| ObjectiveHyperuricemia has become the second-largest metabolic disease after diabetes,which directly threatens people’s life safety.As the main excretory organ of uric acid,the kidney is one of the most vulnerable target organs in hyperuricemia.The deposition of uric acid or urate crystallites in the kidney can cause dysfunction of renal tubular epithelial cells,renal interstitial fibrosis damage,decreased glomerular filtration rate,and renal failure.In recent years,numerous studies have found that mast cells are involved in many kidney diseases and become a promising therapeutic target.Degranulation of mast cells promotes renal renin/Ang Ⅱ production and promotes renal oxidative stress,microvascular damage.Therefore,this experiment explored the preventive and therapeutic effects of the mast cell membrane stabilizer sodium cromoglycate,the Ang Ⅱ receptor antagonist valsartan,and the antioxidant quercetin on uric acid nephropathy,and preliminarily explored the relevant mechanism of action.MethodsThis paper involves three parts of research,the experimental content and methods are as follows:（1）Rat model of uric acid nephropathy prepared by high purine dietSprague Dawley（SD）male rats were randomly divided into the normal control group and uric acid nephropathy group.The uric acid nephropathy group was given100 mg/kg adenine by gavage and fed high purine animal feed containing 10%yeast powder to establish a uric acid nephropathy model,and were continuously intervened for 14 days,21 days,and 28 days,respectively.The control group was given the drug vehicle 1%gum arabic solution by gavage every day,once a day,for 28 consecutive days.Blood and kidney samples were collected 2 h after the last administration.The contents of serum uric acid,creatinine,blood urea nitrogen,CO2 binding capacity,and cystatin C were determined by automatic biochemical analyzer.Renal pathological changes and fibrotic injury by HE and Masson staining.Determination of urinary toxin in the kidney by UPLC-MS/MS.Observation of the distribution and number of mast cells in the kidney by toluidine blue staining.The kits detected the content of kidney glutathione（GSH）,glutathione peroxidase（GSH-Px）,superoxide dismutase（SOD）,and malondialdehyde（MDA）.（2）The preventive and therapeutic effects of sodium cromoglycate and valsartan on uric acid nephropathy caused by high purine dietExperiment 1:SD male rats were randomly divided into the control group,model group,sodium cromoglycate group,and valsartan group.The model group,sodium cromoglycate group,and valsartan group were given 100 mg/kg adenine by gavage and fed high purine animal feed to establish the uric acid nephropathy model.In addition,at the same time of modeling,the sodium cromoglycate group was intraperitoneally injected with 25 mg/kg sodium cromoglycate,and the valsartan group was intragastrically administered with 50 mg/kg valsartan,once a day,for 4consecutive weeks of intervention.The control group was given an equal volume of normal saline and fed normal animal chow in the same way.Experiment 2:SD male rats were randomly divided into 2 groups:the control group and the uric acid nephropathy group.The uric acid nephropathy group was given 100 mg/kg adenine by gavage and fed high purine animal feed for 3 weeks to construct the uric acid nephropathy model,while the control group was fed ordinary feed and purified water.In the 4th week,the rats in the uric acid nephropathy group were redivided into 4 groups:the model group,sodium cromoglycate group,valsartan group,and allopurinol group（positive group）.The sodium cromoglycate group was given intraperitoneal injection of 25 mg/kg sodium cromoglycate,the valsartan group was given 50 mg/kg valsartan by gavage,and the allopurinol group was given 40mg/kg allopurinol by gavage.Both the model group and the control group were intraperitoneally injected with an equal volume of normal saline and intragastrically administered with purified water,once a day,for 3 weeks of continuous intervention.Blood and kidney samples were collected from all the above animals 2 h after the last administration.The contents of serum uric acid,creatinine,blood urea nitrogen,CO2binding capacity and cystatin C were determined by automatic biochemical analyzer.The pathological changes and fibrotic damage of the kidneys were examined by HE and Masson staining.UPLC-MS/MS was used to determine the content of serum various urinary toxins.Determination of renal OAT1,OAT3,URAT1,BCRP,and MRP4 expression by Western blot.The distribution and number of renal mast cells were observed by toluidine blue staining.The kits detected the contents of renal GSH,GSH-Px,SOD,and MDA.Transmission electron microscope was used to observe the mitochondrial microstructure of renal tubular epithelial cells.ELISA kits were utilized to detect renal renin and serum Ang Ⅱ levels.The changes of renal microvessels were observed by CD31 immunofluorescence.