Relationship Between Carnosine,carnosinase And Renal Injury And Its Effect On Mesangial Cells In Diabetic Nephropathy

Background and Objective: Diabetic nephropathy(DN)is one of the most common and serious complications of diabetes mellitus(DM).The main clinical manifestations of DM are albuminuria,hypertension and gradual decline of renal function.In addition,DN is also one of the major causes of end-stage renal disease(ESRD).Approximately 40% of DM patients worldwide could still develop ESRD after 20 years hypoglycemic therapy,and subsequently require renal replacement therapy.The pathogenesis of DN is complex and involves many factors,including hemodynamic disorder,glucose and lipid metabolism disorders,advanced glycosylation end products(AGEs)and advanced glycosylation end products(ALEs)accumulation,renin-angiotensin-aldosterone system(RAAS)activation,oxidative stress reaction,overexpression of various vasoactive substances and cytokines,heredity and so forth.Currently,there are two main overall goals in the treatment of DN: one is to maintain renal function to reduce the risk of ESRD;second is to reduce the risk of cardiovascular events and mortality.The clinical treatment options for DN mainly include the control of blood glucose and blood pressure,and the use of statins as well as RAAS blockers may delay the progress of disease in a certain extent but cannot completely prevent the development of DN.Carnosine is a water-soluble dipeptide composed of ?-alanine and L-histidine,which is widely distributed in human organs.It has a variety of biological activities, including physiological p H balance,chelating metal ions,anti-oxidative stress,inhibition of inflammatory cytokines expression,inhibition of RAAS activity,inhibition of AGEs and ALEs.Due to its characteristics,it plays a protective role in the kidney by participating in a multiple of biological metabolic pathways.Many researches have shown that carnosine and its metabolic enzymes play an important therapeutic effect in many kidney diseases,especially in the treatment of Chronic kidney disease(CKD)which is considered as a global public health problem.However,how carnosine and its metabolic enzymes play a role in DN has kindled our interest.This study aims to explore the relationship between carnosine,carnosinase and renal function in DN patients as well as its effect on mesangial cells of DN,so as to lay a scientific foundation for its clinical application in DN.Methods: Part I: 37 patients from the department of nephrology between January 2019 and January 2020 with renal biopsy pathologic diagnosis of diabetic nephropathy were selected as the DN group.14 patients at the same duration of hospital stay who were pathologically diagnosed as minimal change disease(MCD)were chosen as control group 1,and 20 medical examination subjects from medical center were taken as control group 2.Age,gender,height,weight,body mass index(BMI)and blood pressure of the subjects in each group were collected,then the blood glucose,serum creatinine,urea,uric acid,glomerular filtration rate,24-hour urinary albumin,urinary albumin/creatinine,and urinary protein composition of each group were detected.The ultra-performance liquid chromatography(UPLC)was used to test the serum carnosine content of subjects in each group,and the enzyme linked immunosorbent assay(ELISA)was used to determine the content and activity of Carnosinase 1(CN-1)in serum of all groups.Immunohistochemistry(IHC)was conducted to detect CN-1,Fibrosis-related factors of Fibronectin(FN),Collagen I(Col-I)and IV(COL-IV),tumor necrosis factor-?(TNF-?),Interleukin1?(IL-1?),monocyte chemoattractant protein-1(MCP-1),kidney injury molecule 1(KIM1),and oxidative stress related indicators 8-hydroxy-2'-deoxyguanosine(8-OHd G)and 4-hydroxynonenal(4-HNE)of kidney tissue.In DN group,the correlations between serum CN-1 concentration and clinical indexes as well as renal pathological damage indexes were analyzed.Part II: The mouse mesangial cells SV40 were selected for the experiment.Firstly,the optimal intervention concentration of carnosine was screened by MTT test.The samples were divided into 5 groups: normal control group,mannitol control group,normal dosing group,high glucose model group and model dosing group.Cell supernatant,protein and m RNA were extracted from each group.The secretion of extracellular matrix FN,Col-IV and ?