Calcium Dobesilate For Diabetic Nephropathy:a Basic And Clincal Research

Objective Diabetic kidney disease(DKD)is a leading cause of end-stage renal disease.However,the pathogenesis of DKD remains unclear,and no effective treatments for this disease are available.Endothelial cells(ECs)play critical roles in many physiological functions.The underlying EC dysfunction is often underestimated in cases of DKD.Moreover,the microinflammatory state plays an important role in DKD development and progression.Calcium dobesilate(Ca D)is considered an angioprotective drug that can reduce blood viscosity,platelet activity and capillary permeability as well as alleviate microcirculatory and haemorheologic abnormalities.Ca D is widely used to treat diabetic retinopathy,chronic venous insufficiency,and various microangiopathies.Moreover,recent studies have demonstrated that Ca D exerts protective effects against diabetic nephropathy and gentamicin-induced acute kidney injury.Despite its broad use,very little attention has been devoted to the molecular and cellular mechanisms underlying the vasculoprotective effects of Ca D.Thus,the aim of the present study was to elucidate the molecular and cellular mechanisms underlying the protective effects of Ca D against diabetes-induced endothelial dysfunction and inflammation.Methods1)Human umbilical vein endothelial cells(HUVEC)were cultured with different D-glucose concentrations to determine the effect of HG on HUVEC gene expression.Cells were incubated with HG or mannitol,which served as an osmotic control,for 3 days.HUVECs were also incubated with Ca D(Ca D25 u M?Ca D 50 u M?Ca D 100 u M)for 3 days to determine the effects of Ca D on HUVEC viability.Db/db mice were divided into two groups: control group(saline gavage)and treatment group(Ca D 100mg/kg gavage);db/m mice were randomly divided into two groups,too.Westernblot and real-time PCR were used to detect VEGF,VEGFR,ET-1,p-e NOS/e NOS,PTX3,MCP-1,ICAM expression in mice and HUVECs.HUVEC proliferation was detected by CCK8.A transwell migration apparatus was used to measure HUVEC migration.Permeability assay was determined by FITC-BSA.Apoptosis was measured by flow cytometry.ELISA method for detection of serum cystatin C.HE and PAS staining were used to observe the damage of renal tissue in mice.TUNEL staining was used to observe the apoptosis of renal tissue.The expression of p-e NOS was observed by immunofluorescence staining.The expression of CD34,VEGF and PTX3 were observed by immunohistochemical staining.2)TPCA blocked NF-? B pathway in HUVECs.The expression of PTX3 and p-e NOS/e NOS was detected by westernblot and real-time PCR.In si RNA-PTX3 intervention experiment,westernblot was used to detect PTX3 and p-e NOS/e NOS.Cell apoptosis was measured by flow cytometry.In adenovirus encapsulated si RNA-PTX3 intervention experiment of db/db mice,mice were divided into four groups: db/m,db/m+si RNA,db/db,db/db+si RNA.ELISA method was used for detection of cystatin C.HE and PAS staining were used to observe the damage of renal tissue in mice.TUNEL staining was used to observe the apoptosis of renal tissue.The expression of p-e NOS was observed by immunofluorescence staining.The expression of p-IKBa,PTX3,P65 were observed by immunohistochemical staining.3)Type 2 diabetes patient were randomly divided into the treatment group(take calcium dobesilate,500 mg,3 times a day)and the observation group.The DKD patients were treated for 3 months.Diabetic patients without proteinuria and healthy people were enrolled,too.Clinical data and related biochemical parameters were collected.Endothelial function markers(VEGF,ET-1,e NOS,NO)and inflammatory markers(MCP-1,ICAM,PTX3)were detected.Results1)In vitro,high glucose induced VEGF,VEGFR,ET-1,ICAM,MCP-1,PTX3 upregulation,p-e NOS/e NOS down regulation.Calcium dobesilate alleviated glucose induced endothelial cell proliferation and increased migration capacity,and also alleviated glucose induced permeability and apoptosis.In vivo,calcium dobesilate alleviated glomerular vascular proliferation,renal endothelial dysfunction and inflammation,renal cell apoptosis of db/db mice.Thereby calcium dobesilate reduced urinary albumin and protected renal function of db/db mice.2)In vitro,calcium dobesilate down-regulated the expression of PTX3 and p-IKBa/IKBa,and up-regulated the expression of p-e NOS/e NOS.When TPCA was used,high glucose induced high PTX3 expression decreased,meanwhile the expression of p-e NOS/e NOS increased.After silencing PTX3 gene,the expression of p-e NOS/e NOS also increased.Silencing PTX3 gene can reduce the apoptosis of endothelial cells induced by high glucose.In vivo,calcium dobesilate reduced the expression of PTX3,p-IKBa/IKBa and P65 in the kidney of db/db mice,and increased the expression of p-e NOS/e NOS.After PTX3 gene was silenced,the urine protein and renal function of db/db mice were ameliorated,the glomerular extracellular matrix was decreased and the expression of p-e NOS/e NOS was increased.Renal cell apoptosis of db/db mice was decreased.3)PTX3,NO and Hb Alc were the influence factors of DKD.DKD patients were treated with Ca D.After the treatment,24 hours urinary albumin,24 hours urinary protein significantly decreased,cystatin C based GFR did not change.Patients' inflammatory and endothelial dysfunction markers significantly ameliorated than before.Conclusion It is suggested that Ca D may inhibit the expression of PTX3 by adjusting the IKK/IKB/NF?B pathway,thereby improving the endothelial dysfunction.PTX3 may be a potential therapeutic target for DKD.