（3）The preventive and therapeutic effects of quercetin on uric acid nephropathy caused by high purine dietExperiment 1:SD male rats were randomly divided into the control group,model group,and quercetin group.Rats in the model group and quercetin group were given100 mg/kg adenine by gavage and fed high-purine animal feed to establish a model of uric acid nephropathy.In addition,at the same time of modeling,the quercetin group was given 15 mg/kg quercetin by gavage,once a day,for 4 weeks of continuous intervention.The control group was given an equal volume of normal saline and fed normal animal chow in the same way.Experiment 2:SD male rats were randomly divided into two groups:the control group and the uric acid nephropathy group.The uric acid nephropathy group was given 100 mg/kg adenine by gavage,and fed high purine animal feed for 3 weeks to construct a uric acid nephropathy model.The control group was fed ordinary feed and purified water.In the fourth week,the rats in the uric acid nephropathy group were redivided into two groups:the model group and the quercetin group.The quercetin group was given 15 mg/kg quercetin by intragastric administration,and the model group and control group were given intraperitoneal injection of equal volume of normal saline and intragastric administration of purified water,once a day,for 3weeks of continuous intervention.Blood and kidney samples were collected from all the above animals 2 h after the last administration.The contents of serum uric acid,creatinine,blood urea nitrogen,CO2 binding capacity,and cystatin C were determined by automatic biochemical analyzer.The pathological changes and fibrotic damage of the kidneys were examined by HE and Masson staining.The kits detected the contents of renal GSH,GSH-Px,SOD,and MDA.Observation of mitochondrial microstructure in renal tubular epithelial cells by transmission electron microscope.The changes of renal microvessels were investigated by CD31 immunofluorescence.Results（1）Rat model of uric acid nephropathy prepared by high purine diet.Serum biochemical results showed that compared with the control group,the levels of serum urea nitrogen and creatinine were significantly increased at 14 days,21 days,and 28days after modeling（P<0.01）,serum cystatin C levels were significantly increased at21 and 28 days after modeling（P<0.05/P<0.01）,and serum uric acid level was significantly increased at 14 days,21 days and 28 days after modeling（P<0.05/P<0.01）.The results of HE and Masson staining of kidneys showed that renal tubular dilation,urate crystal accumulation,bright blue collagen fiber deposition,and flocculation were observed in the kidneys at 14 days,21 days,and 28 days after modeling,and with the increase of modeling days,the above-mentioned renal pathological damage was aggravated.When analyzing the content of urinary toxins,it was found that compared with the control group,with the increase of modeling days,the content of urinary toxins in the kidneys increased significantly（P<0.05/P<0.01）.Renal toluidine blue staining showed that the renal mast cells in the uric acid nephropathy model group were mainly distributed in the renal cortex,tubulointerstitium,glomerular and perivascular areas and were clustered,and compared with the control group,the number of mast cells increased significantly at21 and 28 days after modeling（P<0.05/P<0.01）.The results of the kidney oxidative stress test showed that compared with the control group,the levels of GSH in the kidneys were significantly decreased at 14,21,and 28 days after modeling（P<0.01）.At 21 and 28 days after modeling,the levels of GSH-Px in the kidneys were significantly decreased（P<0.05/P<0.01）,at 21 and 28 days after modeling,the levels of renal SOD were significantly decreased（P<0.01）,at 14,21 and 28 days after modeling,the levels of MDA in kidneys were significantly increased（P<0.05/P<0.01）.（2）The preventive and therapeutic effects of sodium cromoglycate and valsartan on uric acid nephropathy caused by high purine diet.Serum biochemical results showed that compared with the model group,the levels of serum urea nitrogen,creatinine,uric acid,and cystatin C were significantly decreased after preventive application of cromoglycate sodium or valsartan（P<0.05/P<0.01）.Serum creatinine and cystatin C levels were significantly decreased after the therapeutic application of cromolyn sodium or valsartan（P<0.05）.After therapeutic application of allopurinol,the level of uric acid was significantly decreased（P<0.01）,while the levels of blood urea nitrogen,creatinine,and cystatin C tended to increase.The results of renal HE and Masson staining showed that compared with the model group,pathological damages such as renal tubular dilatation,urate crystal accumulation,and fibrotic injury were improved after preventive and therapeutic intervention with sodium cromoglycate or valsartan.After therapeutic application of allopurinol,a small number of urate crystals were seen in the kidneys,and pathological damage such as renal tubular dilatation and fibrosis was not significantly relieved.The detection results of serum urea toxin showed that the contents of p-cresol sulfate,indoxyl sulfate,Hippuric acid,N-acetyl-L-arginine,and Pseudouridine were significantly decreased after the intervention of preventive application of sodium cromoglycate and valsartan（P<0.05/P<0.01）.After therapeutic intervention with sodium cromoglycate,the contents of p-cresol sulfate and Pseudouridine were significantly decreased（P<0.05）,and the contents of indoxyl sulfate showed a decreasing trend.After therapeutic intervention with valsartan,the contents of p-cresol sulfate and p-cresol glucuronide were significantly decreased（P<0.05）.After therapeutic intervention with allopurinol,there was no significant difference in urinary toxin content compared with the model group.Western blot results showed that compared with the model group,the expressions of OAT1,OAT3,URAT1,MRP4,and BCRP in the kidneys were significantly increased after preventive application of cromolyn sodium or valsartan（P<0.05/P<0.01）.After therapeutic intervention with sodium cromoglycate,the expressions of OAT1,URAT1,MRP4,and BCRP in the kidneys were significantly increased（P<0.05/P<0.01）,and after the therapeutic application of valsartan,the expressions of OAT3 and MRP4 in the kidneys were significantly increased（P<0.05）.After therapeutic intervention with allopurinol,the