An investigation of the expression of glucose regulated genes μ-crystallin and Molybdenum Cofactor Sulfurase C-Terminal in Diabetic Nephropathy

摘要

Diabetic Nephropathy (DN), a major cause of end stage renal failure, is believed to result from hyperglycaemia-induced pathways in the kidney. We previously identified μ-crystalline (CRYM) and Molybdenum Cofactor Sulfurase C-terminal domaincontaining protein 2 (MOSC2) as two of several hyperglycaemia-induced renal genes in the Goto-Kakizaki (GK) rat, and showed that both were regulated by glucose in vitro in human mesangial cells (HMCs). In the current thesis, we investigated their expression in models of diabetes as well as in circulating cells from DN patients, in order to evaluate these genes as potential biomarkers and to explore their possible roles in the pathophysiology of DN. The expression of CRYM and MOSC were examined in tissues obtained from in vivo mouse models including a streptozotocin (STZ)-induced model of diabetes reversible through islet transplantation, renal cells cultured in normal (5mM) and high (25mM) glucose, and blood samples from diabetic patients with and without nephropathy (n= 97)/ retinopathy (n= 36). mRNA levels were determined using qPCR relative to reference genes, and protein location/abundance was determined using immunofluorescence. Functional analysis included N-hydroxylation assays. Both CRYM and MOSC2 mRNAs increased during hyperglycaemia in the diabetic kidneys of the STZ-induced mice and this increase was attenuated by treatment of diabetes (P0.05). Immunohistochemistry revealed an abundant expression of both proteins in tubular cells, and very low expression in mesangial cells. The hyperglycaemia-induced increase in renal CRYM was specific as we could not demonstrate any change in cardiac CRYM, and similarly no change in expression was found under diabetic conditions in vitro in Molybdenum Cofactor Sulfurase Cterminal domain-containing protein 1 (MOSC1), a homolog of MOSC2. Surprisingly, in high glucose (HG), CRYM and MOSC2 mRNA levels showed a slight decrease in cultured tubular cells, whereas they showed a significant increase in HMCs. Biochemical analysis of MOSC protein showed that hyperglycaemia increased the Nreductive activity of MOSC2 protein. To evaluate CRYM and MOSC mRNA levels as potential biomarkers of DN, we examined their expression in the peripheral blood of diabetic patients. We could detect low levels of CRYM but there was no expression of MOSC2. However, the closely related homolog MOSC1 was expressed in the peripheral blood though its mRNA levels did not change in association with DN. CRYM mRNA levels were 7.4-fold increased in patients with type 2 nephropathy and this effect was strongest in patients with a well controlled nephropathy compared to those with proteinuria. However, we found that circulating CRYM mRNA was reduced in patients with retinopathy. Therefore it is currently unclear if circulating CRYM is associated with nephropathy and its low levels of expression suggest it may not be a useful biomarker. Our data suggest that diabetes leads to an increased expression of renal CRYM and MOSC2 mRNAs, and although there are high levels of both mRNAs in tubular cells, the up-regulation may be taking place in mesangial or other renal cells. Circulating CRYM mRNA levels showed changes in patients with nephropathy and retinopathy but MOSC1 remained unchanged. CRYM or MOSC1/2 are unlikely to be useful biomarkers for DN but may be involved in the pathways that lead to DN.