Immunological detection of nitrosative stress-mediated modified Tamm–Horsfall glycoprotein (THP) in calcium oxalate stone formers

Abstract

The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in hyperoxaluric condition has been proved experimentally. This may result in the formation of the cytotoxic metabolite peroxynitrite, which is capable of causing lipid peroxidation and protein modification. The presence of nitrotyrosine in proteins has been associated with several pathological conditions. The present study investigated the presence of nitrotyrosine in the stone formers Tamm-Horsfall glycoprotein (THP). In vitro nitration of control THP was carried out using peroxynitrite. New Zealand white rabbits were immunized with peroxynitrated THP at 15-day intervals. Antisera collected following the third immunization were assayed for antibody titres using solid-phase ELISA. Antibodies were purified by affinity chromatography. The carbonyl content of control, stone formers and nitrated THP were determined. Western blotting was carried with control, stone formers and nitrated THPs. Immunodiffusion studies demonstrated cross-reaction with nitrated bovine serum albumin. Significant amounts (p < 0.001) of carbonyl content were present in both stone formers and nitrated THPs. Western blot analysis confirmed the presence of nitrated amino acid 3-nitrotyrosine in stone formers, which could bring about structural and functional modifications of THP in hyperoxaluric patients. A cross-reaction with nitrated bovine serum albumin confirms that the raised antibody has certain paratopes similar to the epitope of nitrated protein molecules. Detection of 3-nitrotyrosine in stone formers THP indicates that it is one of the key factors influencing the conversion of THP to a structurally and immunologically altered form during calcium oxalate stone formation.

Radical Scavenging Activity of Neolamarckia Cadamba (Roxb) Bosser in Nephrolithiasis Related Oxidative Stress

“…Free radical scavengers and metabolic inhibition of LPO are necessary for protecting the renal cells against oxalate induced renal cell injury. The MFNC administration in experimental rats have decreased the concentration of intracellular calcium which might be due to the activity of the extract in increasing the cGMP activation and was reported to control the increase in intracellular calcium concentration 34 . The polyphenolic contents of fruits and vegetables are reported to have antioxidant activities and the free radical scavenging activity of the plant extract is attributed to the flavonoid phytochemicals in it 35 .…”

Toxic effects of gentamicin in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg9

“…29 Malondialdehyde (MDA), a major product of lipid peroxidation, has been considered as a reliable marker of oxidative stress. 30 In our present study the method used for the estimation of lipid peroxidation is based on the reaction of malondialdehyde (MDA) with 1-methyl 2-phenylindole at 45 °C. Two molecules of 1-methyl 2-phenylindole react with one molecule of MDA to form a stable chromophore having maximal absorbance at 586 nm.…”

Antibody microarrays for high-throughput, multianalyte analysis

“…There is considerable evidence that PTMs are modified in diseases such as breast cancer and can be used to distinguish cancer stages or for monitoring therapeutic response [15]. Conventional ELISA analysis employing 96-well plates has been widely used for studies on PTMs [62][63][64][65][66].…”