expressions of OAT1,OAT3,MRP4,and BCRP in the kidneys were significantly increased（P<0.05/P<0.01）.The results of renal toluidine blue staining showed that compared with the model group,the number of renal mast cells was significantly decreased after preventive and therapeutic intervention with sodium cromolyn（P<0.05/P<0.01）,while after intervention with valsartan or allopurinol,there was no significant difference.The results of the renal oxidative stress test showed that compared with the model group,the levels of GSH and GSH-Px in the kidneys were significantly increased after preventive application of cromolyn sodium or valsartan（P<0.05/P<0.01）,and the level of MDA was significantly decreased（P<0.05）.And the renal GSH and GSH-Px levels were significantly increased after therapeutic intervention with sodium cromoglycate,valsartan,or allopurinol,respectively（P<0.05）.The results of transmission electron microscopy showed that compared with the model group,the mitochondrial structural damage was significantly improved after prophylactic and therapeutic intervention with sodium cromoglycate or valsartan.However,mitochondrial structural damage was not effectively alleviated after therapeutic intervention with allopurinol.The results of the ELISA kit showed that compared with the model group,after the preventive application of cromolyn sodium or valsartan intervention,the contents of renal renin and serum Ang Ⅱ were significantly decreased（P<0.05/P<0.01）.After the therapeutic application of sodium cromoglycate or valsartan intervention,the serum Ang Ⅱ content was significantly decreased（P<0.05）,and the renal renin content showed a decreasing trend,but there was no statistical difference.There were no significant differences in renal renin and serum Ang Ⅱ levels after therapeutic allopurinol intervention.The results of renal CD31immunofluorescence staining showed that compared with the model,the positive areas of renal CD31 immunofluorescence staining were significantly increased after prophylactic and therapeutic intervention with sodium cromolyn or valsartan and therapeutic application of allopurinol.（3）The preventive and therapeutic effects of quercetin on uric acid nephropathy caused by high purine diet.Serum biochemical results showed compared with the model group,after preventive application of quercetin,the levels of serum creatinine,uric acid,and cystatin C were significantly decreased（P<0.05）,and the CO2binding capacity was significantly increased（P<0.05）.After the therapeutic application of quercetin intervention,the levels of serum creatinine,uric acid,and cystatin C all decreased,but there was no statistical difference.The results of renal HE and Masson staining showed compared with the model group,the pathological damages such as renal tubular dilatation,urate crystal accumulation,and fibrosis injury were significantly improved after preventive application of quercetin.However,after the therapeutic application of quercetin,the above pathological damage could not be effectively alleviated.The results of transmission electron microscopy showed compared with the model group,the damage of mitochondrial structure was significantly improved after preventive application of quercetin,while the damage of mitochondrial structure was not significantly improved after therapeutic application of quercetin.The results of the renal oxidative stress test showed compared with the model group,the levels of GSH and SOD in the kidneys were significantly increased after the preventive application of quercetin（P<0.05）.GSH-Px levels tended to increase,and MDA levels tended to decrease.After the therapeutic application of quercetin intervention,the renal GSH level was significantly increased（P<0.05）,and the GSH-Px level had an increasing trend.The results of renal CD31immunofluorescence staining showed that compared with the model group,the positive area of renal CD31 immunofluorescence staining was significantly increased after the preventive application of quercetin.After therapeutic intervention with quercetin,the positive area of CD31 immunofluorescence staining in the kidney was still significantly reduced.Conclusions（1）In this study,we simulate a high purine diet to construct a model of uric acid nephropathy.The levels of serum uric acid,creatinine,blood urea nitrogen and cystatin C were significantly increased,and the renal function was significantly damaged,and a large amount of urate accumulation and fibrotic damage appeared in the kidneys,indicating the uric acid nephropathy model was successfully established.（2）Uric acid nephropathy induced by high purine diet could lead to the occurrence of renal mast cell infiltration and oxidative stress.（3）Sodium cromoglycate reduced the production of renin/Ang Ⅱ by stabilizing the mast cell membrane,and valsartan reduced the production of Ang Ⅱ by antagonizing the Ang Ⅱ receptor,enhanced the antioxidant capacity of the kidney,relieved the damage to the mitochondrial structure of renal tubular epithelial cells,and extenuated the kidney microvascular system damage,increased the expression of uric acid-related transporters,promoted the excretion of uric acid and urinary toxins,and effectively prevented the occurrence of uric acid nephropathy or had a significant therapeutic effect on uric acid nephropathy.（4）Quercetin could partially improve the damage to the mitochondrial structure of renal tubular epithelial cells and the damage to the renal microvascular system by enhancing the antioxidant capacity of the kidney and relieving uric acid nephropathy injury,but its alleviating effect was limited. |
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