Hyperglycemia activates the renin-angiotensin system and induces epithelial-mesenchymal transition in streptozotocin-induced diabetic kidneys

Figure 1.. (a) Representative hematoxylin and eosin staining, (b) the proportion of the kidney occupied by the cortex, (c) the proportion of the cortex occupied by glomeruli, (d) the glomerular size, and (e) tubular injury score in control gerbils (control) and gerbils with streptozotocin-induced diabetes mellitus (DM). Diabetic kidneys exhibited tubular atrophy, dilatation of the tubular lumen, brush border loss, and increased space (asterisks) between renal tubules. Acidophilic and swollen tubular cells and enlarged podocytes (arrows) were observed in the DM group. Streptozocin-treated gerbils exhibited significantly a larger proportion of the cortex, a smaller proportion of the cortex occupied by glomeruli, larger glomerular size, and higher tubular injury scores than did control gerbils (*p<0.001). Data are expressed as mean±standard deviation (SD).

Figure 2.. Immunohistochemistry of anti-8-hydroxy-2′-deoxyguanosine (8-OHdG), toll-like receptor 4 (TLR4), anti-nuclear factor-κB (NF-κB), image merged of TLR4 and NF-κB, and anti-tumor necrosis factor-α (TNF-α) in the kidney sections of control and diabetes mellitus (DM) gerbils. To compare DM gerbils with control gerbils, a DM gerbil was expected to express these proteins. The 8-OHdG immunoreactivity was detected in the nuclei of podocytes and tubular cells (black arrows). Immunofluorescence reactivity of TLR4 and NF-κB was identified in the cytoplasm of podocytes and tubular cells (white arrows). NF-κB expression was observed in the nuclei of podocytes and tubular cells (white arrowheads), which were purple after treatment with DAPI (for the nucleus in blue). The immunofluorescence of TLR4 and NF-κB was colocalized in the cytoplasm of podocytes and tubular cells and demonstrated a color change from yellow to orange. The immunoreactivity of TNF-α (black arrows) was detected in the nuclei and cytoplasm of podocytes and tubular cells.

Figure 3.. Representative photomicrographs of renin–angiotensin system (RAS) protein expression. The immunoreactivity of angiotensin II (Ang II) and Ang II type 1 receptor (AT1R) is extensively displayed in endothelial cells (white arrows), tubular cells (black arrows), and podocytes (black arrowheads) in diabetic gerbils (diabetes mellitus (DM)). Angiotensin (1–7) (Ang-(1–7))-positive staining cells were discovered in the endothelial cells (white arrows) of control and diabetic gerbils. The immunoreactivity of Ang-(1–7) was observed in the cells of distal tubules (black arrows) of control gerbils. Angiotensin-converting enzyme (ACE) expression was observed in endothelial cells (white arrows) and tubular cells (black arrows); the immunoreactivity was stronger in the diabetic gerbils than in the control gerbils. The control gerbils exhibited prominent ACE2 immunoreactivity, which was exhibited in endothelial cells and in the top of tubular cells (coincident with the brush border) (white arrows) compared with the diabetic gerbils.

Figure 4.. Immunohistochemistry of transforming growth factor-β (TGF-β) and anti-connective tissue growth factor (CTGF) in the kidney sections of control and diabetes mellitus (DM) gerbils. The immunoreactivity of TGF-β and CTGF was detected in the cytoplasm and nuclei of renal tubule cells, podocytes, and mesangial cells of glomeruli (black arrows). The streptozocin-treated diabetic gerbils exhibited higher numbers of TGF-β- and CTGF-positive cells than did the nondiabetic gerbils.

Figure 5.. Immunofluorescence staining for E-cadherin (green), N-cadherin (red), and 4′,6-diamidino-2-phenylindole (DAPI) (blue). The immunofluorescence of E-cadherin and N-cadherin was detected in renal tubule cells. The renal tubules of the control gerbils (control) exhibited abundant E-cadherin and few N-cadherin immunopositive cells. By contrast, the immunofluorescence of N-cadherin was more widely distributed and few E-cadherin immunofluorescent cells were found in the diabetic gerbils. The colocalized E-cadherin and N-cadherin is indicated as yellow to orange in the merged image.

Figure 6.. (a) Representative photomicrographs of Masson’s trichrome staining; (b) grade of interstitial fibrosis; and (c) optical density of collagen in the control gerbils (control) and gerbils with streptozotocin-induced diabetes mellitus (DM). Collagen fibers were mainly deposited in the glomerular mesangium and extracellular tissue of renal tubules in the diabetic gerbils. The streptozocin-treated diabetic gerbils exhibited a significantly higher grade of interstitial fibrosis and a higher optical density of collagen than did the control gerbils (*p<0.001). Data are expressed as mean±standard deviation (SD).

Figure 7.. (a) Representative photomicrographs of immunohistochemistry of anti-α-smooth muscle actin (α-SMA) and the periodic acid-Schiff (PAS) staining and (b) semiquantitative scoring of the glomerular damage in the control gerbils (control) and gerbils with streptozotocin-induced diabetes mellitus (DM). The diabetic kidneys exhibited α-SMA immunoreactivity in podocytes and tubular cells (arrow). PAS photomicrographs indicate the thickened basal membrane of glomerular capillaries, diffused hyalinosis (asterisk), degenerated glomeruli, and thickened Bowman`s capsule (arrow). The streptozocin-treated diabetic gerbils exhibited significantly higher glomerular damage than did the control gerbils (*p<0.001). Data are expressed as mean±standard deviation (SD).