-SMA in each group was detected by western blot and immunofluorescence assay.The expressions of inflammatory cytokines TNF-?,IL-1? and MCP-1 in each group were tested by real-time quantitative PCR.The expressions of TGF-?1,p-Smad3,Smad3,NF-?Bp65 and NF-?BP-p65 pathway proteins were detected by western blot.Finally,the expressions of oxidative stress indexes Nox1,Nox2 and Nox4 were detected by western blot,and the levels of intracellular oxidative stress were tested by DCF and DHE fluorescence.We also transfected and silenced CNDP1 gene with si RNA,and detected the content of carnosine in the supernatant of cells in each group by UPLC,and detected the content of carnosine in the supernatant of cells in each group by UPLC.The expressions of FN,Col-IV,?-SMA,TGF-?1,p-Smad3,Smad3,NF-?Bp65 and NF-?BP-p65 were detected by western blot after silenced,and the m RNA levels of inflammatory cytokines TNF-?,IL-1? and MCP-1 were tested by real-time PCR.Part III: C57/BL mice were intraperitoneally injected with STZ(50mg/kg)for 5 consecutive days to establish a diabetes model.After successful modeling,they were treated with carnosine(1g/ L)and fed for 16 weeks.The experiment was divided into the following 4 groups: normal control group,normal administration group,diabetes model group and model administration group.After the feeding cycle,24-hour urinary protein,serum and renal tissue were collected,and blood glucose,serum creatinine,urea nitrogen,24-hour urinary albumin and renal weight/body weight of mice in each group were gotten.HE?PAS?MASSON staining were applied to observe the pathological structure of kidney tissues in each group.The ultrastructure of renal tissue was observed by transmission electron microscopy.The expressions of FN,Col-IV,?-SMA,TNF-?,IL-1?,MCP-1,TGF-?1,p-Smad3,Smad3,NF-?Bp65 and NF-?Bp-p65 in renal tissues of mice in each group were detected by IHC and western blot.The m RNA levels of inflammatory cytokines TNF-?,IL-1? and MCP-1 in renal tissues of mice in each group were detected by real-time PCR.Results: Part I: The serum concentration and activity of CN-1 were the highest and the serum carnosine content was the lowest in DN patients with large proteinuria group.Serum CN-1 concentration was positively correlated with uric acid(UA)and creatinine(SCR)in DN group,and negatively correlated with estimated glomerular filtration rate(e GRF).Moreover,the serum concentration of CN-1 has positive correlation with 24 hours urinary albumin creatinine ratio(24h-U-PRO/CRE),urinary albumin and creatinine ratio(Alb/CRE),urinary transferrin(TRF),retinol binding protein(RBP),N-acetyl amino glycosidase(NAG),immunoglobulin G(Ig G),urine Cystatin-C(Cys-C)and ?2-microglobulin(?2-MG)and ?1-microglobulin(?1-MG).In the renal tissue structure injury,serum CN-1 in DN combined with large amount of proteinuria group was also positively correlated with renal fibrosis,oxidative stress,renal injury indexes and other indicators.Part II: MTT assay indicated that carnosine could inhibit the proliferation of glomerular mesangial cells in the presence of high glucose.Western blot results manifested that the expression of extracellular matrix FN,Col-IV and ?-SMA in mesangial cells was significantly increased under the stimulation of high glucose,and the secretion of extracellular matrix was inhibited after carnosine intervention.Real-time PCR results confirmed that high glucose could induce the activation of inflammatory cytokines TNF-?,IL-1? and MCP-1 in mesangial cells,while carnosine could reduce the m RNA expression of inflammatory cytokines.Western blot showed that the expression of NOX1,NOX2 and NOX4 in mesangial cells increased under the stimulation of high glucose,while the expression of NOX1,NOX2 and NOX4 was inhibited after the intervention of carnosine,and oxidative stress was alleviated.Similarly,the effects of carnosine on reactive oxygen species and superoxide in mesangial cells stimulated by high glucose were verified by DCF fluorescence probe and DHE fluorescence staining.The fluorescence staining results showed that carnosine could reduce the ROS level of cells induced by high glucose and reduce the generation of superoxide.Western blot results suggested that high glucose stimulation promoted the expression of TGF-?1 and activated the phosphorylation level of Smad3.After the intervention of carnosine,the secretion of TGF-?1 was significantly reduced and the phosphorylation of Smad3 was restrained.Finally,the results of Western blot manifested that carnosine could reduce the phosphorylation level of the key protein P65 in NF-?B pathway under high glucose stimulation,thus inhibiting inflammation.UPLC-MS/MS results showed that after CNDP1 silenced,carnosine content in the supernatant of SV40 cells was decreased under the stimulation of high glucose,which also verified that inhibition of CN-1 expression could reduce its degradation of endogenous carnosine.After CNDP1 silenced,Western blot results showed that the secretion of extracellular matrix FN,Col-IV,?