Effects of oxalate exposure on Madin-Darby canine kidney cells in culture: renal prothrombin fragment-1 mRNA expression

Abstract

It has been suggested that renal tubular cell damage induced by oxalic acid, one of the components of urinary calculi, may be involved in a variety of ways in the development of urolithiasis. During our study on a calculus related protein, renal prothrombin fragment-1 (RPTF-1), we noted that this is an inflammation related substance that mediates an acute inflammatory reaction, one of the original roles of prothrombin. RPTF-1 is a part of prothrombin that is a coagulation factor known to be expressed in the renal tubule. We examined whether oxalic acid may cause cytotoxic effects on tubular epithelial cells and whether such chemical stimulation may promote the translation of RPTF-1 mRNA into RPTF-1 proteins. We used Madin-Darby canine kidney (MDCK) cells derived from the distal tubule of a dog kidney. In this study, the effects of oxalic acid in culture solution at different concentrations on cytotoxicity were assessed using a MTT assay. The location of active oxygen species was identified using dichlorofluorescein diacetate. After the prothrombin sequence of RPTF-1 was confirmed in MDCK cells, RPTF-1 mRNA expression was determined by RT-PCR. The gene sequence of the same promoter area was ligated, and a luciferase sequence was inserted downstream of the vector. The target sequence was transfected into MDCK cells and the relation between oxalic acid and prothrombin promoter was examined. In addition, the variable expression of RPTF-1 mRNA was quantitatively compared depending on oxalic acid concentrations using real-time PCR. When cytotoxicity was investigated, cells were not damaged but, by contrast, were stimulated and activated under oxalic acid below a certain concentration. The relation between cytotoxicity on the cultured MDCK cell membrane and active oxygen species was confirmed. Luminescence in MDCK cells containing the luciferase gene was detected by the addition of oxalic acid, which activated the prothrombin promoter. A part of the prothrombin gene sequence in the MDCK cells was detected and an increase in the expression of RPTF-1 mRNA in MDCK cells by the addition of oxalic acid was confirmed using real-time PCR. Increased expression of prothrombin by adding oxalic acid has already been demonstrated in previous studies. In this study, however, RPTF-1 mRNA was promoted by oxalic acid and a direct association between oxalic acid and RPTF-1 is indicated. This finding shows that increased oxalic acid in urine induces the expression of RPTF-1 in tubular epithelial cells and thereby causes the generation of active oxygen species.

Inhibitions of urinary oxidative stress and renal calcium level by an extract of Quercus salicina Blume/Quercus stenophylla Makino in a rat calcium oxalate urolithiasis model

“…Oxalate toxicity is mediated in part by activation of lipid signaling pathways, which in turn can disrupt mitochondrial function by increasing the production of reactive oxygen species such as O 2 ‐ , hydrogen peroxide, singlet oxygen and hydroxyl radicals. Reactive oxygen species mediate cell injury by modifying lipids, proteins, and DNA, and this can result in the promotion of calcium oxalate crystal formation 3,11,15 . Oxidative damage is therefore considered to play an important part in stone formation.…”

Kidney Stone Disease: An Update on Current Concepts

“…Following renal tubular cell injury, cellular degradation produces numerous membrane vesicles which are nucleators of calcium crystals as supported by in vitro and in vivo studies [ 41 ]. Injured cells release substances like renal prothrombin fragment-1 or other anionic proteins which induce COM crystal agglomeration [ 68 ]. Reactive oxygen species is thought to be one of the factors involved in renal cell injury [ 69 ].…”

Atorvastatin inhibits renal inflammatory response induced by calcium oxalate crystals via inhibiting the activation of TLR4/NF‐κB and NLRP3 inflammasome

“…Recent evidence revealed that the injury of renal tubular epithelial (HK‐2) cells is closely related to the early process in renal calculi formation. Previous studies showed that high concentrations of oxalic acid and calcium oxalate crystals led to the injury, apoptosis, and necrosis of HK‐2 cells . Oxidative stress and inflammation play pivotal roles in this process and has been considered as an important link to the formation of intrarenal crystals.…”