Altered Expression of EMT-Related Factors Snail, Wnt4, and Notch2 in the Short-Term Streptozotocin-Induced Diabetic Rat Kidneys

Abstract

Background: The aim of this study was to determine the expression of epithelial to mesenchymal transition (EMT)-related transcription factors Snail, Wnt4, and Notch2 with key roles in renal fibrosis, in different renal areas of diabetic rats: glomeruli (G), proximal and distal convoluted tubules (PCT; DCT). Methods: Male Sprague Dawley rats were instilled with 55 mg/kg streptozotocin (diabetes mellitus type I model, DM group) or citrate buffer (control group). Kidney samples were collected 2 weeks and 2 months after DM induction and processed for immunohistochemistry. Results: Diabetic animals showed higher Wnt4 kidney expression both 2 weeks and 2 months post-DM induction, while Snail expression significantly increased only 2 weeks after DM initiation (p < 0.0001). We determined significantly higher expression of examined EMT-related genes in different kidney regions in diabetic animals compared with controls. The most substantial differences were observed in tubular epithelial cells in the period of 2 weeks after induction, with higher Snail and Wnt4 expression in PCT and increased Snail and Notch2 expression in DCT of diabetic animals (p < 0.0001; p < 0.001). Conclusion: The obtained results point to the EMT-related factors Snail, Wnt4, and Notch2 as a potential contributor to diabetic nephropathy development and progression. Changes in their expression, especially in PCT and DCT, could serve as diagnostic biomarkers for the early stages of DM and might be a promising novel therapeutic target in this condition.

Keywords: Notch2; Wnt4; diabetic nephropathy; epithelial to mesenchymal transition (EMT); snail.

Figure 1

Immunohistochemical expression patterns of Snail (a) and Wnt4 (b) markers in the kidneys of diabetic (DM) and control (C) rat groups at 2 weeks (2w) and 2 months old (2M). Positive cells were observed as cytoplasmic or membrane staining (arrows) through the renal cortex. (c) DAPI nuclear staining. Co-expression of these markers is indicated with arrowheads (d). g—glomerulus, pct—proximal convoluted tubules, dct—distal convoluted tubules. The scale bar is 20 μm, which refers to all images.

Figure 2

Percentage of Snail (a), Wnt4 (b), and Notch2 (c) positive cells in diabetic (DM) and control (C) rat groups at 2 weeks (2w) and 2 months (2M). Data presented as mean ± SD, unpaired t-test. * p < 0.05, **** p < 0.0001.

Figure 3

Percentage of Snail (a), Wnt4 (b), and Notch2 (c), positive cells in nephron substructures between diabetic (DM) and control (C) rat groups at the periods of 2 weeks (2w) and 2 months (2M). Data presented as mean ± SD, two-way ANOVA. g—glomerulus, pct—proximal convoluted tubules, dct—distal convoluted tubules. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 4

Immunohistochemical expression patterns of Snail (a) and Notch2 (b) markers in the kidneys of diabetic (DM) and control (C) rat groups at 2 weeks (2w) and 2 months old (2M). Positive cells were observed as cytoplasmic or membrane staining (arrows) through the renal cortex. DAPI nuclear staining (c). Co-expression of these markers is indicated with arrowheads (d). g—glomerulus, pct—proximal convoluted tubules, dct—distal convoluted tubules. The scale bar is 20 μm, which refers to all images.

Figure 5

Immunohistochemical expression patterns of vimentin (a) and α-SMA (b) markers in the kidneys of diabetic (DM) and control (C) rat groups at 2 weeks (2w) and 2 months old (2M). DAPI nuclear staining (c). Co-expression of these markers is indicated with arrowheads (d). g–glomerulus, pct–proximal convoluted tubules, dct–distal convoluted tubules. Percentage of vimentin (e) and α-SMA (f) positive cells in kidney cortical structures between diabetic (DM) and control (C) rat groups at the periods of 2 weeks (2w) and 2 months (2M). Data presented as mean ± SD, two-way ANOVA. g—glomerulus, pct—proximal convoluted tubules, dct—distal convoluted tubules. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The scale bar is 20 μm, which refers to all images.