Development Of Novel Chronic Kidney Disease Models Based On Ochratoxin A And Study On The Mechanism And Application

Chronic kidney disease(CKD)is a common and frequently occurring disease in humans,dogs and cats,with high mortality and high treatment cost.It has attracted more and more attention in the field of human medicine and veterinary medicine.A good animal model is the prerequisite for the study of CKD.Chronic cyclosporine A(CsA)nephropathy model and adriamycin(ADR)nephropathy model are the two common models of CKD model.But studies found that high dosages and long-time using CsA or ADR could bring other side effects to the mice,such as: stability,mortality,cardiotoxicity,hepatotoxicity,nerve toxicity and the injection site severe ulcer,etc.Ochratoxin A(OTA)is a mycotoxin produced by Penicillium and Aspergillus,and its target organ is kidney.Hence,we hypothesized that the combination of low dose OTA and the lower-dose CsA or ADR could develop two novel models of chronic kidney disease with less side effects caused by high-dose CsA or ADR.In addition,based on the roles of Icariin(ICA)and organic selenium(SeMet)in regulating autophagy,antioxidant and anti-fibrosis,we applied the novel model to further propose that ICA and SeMet can alleviate CsA and OTA induced renal injury,providing a new nutrient-assisted therapy strategy for CKD.In order to verify the hypothesis,this study performed experiments in vitro and in vivo,using mice,human renal tubular epithelial cells(HK-2),and glomerular mesangial cells(HMC)to explore the development of chronic kidney disease models with less side effects induced by OTA and CsA or OTA and ADR,respectively,and the possible potential mechanism.Finally,the protective effect of ICA and SeMet on renal injury in our novel chronic tubular nephropathy model and its potential mechanism were explored in vivo and in vitro.Experiment Ⅰ.Study on the development and mechanism of a novel mice model of chronic tubular nephropathy based on OTA and CsATo explore whether OTA and CsA can be used to develop a mice model of chronic tubular nephropathy with less side effects,male C57 BL/6 mice aged 6-8weeks were selected for the experiment.Firstly,the effects of different dosages of OTA(0.25 mg/kg and 0.5 mg/kg,intraperitoneal injection every other day for 28 d)on kidney injury in mice were investigated.The results showed that 0.25 mg/kg OTA had no significant damage to kidney in mice,so 0.25 mg/kg OTA was selected in the subsequent experiments.In the following study,mice were treated with 0.25 mg/kg OTA combined with 20 mg/kg CsA,compared with those treated with 30 mg/kg CsA in the traditional model group.OTA and CsA were given by intraperitoneal injection,and the test period was 28 d.The effects of 0.25 mg/kg OTA combined with 20mg/kg CsA on renal injury in mice were assessed by serum biochemistry,urine gravity,urine protein,tissue staining,q RT-PCR,western blotting and other methods.Results showed that 0.25 mg/kg OTA combined with 20 mg/kg CsA successfully developed a model of chronic tubular nephropathy,while reducing the side effects associated with high dose and prolonged use of CsA,similar to the traditional model group.In addition,activation of TLR4 and autophagy-related proteins was found in the renal tissue of a new model of chronic tubular renal disease.To further demonstrate the underlying mechanism,we applied TLR4 knockout mice,and the results showed that OTA and CSA-induced renal injury and elevated expression of autophagy related proteins were significantly alleviated in the renal tissue of TLR4 knockout mice.This study showed that low dosage of OTA combined with lowerdosage of CsA could develop a chronic tubular nephropathy model through TLR4-mediated autophagy,and the side effects of other organs in this model were significantly less than those in the traditional CsA nephropathy model.This provides a new direction for exploring the pathogenesis and screening of therapeutic drugs for chronic tubular nephropathy.Experiment Ⅱ.Study on the combined toxicity of OTA and CsA to renal tubular epithelial cells and its mechanismThe target organ of OTA is the kidney,and both OTA and CsA are nephrotoxic.In order to explore whether OTA with non-toxic concentration and CsA with lower concentration can jointly induce renal tubular epithelial cells(HK-2)cytotoxicity,this study firstly treated cells with different concentrations of OTA and CsA for 48 h,and detected cell viability by MTT and LDH methods.OTA(0.5 μg/m L)and CsA(2,4,6μg/m L)were selected for the following experiment.Secondly,western blotting,q RTPCR and immunofluorescence methods were used to detect the effects of OTA and lower-concentration of CsA in combination on intracellular apoptosis,oxidative stress and expression of renal fibrosis related proteins.The results showed that the combination of 0.5 μg/m L OTA and 6 μg/m L CsA induced apoptosis,oxidative stress,and increased the expression level of renal fibrosis related proteins in HK-2 cells.Meanwhile,we found that OTA and CsA jointly activated the expression of TLR4 and autophagy related proteins in HK-2 cells,and reduced the expression of autophagy inhibitor-related pathway proteins.To further explore the underlying mechanism,autophagy inhibitor 3-MA and RNA interference techniques were used.The results showed that the expression of genes and proteins related to renal fibrosis induced by OTA and CsA were reversed after the addition of 3-MA.Subsequently,TLR4 expression was interfered by si RNA,and cytotoxicity and autophagy