-SMA in mesangial cells induced by high glucose was restrained,and the expression of TGF-?1 and phosphorylation of Smad3 were also significantly reduced.In addition,results of Real-time PCR and Western blot indicated that CNDP1 silences inhibited the release of inflammatory cytokines TNF-?,IL-1?,and MCP-1,and decreased the phosphorylation of key protein P65 in the NF-?B pathway in mesangial cells.Part III: Sixteen weeks after STZ-induced DM mice were successfully established,we found typical DM symptoms such as polydipsia,polyphagia,polyuria and weight loss in DM mice.Compared with the control group,the vitality of DM mice decreased obviously,and hair lost luster.The symptoms of polydipsia,polyphagia and polyuria of DM mice treated with carnosine were improved compared with those of the model group,and the weight loss was not noticeable,the vitality was fair,and the hair was glosser than that of the model group.The blood glucose,24 h urinary albumin,renal weight/body weight,serum creatinine and urea nitrogen of mice treated with carnosine were improved compared with those in DM group.The results of HE,PAS and Masson staining showed that after carnosine treatment,the renal mesangial dilatation index and renal tubular injury index were decreased,and the degree of fibrosis was decreased compared with DM mice.The kidney tissue of mice in each group was observed under transmission electron microscopy.Compared with the NC group,the mesangial cells in DM group were proliferated with swelling,mesangial matrix was proliferated,local electron dense deposition was observed,and the basement membrane was obviously thickened.The number of podocytes decreased and there were foot process fusion.The hyperplasia of mesangial cells,thickness of basement membrane and foot process fusion in renal tissue of DM mice treated with carnosine were reduced compared with those in DM group.IHC and Western blot results showed that the expressions of FN,Col-IV,?-SMA in renal tissue of mice in DM group were significantly higher than those in NC group,while the expressions of FN,Col-IV,?-SMA in renal tissue were decreased after carnosine treatment.In addition,TGF-?1 expression and phosphorylation of Smad3,a key protein in the TGF-?1/Smad3 pathway,were significantly increased in the kidney tissues of DM mice,while expressions of TGF-?1 and phosphorylated Smad3 were obviously decreased in DM mice treated with carnosine compared with model group.Real-time PCR results indicated that carnosine could obviously reduce the m RNA expression of TNF-?,MCP-1 and IL-1? induced by high glucose.IHC results also demonstrated that carnosine reduced the release of TNF-?,MCP-1,and IL-1? in renal tissue of DM mice.Western blot confirmed that carnosine can reduce the protein levels of TNF-? and IL-1? in renal tissue induced by DM,as well as reduce the phosphorylation level of P65,a key protein in NF-?B pathway.Finally,Western blot confirmed the oxidative stress response mediated by NOX1,NOX2 and NOX4 members of the NOX family in the kidney tissue of DM mice,and carnosine treatment could inhibit the expression of NOX1,NOX2 and NOX4,thus alleviating the oxidative stress injury of the kidney.Conclusion:(1)In DN,with the progression of the disease,the content of serum carnosine decreases,while the content and activity of CN-1 gradually increase.The concentration of serum CN-1 is not only correlated with renal function indexes and urinary protein components,but also correlated with renal fibrosis,renal tubular injury and renal tissue oxidative stress injury,suggesting that CN-1 is involved in the development of DN.(2)In vitro studies have indicated that exogenous carnosine can protect glomerular mesangial cells stimulated by high glucose through TGF-?1/Smad3 and NF-?B pathways after intervention.Furthermore,the silencing of CNDP1 gene increased the endogenous carnosine content,which also had a certain protective effect on mesangial cells.(3)In vivo studies have manifested that exogenous carnosine therapy can effectively improve renal function and structural damage caused by DM,inhibit the secretion of extracellular matrix,inflammatory response and oxidative stress injury of renal tissue,so it has been shown a good therapeutic potential.