The Role Of CLEC14A In Acute Kidney Injury And Renal Fibrosis

Objective:Acute kidney injury(AKI)and chronic kidney disease(CKD)are common clinical syndromes that cause substantial morbidity and mortality.They are important pathogenic factors of ESRD,and have become important public health problems worldwide.More and more studies have shown that AKI can transition to CKD through vascular injury,inflammatory response,tubulointerstitial fibrosis and other different pathways,and eventually lead to ESRD.Renal fibrosis is the common pathway leading to ESRD.To study the common regulatory mechanism of AKI and CKD and to block the development of fibrosis is the key to find a treatment strategyCLEC14A(c-type lectin family 14 member A)is a new pattern recognition receptor(PRRs),belonging to the 14th family in the c-type lectin superfamily,the thrombolytic regulatory protein family.Other members of the same family include CD93,THBD(thrombomodulin or CD141)and CD248(endosialin or tumor marker-1).They are all type I transmembrane proteins and have similar domain architecture.The extracellular domain consists of a CTLD,many EGF-like domains and a sushi-like domain.These cell surface proteins yet mediate a diverse range of cellular functions,including but not restricted to angiogenesis,inflammation and cell adhesion.CLEC14A has been shown to promote angiogenesis and enhance cell adhesion,but its role in the kidney has not been reported.Therefore,this study will further study the role of CLEC14A in AKI and renal fibrosisMethods and Results:1 The expression of CLEC14A in the kidney with ischemia/reperfusion injury1.1 Detected the expression of CLEC14ACBA/J wild-type(WT)male mice and Clec14a-/-male mice aged 8-12 weeks were randomly divided into two groups,control group and model group.Anesthetize the mice and open the abdominal cavity.Then perform right uninephrectomy,and clip the contralateral renal pedicles using microaneurysm clamps for 35min.The expression of CLEC14A in the kidney was detected by various methods.The results showed that the expression level of CLEC14A in IRI kidney was significantly decreased1.2 Different approaches to mimic hypoxia condition and detect the expression of CLEC14AImmortalized human renal proximal tubule(HK-2)cells were cultured as described.Three approaches to mimic hypoxia condition including chemical anoxia/recovery induced by incubating HK-2 cells with AA/ADG for 60min,oxygen-glucose deprivation and CoCl2 treatment for 24h.WB was used to detect the expression of CLEC14A in HK-2 cells under these conditions.The results showed that the expression level of CLEC14A was significantly decreased.2 The role of CLEC14A in the IRI induced AKI2.1 Assessed the renal function of WT and Clecl4a-/-miceDetected Serum creatinine(Scr)and blood urea nitrogen(BUN)of WT and Clecl4a-/-mice.The results showed that Scr and B UN levels of Clecl4a-/-mice in the model group were significantly higher than WT mice.2.2 Examined the histology damage of the kidney in WT and Clecl4a-/-miceHE staining was used to detect renal morphological damage in mice.Detected kidney cell death by TUNEL.The expression of kidney injury molecule-1(kim-1)was detected by immunofluorescence.Compared with WT mice,the renal morphological damage in Clec14a-/-model group was significantly aggravated,the expression level of Kim-1 and the number of cell death were significantly increased2.3 Effect of CLEC14A deficiency on IRI induced inflammatory response in the kidneyDetect the markers of neutrophil and macrophage,Ly6B and CD68 by IF.Compared with WT mice,neutrophil and macrophage accumulation was further increased in the kidney from Clec14a-/-mice.3 The pathophysiological role of CLEC14A in the AKI induced by cisplatin3.1 Established mouse model and detected the expression of CLEC14ACBA/J wild-type(WT)male mice and Clecl4a-/-male mice aged 8-12 weeks were selected for modeling.Mice were intraperitoneally injected with cisplatin at 30mg/kg to construct the AKI model.Detected the expression level of CLEC14A in the mice kidney by WB and IHC.The results showed that the expression level of CLEC14A in kidney tissues of cisplatin-induced AKI model mice was significantly decreased.3.2 The role of CLEC14A in cisplatin-induced AKIHE staining was used to detect renal morphological damage in mice.The results showed that CLEC14A deficiency exacerbated kidney damage with cisplatin injection.4 The role of CLEC14A in the mice model of unilateral ureteral obstruction(UUO)4.1 Detecting the expression of CLEC14A in the mice kidney with UUOCBA/J wild type(WT)male mice and Clecl4a-/-male mice aged 8-12 weeks were used for modeling.Anesthetized the mice and opened the abdominal cavity,then ligated the unilateral ureter with sutures.CLEC14A expression in renal tissues was detected by Real-time PCR,WB,and IHC.The results showed that the expression level ofCLEC14A was significantly decreased in renal tissues of mice with UUO.4.2 Detecting the renal fibrosis indicators in WT and Clecl4a-/-mice Detected the expression of collagen 1,vimentin,and α-SMA by Western Blot(WB).The mice kidney paraffin sections were stained with Masson trichrome staining and Sirius red staining.The results showed that Clecl4a-/-mice had more severe renal fibrosis than WT mice.4.3 Histology examination of the WT and Clec14a-/-mice kidneyHE staining was used to detect renal morphological damage in mice.Compared with WT mice,the renal morphological damage in the Clec14a-/-model group was significantly aggravated.4.4 Effect of CLEC14A deficiency on UUO induced inflammatory response in the kidneyDetected the markers of neutrophil and macrophage,Ly6B and CD68 by IF.Compared with WT mice,neutrophil and macrophage accumulation was further increased in the kidney from Clecl4a-/-mice.5 The mechanism of CLEC14A in unilateral ureteral obstruction model5.1 Detecting the expression of TFAM in the kidney of WT and Clecl4a-/-miceDetected the expression of TFAM in the kidney of WT and Clec14a-/-mice after UUO by WB and IHC.CLEC14A deficiency downregulated the protein level of TFAM in the mice kidney with UUO.5.2 Effects of CLEC14A deficiency on mitochondrial functionDetected the expression of COX Ⅳ,ATP5A,NDUFB8,SDHB and UQCRC2 in the kidney of WT and Clec14a-/-mice after UUO by WB and IHC.CLEC14A deficiency downregulated the protein level of them in the kidney with UUO5.3 Detecting the expression of STING in WT and Clecl4a-/-miceDetected the expression of STING in the kidney of WT and Clecl4a-/-mice after UUO by WB and IHC.CLEC14A deficiency upregulated the protein level of STING in the kidney with UUOConclusions:1.This study was the first to clarify the pathophysiological role of CLEC14A in kidney disease and found that the expression of CLEC14A significantly reduced in AKI and renal fibrosis.CLEC14A deficiency aggravates renal damage in AKI and renal fibrosis by promoting inflammatory response,renal cell death,and interstitial fibrosis2.We demonstrated that the role of CLEC14A in AKI and renal fibrosis may depend on its positive regulation of TFAM,thus affecting mitochondrial function and STING signaling pathway.This study is conducive to further elucidate the molecular mechanism of AKI and renal fibrosis,and to provide certain theoretical and experimental basis for CLEC14A as a therapeutic target of kidney disease.