Effect of a low-protein diet on expression of non-muscle type myosin heavy-chain isoforms in glomeruli of rats with puromycin aminonucleoside nephrosis

Abstract

We reported that an embryonic type of non-muscle-type myosin heavy-chain isoform (SMemb) may be a molecular marker for phenotypic alteration in initial glomerular injury and that methyl-prednisolone has no effect on SMemb expression in glomeruli of rats with puromycin aminonucleoside (PAN) nephrosis. The present study was designed to assess whether SMemb mRNA and protein expression in glomeruli are affected by a low-protein diet in rats with PAN-induced nephrosis and in control rats. Rats were divided into four groups: group 1, PAN-injected rats fed a standard diet containing 22% protein; group 2, PAN-injected rats fed a low-protein diet containing 6% protein, starting on the day of PAN injection; group 3, control rats fed a standard diet; group 4, control rats fed a low-protein diet for the same period. We prepared glomerular RNA and performed Northern blot analysis and immunohistochemistry in all groups. Glomerular SMemb mRNA increased on days 2 and 4 (prior to and soon after the onset of proteinuria), but declined on day 8 (the peak of proteinuria). Myosin heavy-chain protein expression was evaluated immunohistochemically by use of three antibodies against SM1, SM2, and SMemb. SM1 and SM2 were absent from the glomeruli of rats with PAN nephrosis until day 20. The SMemb isoform was barely detectable in normal glomeruli, but substantial amounts of SMemb were demonstrated in the glomeruli of rats with PAN nephrosis. In the latter condition, the number of SMemb-positive glomerular epithelial cells increased on days 3 and 4, then decreased in subsequent days. Moreover, some mesangial cells became SMemb-positive transiently, returning to barely detectable levels on day 20. In addition, alpha-smooth-muscle actin, type I and III collagens were absent from the glomeruli of rats with PAN nephrosis until day 20. Urinary protein excretion was markedly suppressed by the 6% protein diet in PAN nephrosis. The low-protein diet reduced the increased mRNA expression of SMemb as well as the increased number of SMemb-positive cells in the glomeruli of rats with PAN nephrosis. However, the low-protein diet did not affect SMemb mRNA and protein levels in the glomeruli of control rats. In rats with PAN nephrosis, findings suggest that restriction of dietary protein leads to a reduction in glomerular SMemb expression.