Characterization of glomerular extracellular matrix in IgA nephropathy by proteomic analysis of laser-captured microdissected glomeruli

Background IgA nephropathy (IgAN) involves mesangial matrix expansion, but the proteomic composition of this matrix is unknown. The present study aimed to characterize changes in extracellular matrix in IgAN. Methods In the present study we used mass spectrometry-based proteomics in order to quantitatively compare protein abundance between glomeruli of patients with IgAN (n = 25) and controls with normal biopsy findings (n = 15). Results Using a previously published paper by Lennon et al. and cross-referencing with the Matrisome database we identified 179 extracellular matrix proteins. In the comparison between IgAN and controls, IgAN glomeruli showed significantly higher abundance of extracellular matrix structural proteins (e.g periostin, vitronectin, and extracellular matrix protein 1) and extracellular matrix associated proteins (e.g. azurocidin, myeloperoxidase, neutrophil elastase, matrix metalloproteinase-9 and matrix metalloproteinase 2). Periostin (fold change 3.3) and azurocidin (3.0) had the strongest fold change between IgAN and controls; periostin was also higher in IgAN patients who progressed to ESRD as compared to patients who did not. Conclusion IgAN is associated with widespread changes of the glomerular extracellular matrix proteome. Proteins important in glomerular sclerosis or inflammation seem to be most strongly increased and periostin might be an important marker of glomerular damage in IgAN.

Negative regulation of Smad1 pathway and collagen IV expression by store-operated Ca2+ entry in glomerular mesangial cells

Collagen IV (Col IV) is a major component of expanded glomerular extracellular matrix in diabetic nephropathy and Smad1 is a key molecule regulating Col IV expression in mesangial cells (MCs). The present study was conducted to determine if Smad1 pathway and Col IV protein abundance were regulated by store-operated Ca2+ entry (SOCE). In cultured human MCs, pharmacological inhibition of SOCE significantly increased the total amount of Smad1 protein. Activation of SOCE blunted high-glucose-increased Smad1 protein content. Treatment of human MCs with ANG II at 1 µM for 15 min, high glucose for 3 days, or TGF-β1 at 5 ng/ml for 30 min increased the level of phosphorylated Smad1. However, the phosphorylation of Smad1 by those stimuli was significantly attenuated by activation of SOCE. Knocking down Smad1 reduced, but expressing Smad1 increased, the amount of Col IV protein. Furthermore, activation of SOCE significantly attenuated high-glucose-induced Col IV protein production, and blockade of SOCE substantially increased the abundance of Col IV. To further verify those in vitro findings, we downregulated SOCE specifically in MCs in mice using small-interfering RNA (siRNA) against Orai1 (the channel protein mediating SOCE) delivered by the targeted nanoparticle delivery system. Immunohistochemical examinations showed that expression of both Smad1 and Col IV proteins was significantly greater in the glomeruli with positively transfected Orai1 siRNA compared with the glomeruli from the mice without Orai1 siRNA treatment. Taken together, our results indicate that SOCE negatively regulates the Smad1 signaling pathway and inhibits Col IV protein production in MCs.

Rosiglitazone reduces renal and plasma markers of oxidative injury and reverses urinary metabolite abnormalities in the amelioration of diabetic nephropathy

Recent studies suggest that thiazolidinediones ameliorate diabetic nephropathy (DN) independently of their effect on hyperglycemia. In the current study, we confirm and extend these findings by showing that rosiglitazone treatment prevented the development of DN and reversed multiple markers of oxidative injury in DBA/2J mice made diabetic by low-dose streptozotocin. These diabetic mice developed a 14.2-fold increase in albuminuria and a 53% expansion of renal glomerular extracellular matrix after 12 wk of diabetes. These changes were largely abrogated by administration of rosiglitazone beginning 2 wk after the completion of streptozotocin injections. Rosiglitazone had no effect on glycemic control. Rosiglitazone had similar effects on insulin-treated diabetic mice after 24 wk of diabetes. Podocyte loss and glomerular fibronectin accumulation, other markers of early DN, were prevented by rosiglitazone in both 12- and 24-wk diabetic models. Surprisingly, glomerular GLUT1 levels did not increase and nephrin levels did not decrease in the diabetic animals; neither changed with rosiglitazone. Plasma and kidney markers of protein oxidation and lipid peroxidation were significantly elevated in the 24-wk diabetic animals despite insulin treatment and were reduced to near-normal levels by rosiglitazone. Finally, urinary metabolites were markedly altered by diabetes. Of 1,988 metabolite features identified by electrospray ionization time of flight mass spectrometry, levels of 56 were altered more than twofold in the urine of diabetic mice. Of these, 21 were returned to normal by rosiglitazone. Thus rosiglitazone has direct effects on the renal glomerulus to reduce reactive oxygen species accumulation to prevent type 1 diabetic mice from development of DN.