect is independent of blood pressure.3.Hcy can induce the decrease of nephrin expression in podocytes by enhancing local oxidative stress in the kidney,and it can cooperate with hypertension to aggravate kidney damage.PART TWOEffect and mechanism of different doses of folic acid on renal damage induced by hyperhomocysteinemia in hypertensive ratsObjectiveFurther explore the mechanism of Hcy involved in podocyte injury,and clarify the effect and mechanism of different doses of folic acid intervention on Hcy-induced renal damage in hypertensive rats.MethodsThe SHRs were randomized into a control group,HHcy group,HHcy+low dose folic acid group(HHcy+LFA group)and HHcy+high dose folic acid group(HHcy+HFA group),with eight animals per group.All animals were given intraperitoneal injections of drugs for 12 weeks,and were given gavage treatment at the same time from the 5th week of the experiment for 8 weeks.The specific method is as follows:1)control group:physiological saline(PS,5 ml/kg,twice a day)was intraperitoneally injected for 12 weeks and PS(0.5 ml/d)was gavage for 8 weeks;2)HHcy group:2%DL-Hcy(5 ml/kg,twice a day)was intraperitoneally injected for 12 weeks and PS(0.5 ml/d)was gavage for 8 weeks;3)HHcy+LFA group:2%DL-Hcy(5 ml/kg,twice a day)was intraperitoneally injected for 12 weeks and FA(0.4 mg/kg/d)was gavage for 8 weeks;and 4)HHcy+HFA group:2%DL-Hcy(5 ml/kg,twice a day)was intraperitoneally injected for 12 weeks and FA(4 mg/kg/d)was gavage for 8 weeks.Blood pressure and plasma Hcy level were measured at week 4,8 and 12,respectively.At the 12th week,vena cava blood and urine of rats were collected for plasma creatinine,serum MDA,SOD,UACR and GFR detection.PAS staining was performed on the renal tissue sections to observe the glomerular damage and calculate the glomerulosclerosis index(GSI).The ultrastructure of podocytes was observed by electron microscopy.Expression of NADPH oxidase subunits NOX2 and NOX4 in the kidney was detected by qRT-PCR and western blot.Immunohistochemical staining and qRT-PCR were used to detect the expression of nephrin,Akt,Bcl-2 and Bax in renal tissues.The apoptosis of podocytes was detected by TUNEL assay.Results1.On the basis of the animal model of hypertension combined with HHcy,different doses of folic acid were given intervention,and it was found that both high and low doses of folic acid can effectively reduce the plasma Hcy level.However,compared with low-dose FA,high-dose FA did not show a better effect in reducing plasma Hcy,and increasing the dose of folic acid did not show a reasonable dose-effect relationship.In addition,after the intervention of Hcy and folic acid,the systolic and diastolic blood pressure of hypertensive rats did not change significantly.2.Folic acid can significantly reduce albuminuria and improve glomerular filtration rate in hypertensive rats complicated with HHcy.There was no significant difference in the improvement of urinary protein and glomerular filtration rate in high dose folic acid compared with low dose folic acid.Even low dose folic acid can protect Hcy-induced renal damage in hypertensive rats.3.Kidney PAS staining showed that HHcy cooperated with hypertension to aggravate glomerular structural damage,manifested as glomerular capillary loops were significantly wrinkled and thickened,glomerular extracellular matrix increased,and GSI increased.After folic acid intervention,the glomerular extracellular matrix and the GSI decreased,but there was no significant improvement in glomerular structure in the high-dose folic acid group compared with the low-dose group..4.Folic acid increased serum SOD level and decreased serum MDA level in hypertensive rats complicated with HHcy,but there was no significant difference in the effect of high and low doses of folic acid.Low doses of folic acid can improve the chronic oxidative stress caused by Hcy.5.The qRT-PCR results showed that folic acid inhibited the upregulation of Hcy-induced NOX2 and NOX4 mRNA expression in renal tissue.Western blot analysis showed that folic acid inhibited the upregulation of Hcy-induced NOX2 and NOX4 protein expression in renal tissue.Moreover,compared with the low-dose folic acid,the high-dose folic acid was more effective in reducing the expression of NOX2,but Hcy levels were not lower in the high-dose folic acid group than in the low-dose group,indicating that folic acid exerted an antioxidant effect independent of Hcy.6.Immunohistochemical staining and qRT-PCR analysis showed that folic acid intervention upregulated the expression of nephrin on the podocyte surface of hypertensive rats complicated with HHcy.However,compared with the low dose folic acid,the high dose folic acid had no obvious advantage in increasing nephrin.7.The ultrastructural changes of podocytes were observed by transmission electron microscopy.The glomerular basement membrane of the control group(hypertensive)was thickened,and partial fusion of podocyte foot processes was observed.However,the brush-like structure of podocyte foot process disappeared and was widely fused in hypertensive rats complicated with HHcy.After the intervention of folic acid,with the recovery of nephrin expression,the ultrastructure of the foot process was significantly improved.8.The qRT-PCR results showed that Hcy down-regulated the mRNA expression levels of Akt and anti-apoptotic factor Bcl-2,and up-regulated the mRNA expression level of pro-apoptotic factor Bax,and the ratio of Bcl-2/Bax decreased.After folic acid intervention,the mRNA expression of Akt and Bcl-2 in renal tissue increased,while the mRNA expression of Bax decreased,and the ratio of Bcl-2/Bax increased.Immunohistochemical results also showed that HHcy reduced the expression of activated Akt and Bcl-2 proteins and decreased the expression of Bax protein in the glomerulus of hypertensive rats.The intervention of folic acid can counteract the above-mentioned changes induced by HHcy.However,there was no significant difference in the expression of Akt,Bcl-2 and Bax between the high and low dose folic acid groups.9.TUNEL assay was used to analyze the apoptosis of podocytes.The results showed that Hcy increased the number of TUNEL positive cells in hypertensive rats,and the apoptosis of podocytes decreased after treatment with folic acid.However,there was no significant difference between high dose and low dose of folic acid on podocyte apoptosis.Conclusions1.Hcy induces the reduction of nephrin expression in podocytes by enhancing renal oxidative stress,then down-regulates the Akt/Bcl-2 signaling pathway,participates in the structural destruction and apoptosis of podocytes,and synergically aggravates renal damage in hypertension.2.Folic acid inhibited apoptosis of podocytes by reversing oxidative stress induced by Hcy and up-regulating nephrin/Akt/Bcl-2 signaling pathway in podocytes.3.Even low dose of folic acid can improve HHcy-related hypertensive renal damage by reducing Hcy levels and independent antioxidant effects.