Tohru Umekawa1, Nasser Chegini, Saeed R Khan. 1. Department of Pathology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida 32610-0275, USA.
Abstract
BACKGROUND: Crystals of calcium oxalate monohydrate (COM) and excess oxalate ions (OX) stimulate an array of responses inducing localized injury and inflammation in the kidneys. These inflammatory responses are key regulators of development of nephrolithiasis. We propose that monocyte chemoattractant protein-1 (MCP-1), a chemokine with potent chemotactic activity for monocytes/macrophages, is a mediator of local inflammatory responses to COM and OX-induced injury. To test this hypothesis, the effects of COM and OX on the expression of MCP-1 mRNA and protein by NRK52E rat renal tubular cells were investigated. METHODS: Confluent cultures of NRK52E cells were exposed to COM (33 to 267 microg/cm2) or OX (125 to 1000 micromol/L, estimated free oxalate levels of 65.8 to 540 micromol/L) and catalase (400 or 2000 U/mL), a free radical scavenger that protects the cells against detrimental effects of COM and OX, for 1 to 48 hours under serum free conditions. The conditioned media were collected and total cellular RNA isolated from the cells and subjected to enzyme-linked immunosorbent assay (ELISA) and semiquantitative polymerase chain reaction (PCR) to determine the expression of MCP-1 protein and mRNA, respectively. RESULTS: NRK52E cells express MCP-1 mRNA and protein, and the level of their expression significantly increases following treatments with COM and OX in a time and concentration dependent manner. MCP-1 mRNA expression and protein production increased more significantly after exposure to COM than to OX. These responses were significantly reduced following treatments with catalase (2000 U/mL). CONCLUSIONS: NRK52E cells express MCP-1 mRNA and protein, and their levels are altered following COM and OX exposure. Since catalase treatment reduced MCP-1 expression, free radicals may be involved in the up-regulation of MCP-1 production by the epithelial cells. The results suggest that elevated expression of MCP-1, which is often associated with local inflammatory response, may mediate similar reactions including attraction of macrophages seen around the interstitial crystals during the early stages of nephrolithiasis.
BACKGROUND: Crystals of calcium oxalate monohydrate (COM) and excess oxalate ions (OX) stimulate an array of responses inducing localized injury and inflammation in the kidneys. These inflammatory responses are key regulators of development of nephrolithiasis. We propose that monocyte chemoattractant protein-1 (MCP-1), a chemokine with potent chemotactic activity for monocytes/macrophages, is a mediator of local inflammatory responses to COM and OX-induced injury. To test this hypothesis, the effects of COM and OX on the expression of MCP-1 mRNA and protein by NRK52E rat renal tubular cells were investigated. METHODS: Confluent cultures of NRK52E cells were exposed to COM (33 to 267 microg/cm2) or OX (125 to 1000 micromol/L, estimated free oxalate levels of 65.8 to 540 micromol/L) and catalase (400 or 2000 U/mL), a free radical scavenger that protects the cells against detrimental effects of COM and OX, for 1 to 48 hours under serum free conditions. The conditioned media were collected and total cellular RNA isolated from the cells and subjected to enzyme-linked immunosorbent assay (ELISA) and semiquantitative polymerase chain reaction (PCR) to determine the expression of MCP-1 protein and mRNA, respectively. RESULTS: NRK52E cells express MCP-1 mRNA and protein, and the level of their expression significantly increases following treatments with COM and OX in a time and concentration dependent manner. MCP-1 mRNA expression and protein production increased more significantly after exposure to COM than to OX. These responses were significantly reduced following treatments with catalase (2000 U/mL). CONCLUSIONS: NRK52E cells express MCP-1 mRNA and protein, and their levels are altered following COM and OX exposure. Since catalase treatment reduced MCP-1 expression, free radicals may be involved in the up-regulation of MCP-1 production by the epithelial cells. The results suggest that elevated expression of MCP-1, which is often associated with local inflammatory response, may mediate similar reactions including attraction of macrophages seen around the interstitial crystals during the early stages of nephrolithiasis.
Authors: Robin S Chirackal; Muthuvel Jayachandran; Xiangling Wang; Samuel Edeh; Zejfa Haskic; Majuran Perinpam; Timothy M Halling; Ramila Mehta; Marcelino E Rivera; John C Lieske Journal: Am J Physiol Renal Physiol Date: 2019-08-28