Figure 1.

Nox5 is expressed in human diabetic kidney biopsies and human podocytes. Characterization of Nox expression in human diabetic kidney and podocytes. (A) Immunofluorescence for Nox5 (green) and TGF-β (red) expression in nondiabetic and diabetic human kidney biopsies. Scale bar, 50 μm. (B) Costaining of human diabetic kidney for Nox5 (red) and nephrin (green) expression (4′,6-diamidino-2-phenylindole as a nuclear counterstain). (C) RT-PCR analyzing expression of Nox1, -2, -3, -4, and -5 in hPOD lysates. (D) Expression of Nox5 splice variants-α (327 bp), -β (270 bp), -δ (354 bp), and -γ (404 bp) in hPOD, human embryonic kidney (HEK) cells, and human proximal tubule epithelial cells (PTECs). Spleen and testis served as positive controls. (E) SYBR green-based quantitative PCR showing relative fold change of Nox5 expression in vehicle (white columns)- and AngII (500 nM; black columns)-treated human podocytes compared with untreated controls. Values are expressed as mean±SEM (n=3). *P=0.03; **P=0.008. (F) SYBR green-based quantitative PCR showing relative fold change of Nox5 expression in HG-treated (black columns) human podocytes compared with low-glucose (LG) controls (white columns) at 2 and 10 hours after stimulation. Values are expressed as mean ± SEM (n=3). (G) Western blot for Nox5 (86 kDa) on human podocyte lysates comparing untreated controls (cont) with vehicle (veh)- and AngII (500 nM)-treated cells at various time points after stimulation. β-Actin served as loading control. (H) Western blot for Nox5 (86 kDa) on human podocyte lysates comparing controls with HG-treated cells. β-Actin served as loading control.