A simple method to identify NET-positive cells in isolated glomeruli

Background. Reactive oxygen radicals are probably involved in the pathogenesis of human and experimental models of renal disease, yet current methods are inadequate to quantify and identify the cells producing reactive oxygen radicals. Methods and results. We used the nitroblue tetrazolium reaction to determine superoxide anion production in glomerular cells in phorbol myristate-stimulated glomerular suspensions and in isolated glomeruli from rats with nephrotoxic nephritis, ureteral obstruction, and puromycin aminonucleoside nephrosis. We were also able to identify these nitroblue tetrazolium + cells using specific appropiate antibodies. When the technique was tested in conditions known to increase reactive oxygen radicals, as phorbol myristate-stimulated glomeruli and glomeruli from animals with nephrotoxic nephritis and ureteral obstruction, increased number of nitroblue tetrazolium + cells were found. These cells were identified as glomerular intrinsic cells (Thy-1 +) or infiltrating leukocytes (leukocyte common antigen+ or antineutrophil +). Conclusion. This method may be useful to determine cells participating in glomerular damage induced by reactive oxygen radicals.