케모카인 수용체 5 조절을 통한 급성 신손상 모델 연구

학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의과학과, 2018. 2. 김연수.The CC chemokine receptor 5 (CCR5) is an important regulator of macrophage trafficking in the kidney in response to inflammation and immunity. Thus, we investigated the role of CCR5 in the pathogenesis of experimental ischemic reperfusion injury (IRI). Bilateral renal artery pedicle clamping for 30 minutes followed by reperfusion was performed on C57BL/6 mice (B6) and B6.CCR5-deficient (B6.CCR5-/-) mice. We performed an adoptive transfer of lipopolysaccharide (LPS)-treated RAW cells following macrophage depletion in mice. B6.CCR5-/- mice showed less aggravated IRI regarding apoptosis of tubular epithelial cells and creatinine (Cr) compared to that in B6 wild-type mice. CXCR3 expression on CD11b+ cells and inducible nitric oxide synthase (iNOS) were more attenuated in B6.CCR5-/- mice than in B6 wild-type mice. Conversely, the B6.CCR5-/- mice showed increased numbers of arginase-1 and CD206-expressing macrophages. Macrophage-depleted wild-type mice showed more severe injury than B6.CCR5-/- mice after the transfer of M1 macrophages. The adoptive transfer of LPS-treated RAW cells, which constitutively express iNOS for M1 tendency, reversed the functional protection against IRI in wild-type mice, but not in B6.CCR5-/- mice. When CCR5 was knocked out in macrophages, bone-marrow-derived macrophages showed M2 macrophage activation, and the migration of bone-marrow-derived macrophages from wild-type mice toward the primary tubular epithelial cells with recombinant CCR5 increased. Moreover, the CCR5 blockade inhibited the migration of macrophages. Expression of phospho-CCR5 in the renal tissue of patients with transplant and glomerulonephritis was increased, showing a positive correlation with acute tubular necrosis severity. These findings show that CCR5 deficiency favors M2 macrophage activation and may be a strategy for treating acute kidney injury by blocking CCR5.Introduction 1 Results 3 Discussion 10 Materials and Methods 40 Reference 46 Abstract (Korean) 53Docto

Multi-sample mass spectrometry-based approach for discovering injury markers in chronic kidney disease

Urinary proteomics studies have primarily focused on identifying markers of chronic kidney disease (CKD) progression. Here, we aimed to determine urinary markers of CKD renal parenchymal injury through proteomics analysis in animal kidney tissues and cells and in the urine of patients with CKD. Label-free quantitative proteomics analysis based on liquid chromatography-tandem mass spectrometry was performed on urine samples obtained from 6 normal controls and 9, 11, and 10 patients with CKD stages 1, 3, and 5, respectively, and on kidney tissue samples from a rat CKD model by 5/6 nephrectomy. Tandem mass tag-based quantitative proteomics analysis was performed for primary cultured glomerular endothelial cells (GECs) and proximal tubular epithelial cells (PTECs) before and after inducing 24-h hypoxia injury. Upon hierarchical clustering, out of 858 differentially expressed proteins (DEPs) in the urine of CKD patients, the levels of 416 decreased and 403 increased sequentially according to the disease stage, respectively. Among 2965 DEPs across 5/6 nephrectomized and sham-operated rat kidney tissues, 86 DEPs showed same expression patterns in the urine and kidney tissue. After cross-validation with two external animal proteome datasets, 38 DEPs were organized; only 10 DEPs, including serotransferrin, gelsolin, poly ADP-ribose polymerase 1, neuroblast differentiation-associated protein AHNAK, microtubule-associated protein 4, galectin-1, protein S, thymosin beta-4, myristoylated alanine-rich C-kinase substrate, and vimentin were finalized by screening human GECs and PTECs data. Among these ten potential candidates for universal CKD marker, validation analyses for protein S and galectin-1 were conducted. Galectin-1 was observed to have a significant inverse correlation with renal function as well as higher expression in glomerulus with chronic injury than protein S. This constitutes the first multisample proteomics study for identifying key renal-expressed proteins associated with CK