| Objective: Acute kidney injury?AKI?is a common clinical disease,especially the incidence,with increasing trend,and the mortality of drug-induced acute renal injury are higher.AKI is also one of the indispensable causes of chronic kidney disease?CKD?.Statistics show that AKI's incidence in foreign general hospitalized patients and critically ill patients were as high as 5% and 20% to 30% respectively;the incidence in Chinese admitted patients was 11.6%,however,40% of these patients with acute renal injury related to drugs administration,which led to extended hospital stay and increased treatment costs.At present,there is no specific method for the diagnosis and treatment of acute renal injury.The main pathogenesis of the drugs induced AKI is considered as necrotic inflammatory response caused by renal tubular epithelial cell necrosis.Mild renal tubular injury can be adaptively repaired through the tubular cell regeneration;sustained injury or severe injury will cause kidney fibrosis via maladaptive repair and eventually progress to chronic kidney disease.A large number of studies have demonstrated that MyD88 molecule is the most crucial adaptor involved in TLRs / NF-?B pathway during the whole process of necro-inflammation,fibrosis and regeneration repair,and also plays an indispensable role in cell necrosis and inflammatory responses,cell regeneration,dedifferentiation and phenotypic transformation.Interetingly,we found all studies on MyD88 proteins or genes are based on the technology of protein antagonism or gene knockout.However,due to the specificity of renal function and structure,there are various types of renal cells.These research methods are difficult to accurately describe the role of different types of cells in the pathogenesis of acute kidney injury.In the meantime,considering the kidney TECs are one of the most important tissue cells in ATN and AKI,and more importantly ATN is the early major pathological changes in drug-induced AKI.Therefore,The purpose of this study was to further clarify the effect of MyD88 gene on renal function and morphology of drug-induced AKI,as well as the impact on damage,repair related markers and its possible mechanism.What's more,aiming to generate a new thinking for AKI research,and a theoretical update for MyD88 which is expected to be a new target in AKI diagnosis and treatment.Methods: In term of the principle of Cre / Lox P gene knockout technique,we crossed a mouse containing Kidney-specific Cre recombinase promoter with another mouse containing two lox P sites flanking Exon 3 of MyD88 gene.Maintaining male MyD88f/fKsp-Cre+ pups by genotyping for further experiments.Meanwhile,confirmation of knockout MyD88 gene in renal tubular epithelial cells.Intraperitoneal injection of folic acid?250 ?g/g body weight?at 68 weeks of age to establish a stable acute kidney injury mouse model.The experimental mice were divided into WT group and MyD88f/fKsp-Cre+ group.Collection of serum,urine samples and kidney samples of baseline,the 2nd,7th,14 th and 28 th day after injection of folic acid.Measurement of renal function and pathology changes,and detection of the biomarkers associated to kidney damage and repairing.At the same time,cell senescence was measured on the second day after FA injection by cell senescence of ?-galactosidase and flow cytometry?Results: 1.The relative expression of MyD88 m RNA in MyD88f/fKsp-Cre+ mouse tubular epithelial cells was significantly lower than that in macrophages and WT mice(0.041 ± 0.027,0.156 ± 0.003 and 0.153 ± 0.043,respectively.The difference was statistically significant.Similarly,the relative expression of MyD88 protein in MyD88f/fKsp-Cre+ mouse tubular epithelial cells was significantly lower than that in macrophages and WT mice?0.145 ± 0.0498,0.737 ± 0.190 and 0.685 ± 0.083,respectively?,with statistical significance.The mortality of FA-AKI in WT group was higher than that of MyD88f/fKsp-Cre+ group,and the survival curves of the two groups were analyzed by Log rank test,P = 0.091,no statistical significance.2.The maximum level of serum creatinine in WT group and MyD88f/f Ksp-Cre+ group was 1.373 ± 0.941 mg/d L and 0.474 ± 0.257 mg/d L,respectively,on the second day after FA injection,the difference was statistically significant.Urinary microalbumin and urinary creatinine concentration?ACR?also reached a peak at day 2 after FA injection,distribution was 137.699 ± 97.542 mg/g and 180.800 ± 132.665 mg/g,the difference was not statistically significant.3.Both H&E staining and PAS staining showed that the renal tubular epithelial cells injury and the brush loss,as well as inflammatory cell infiltration in WT-d28 group were more severe than those in MyD88f/fKsp-Cre+ group.Masson's Trichrome staining showed that the percentage of fibrosis area?%?in WT group and MyD88f/fKsp-Cre+ group was 10.380 ±7.195 and 4.169 ± 4.489,the difference was statistically significant.The ratio of total collagen and total protein in the kidney was 0.062 ± 0.031 and 0.041 ± 0.012,respectively,with statistically significance.4.The FA-AKI-related biomarkers of WT-d28 and MyD88f/f-d28 were detected by immunofluorescence.The quantification method was as follows: the percentage of the fluorescence area in the total area?%?or the percentage of the positive cells in total cell number?%?.The results showed that the markers of renal tubular injury NGAL were 1.227 ± 1.192 and 0.461 ± 0.419,KIM-1 were 1.096 ± 0.837 and 0.251 ± 0.302,respectively.The DNA damage markers ?-H2 AX were 0.444 ± 0.694 and 0.692 ± 0.495;The cell proliferation markers Ki67 were 1.937 ± 1.513 and 2.464 ± 1.349;Fibrosis formation related markers FSP1 were 3.276 ± 2.320 and 2.290 ± 1.573,CD68 were 3.926 ± 2.054 and 2.284 ± 1.041,?-SMA were 0.343 ± 0.749 and 0.601 ± 0.457,F4/80 were 1.020 ± 0.902 and 0.568 ± 0.644 and CD31 were 0.649 ± 0.447 and 1.478 ± 1.220,respectively.The above markers were statistically significant in the two groups.The apoptosis related TUNEL were 0.015 ± 0.012 and 0.014 ± 0.017;Caspase3 were 0.003 ± 0.004 and 0.004 ± 0.003,respectively,not statistically significant.The expression of H3K9me3 were 45.361 ± 35.506 and 55.844 ± 29.633,respectively,no statistical significance.5.The relative expression of KIM-1,NGAL,IL-1? and IL-8 m RNA in WT-d28 group and MyD88f/f-d28 group is statistically significant,and MMP-3,IL-1? and IL-6 differences is not statistically significant.6.The percentages of senescent cells?%?in WT-d2 group and MyD88f/f-d2 group were 7.853 ± 2.382 and 3.02 ± 0.559 respectively,the difference was statistically significant.Conclusions:1.MyD88f/fKsp-Cre mice can be obtained by crossing MyD88f/f mice with Ksp-Cre mice to achieve the purpose of knockout of MyD88 gene in renal tubular epithelial cells.FA-induced AKI model conforms to the clinicopathological process.Mouse mortality and the survival curve of WT group and MyD88f/fKsp-Cre group meet reserach requirement.2.Specifically knockout of renal tubular MyD88 gene can effectively improve renal function and protect the integrity of renal tissue structure,and reduce tissue damage and the degree of fibrosis,is conducive to the prognosis of AKI.3.Specifically knockout MyD88 gene of renal tubule can directly reduce renal tubular injury and promote cell proliferation.At the same time,by reducing the expression of inflammatory cytokines,phenotype transformation of renal parenchymal cells and the percentage of secescence cells,to achieve the role of reducing renal fibrosis. |
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