activation induced by 0.5 μg/m L OTA and 6 μg/m L CsA were significantly inhibited.The results of these experiments proved that OTA at non-toxic concentration combined with CsA at lower concentration could induce cytotoxicity in HK-2 cells.Experiment Ⅲ.Study on the development and mechanism of a novel mice model of chronic glomerular nephropathy based on OTA and ADRADR glomerular nephropathy model is one of the common models of chronic kidney disease,but the application of this model is limited due to high dosage ADRinduced side effects.In our previous study,we found that the target organ of OTA is kidney,which can induce nephrotoxicity.Therefore,in order to explore the improvement effect of low dosage of OTA on chronic ADR nephropathy model,BALb/c mice were firstly divided into five groups: control group,low dosage of OTA group,lower-dosage of ADR group,low dosage of OTA and lower-dosage of ADR jointed group,and the traditional ADR model group.OTA was intraperitoneal injection,ADR was single injection of tail vein,and the test period was 14 d.After the experiment,organ index calculation,tissue staining,electron microscopy,western blotting and q RT-PCR were performed.Consistent with the traditional ADR model group,the results showed that low dose OTA and lower-dose ADR in combination could significantly induce glomerular injury to develop a new model of chronic glomerular nephropathy.In addition,compared with the traditional model group,the mice in the novel glomerular nephropathy model showed significant improvement in other side effects,including the degree of tail ulceration,myocardial hypertrophy,and fibrosis.OTA and ADR joint treatment also inhibited the activation of TFEB,leading to autophagy imbalance.To further explore the underlying mechanism,we validated the results with TFEB activator.It showed that OTA and ADR-induced glomerular injury was caused by the TFEB inhibition,and TFEB activator further confirmed that autophagy disorders were mediated by TFEB.In conclusion,OTA and ADR in combination developed a novel model of chronic glomerular nephropathy with fewer side effects via TFEB mediated autophagy imbalance.This provided a new orientation for studying the pathogenesis and treatment of chronic glomerular nephropathy.Experiment Ⅳ.Study on the combined toxicity of OTA and ADR to renal mesangial cells and its mechanismADR is an antibiotic,which is generally used in the study of glomerular toxicity due to its nephrotoxicity.In order to prove the effect of non-toxic OTA combined with ADR at a lower concentration on glomerular mesangial cells(HMC),non-toxic OTA(2.5 μM)and ADR(0.05,0.1,0.25 μM)were selected by MTT and LDH methods for cell viability and apoptosis.The results showed that the combination of OTA with non-toxic concentration could significantly reduce the concentration of ADR inducing cytotoxicity and apoptosis from 0.5 μM to 0.1 μM in vitro.In addition,the combination of 2.5 μM OTA and 0.1 μM ADR significantly induced oxidative stress and DNA damage in HMC cells,and increased the expression of renal fibrosis related proteins.In addition,the combination of OTA and ADR also promoted the expression of TGF/Smad pathway-related proteins,induced autophagy disorders and inhibited TFEB activation.In order to further explore the mechanism,we conducted experiments using the inhibitors of Smad2,Smad3,autophagy and the activator of TFEB.It showed that OTA and ADR induced cytotoxicity through TGF/Smad2/Smad3 pathway in HMC cells.Moreover,TFEB-mediated dysregulation of autophagy can be regulated by Smad3 rather than Smad2 in vitro.Subsequently,Smad3 inhibitor treatment induced autophagy flux blockade again after interfering with TFEB expression.This study demonstrated that non-toxic concentration of OTA combined with lower concentration of ADR could induce cytotoxicity through Smad3/ TFEB-mediated autophagy in HMC cells.Experiment Ⅴ.Study on the combined protective effect of ICA and SeMet on renal injury in the novel mice model of chronic kidney disease and its mechanismBoth ICA and SeMet have antioxidant and autophagy regulation effects,but their combined protective effect on chronic kidney disease is rarely studied.In this study,we applied the chronic kidney disease model developed by OTA and CsA in the previous study to explore the protective effects of ICA and SeMet on chronic kidney disease.Firstly,mice were divided into control group,model group,ICA group,SeMet group and ICA+ SeMet group in vivo.HK-2 cell line was used in vitro.The results of renal index calculation,tissue staining,serum biochemistry,urine gravity,urine protein,western blotting and q RT-PCR showed that ICA and SeMet could improve cell apoptosis,histopathological injury and collagen fiber deposition by reducing serum urea nitrogen,creatinine,urine protein and urine gravity and decreased expression levels of renal fibrosis related proteins to ameliorate chronic kidney disease.Besides,ICA and SeMet in combination had better renal protection on most indicators.In addition,ICA and SeMet inhibited the activation of TLR4/NFκB pathway in the kidney of model mice.In order to further explore the underlying mechanism,we overexpressed TLR4 and found that the overexpression of TLR4 could reduce the combined protective effects of ICA and SeMet on HK-2 cells.Therefore,ICA in combination with SeMet can effectively improve OTA and CsA induced chronic kidney disease by blocking the TLR4/NFκB pathway,providing new insights into the exploration of adjunctive therapies for chronic kidney disease.