The role of nitric oxide in reflux nephropathy

Reflux nephropathy (RN) is recognized as a major cause of end-stage renal failure in children and young adults. Inhibition of nitric oxide (NO) exacerbates and enhanced production ameliorates tubulointerstitial fibrosis (TIF) in experimental obstructive uropathy. NO is synthesised by NO synthase (NOS), three distinct isoforms of which have been identified: inducible (iNOS), endothelial (eNOS), and neuronal (nNOS). It has been reported that iNOS induces immunologic injury to glomerular cells and enhances accumulation of extracellular matrix in the glomerulus and tubulointerstitial space. Furthermore, it has been suggested that nNOS and eNOS have beneficial effects in ameliorating TIF. We investigated the expression of different isoforms of NOS in severe refluxing kidneys in order to further understand the pathogenesis of RN in kidney specimens from nine children with severe RN obtained at nephrectomy. Control material included normal kidney specimens from three adult patients undergoing partial nephrectomy for small kidney tumours. Histochemistry for NO was performed using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase. Single-label immunofluorescence histochemistry was carried out using polyclonal antibodies to nNOS, iNOS, eNOS, and transforming growth factor (TGF)-ß1 employing laser-scanning confocal microscopy. The TUNEL method was used to assess tubular apoptosis. Strong NADPH staining was observed in the proximal tubules of RN kidneys compared to controls, where there was weak staining. Control kidneys demonstrated weak immunoreactivity for iNOS in the proximal tubules and a lack of immunoreactivity for nNOS and eNOS. RN kidneys demonstrated strong immunoreactivity for nNOS in the tubulointerstitial space, for eNOS in the glomerulus, and for iNOS in the glomerulus and proximal tubules. Strong immunoreactivity for TGF ß1 was seen in the glomerulus and proximal tubules identical to iNOS. Increased immunoreactivity for iNOS and TGF-ß1 strongly correlated with the severity of apoptosis in RN. Our data demonstrate that NO derived from nNOS, iNOS, and eNOS is strongly expressed in RN. The selective shunting of NO via iNOS may induce renal fibrosis in RN. The upregulation of nNOS and eNOS in RN appears to be a compensatory mechanism of ameliorating TIF.