Overcoming refractory massive proteinuria remains a clinical and research issue in diabetic nephropathy. This study was designed to investigate the pathogenesis of massive proteinuria in diabetic nephropathy, with a special focus on podocyte autophagy, a system of intracellular degradation that maintains cell and organelle homeostasis, using human tissue samples and animal models. Insufficient podocyte autophagy was observed histologically in patients and rats with diabetes and massive proteinuria accompanied by podocyte loss, but not in those with no or minimal proteinuria. Podocyte-specific autophagy-deficient mice developed podocyte loss and massive proteinuria in a high-fat diet (HFD)-induced diabetic model for inducing minimal proteinuria. Interestingly, huge damaged lysosomes were found in the podocytes of diabetic rats with massive proteinuria and HFD-fed, podocyte-specific autophagy-deficient mice. Furthermore, stimulation of cultured podocytes with sera from patients and rats with diabetes and massive proteinuria impaired autophagy, resulting in lysosome dysfunction and apoptosis. These results suggest that autophagy plays a pivotal role in maintaining lysosome homeostasis in podocytes under diabetic conditions, and that its impairment is involved in the pathogenesis of podocyte loss, leading to massive proteinuria in diabetic nephropathy. These results may contribute to the development of a new therapeutic strategy for advanced diabetic nephropathy.

To evaluate an interaction between vasoconstrictive (Ang II) and vasodilating (ANP) peptides, we examined the effect of Ang II on ANP-induced accumulation of cGMP in cultured glomerular mesangial cells. ANP rapidly increased intracellular cGMP levels, with a peak stimulation at one minute in the absence of IBMX and at ten minutes in the presence of IBMX. The ANP-induced cGMP accumulation was significantly inhibited when the cells were treated with Ang II simultaneously with ANP for one minute in the absence of IBMX. This inhibitory effect of Ang II was completely abolished by IBMX and significantly reduced in calcium-free media or by W7, but not affected by H7. Similar inhibitory effect was observed when cells were treated with A23187 but not with TPA for one minute. In the presence of IBMX, Ang II inhibited ANP-induced cGMP accumulation when cells were treated with Ang II for 15 minutes prior to the stimulation by ANP. This inhibition by Ang II was blocked by H7. ANP-induced increase in particulate guanylate cyclase activity was significantly reduced in the cells treated with Ang II or TPA. This reduction of enzyme activity was also prevented by H7. These results indicate that Ang II inhibits ANP-induced cGMP accumulation in cultured glomerular mesangial cells through at least two mechanisms; one is the activation of calcium-dependent, calmodulin-stimulated cyclic nucleotide phosphodiesterase in the initial phase, and the other is the inhibition of guanylate cyclase resulting from protein kinase C activation in the maintenance phase.

Summary. Type IV collagen production by cultured glomerular mesangial cells and the effect of glucose on it were evaluated in order to explore the possible contribution of mesangial cells to the accumulation of type IV collagen in mesangial matrix typically seen in diabetes. Type IV collagen was measured quantitatively by enzyme-linked immunosorbent assay. The majority of type IV collagen was secreted into culture media and secreted-type IV collagen increased with cell growth in early log phase and decreased in late log phase and after confluency. By exposing the cells to high concentrations of glucose (27.8 retool/l), both secreted-and cellassociated-type IV collagens increased significantly compared with the cells cultured under normal glucose concentrations (5.6 mmol/1) or under equivalent concentrations of mannitol, resulting in a significant increase in total type IV collagen accumulation from 32.1+6.4 (under 5.6 retool/1 glucose) to 51.0 _+ 4.6 gg/dish (mean + SD, n = 4) on day4, from 113.6+6.6 to 156.8+7.1 on day6, from 248.5 + 15.2 to 310.0 + 12.6 on day 8 and from 372.4 + 14.8 to 507.9 + 17.2 on day 12. These results indicate the importance of glucose-induced alteration of mesangial cell function in the development of diabetic mesangial expansion.

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Although the enhanced activity of the polyol pathway has been detected in diabetic glomeruli, the intraglomerular localization of this pathway has not yet been well defined. In this study, we attempted to identify aldose reductase, a key enzyme of the polyol pathway, in cultured rat mesangial cells and to characterize the properties of this enzyme using enzymological and immunological methods. When the aldose reductase (DL-glyceraldehyde-reducing) activity was analyzed in mesangial cell extract, the Lineweaver-Burk plot showed concave downward curvature, and the Michaelis constant was 0.83 mM DL-glyceraldehyde, and this activity was noncompetitively inhibited by an aldose reductase inhibitor, ICI-128,436. The enzyme activity was enhanced by the addition of sulfate ion and partially suppressed by barbital. The enzyme cross-reacted with the antisera against rat lens and testis aldose reductases on Ouchterlony plate, and migrated to the region of molecular weight of about 36,500 Da on Western blotting. The presence of aldose reductase mRNA was also confirmed by Northern analysis using cDNA for rat aldose reductase, 10Q. From these results, it was concluded that the aldose reductase may exist in rat glomerular mesangial cells and may play a role in the development of diabetic glomerulopathy, though the coexistence of aldehyde reductase(s) may not be fully ruled out.