Effects of insulin and metformin on fetal kidney development of streptozotocin-induced gestational diabetic albino rats

Abstract

Gestational diabetes mellitus is one of common medical complications of pregnancy. Hyperglycemia in utero impairs renal development and produces renal anomalies. Metformin has antioxidant properties and better glycemic control. Aim: assessment insulin and metformin effects on renal development of streptozotocin-induced gestational diabetic albino rats. Sixty virgin female albino rats were used. Once pregnancy confirmed, animals were randomly assigned into control, metformin, diabetic, diabetic plus insulin, diabetic plus metformin and diabetic plus insulin and metformin treated groups. Rats were sacrificed on the 20th day of gestation; fetuses were extracted and weighted. Fetal kidneys were extracted prepared for light, morphometric and electron microscopic examination. Diabetic followed by diabetic plus metformin treated groups revealed retardation of glomerular development in the cortical and Juxtaglomerular zones with a significant increase in the early immature glomerular stages and immature to mature glomerular ratio compared to other groups. Diabetic group also showed morphometric changes, shrunken and empty glomeruli, vacuolar degeneration and hemorrhage. Diabetic plus metformin group showed minimal improvement while diabetic plus insulin and diabetic plus insulin and metformin groups showed developmental, histopathological and morphometric improvement with best results in the combination group. Gestational diabetes mellitus (GDM) possess deleterious effects on fetal kidney development. Insulin improves the glycemic state and decreases GDM effects on fetal kidneys. Metformin produces mild protection while the combination of insulin and metformin produces the best glycemic control and protect fetal kidneys.

Keywords: Albino rat; Gestational diabetes mellitus; Insulin; Kidney development; Metformin.

Fig. 1. Values of maternal random blood sugar throughout gestation in the studied groups. +ve, positive control; −ve, negative control.

Fig. 2. Photomicrographs sections in the fetal kidney. (A) Control group showing the renal corpuscle with the parietal layer of Bowman's capsule (dashed arrow), mature glomerulus (G) and preserved corpuscular space (double head arrow), proximal (P) and distal (D) convoluted tubules. (B, C) Diabetic group showing shrunken glomerulus (G), increased renal corpuscular space (double head arrow), vacuolations (V), empty renal corpuscles (E), degenerated dilated tubules (T), shedding of brush border of proximal tubules (P), degenerated epithelium of distal tubule (D), hemorrhage (H) and pyknotic nuclei (K). (D) Diabetic plus insulin-treated group showing intact mature glomeruli (G) surrounded by Bowman's capsule (dashed arrow) with preserved corpuscular space (double head arrow), intact proximal (P) and distal tubules (D). Notice the presence of mildly shrunken glomerulus (G^*), pyknotic nuclei (K), increased corpuscular space (double head dashed arrow), degenerated proximal (P^*) and distal convoluted tubules (D^*) and areas of vacuolation (V). (E) Diabetic plus metformin-treated group showing shrunken mature glomerulus (G), increased renal corpuscular space (double head arrow), dilated and destructed renal tubules (T), and vacuolation (V). (F) Diabetic plus insulin and metformin-treated group showing intact glomeruli (G), Bowman's capsule (dashed arrow), and preserved corpuscular space (double head arrow), intact proximal (P) and distal (D) convoluted tubules. Notice the presence of areas of vacuolation in the glomerulus (V) and tubules (T) (H&E staining, ×400).

Fig. 3. Periodic acid-Schiff stained sections in the fetal kidney. (A) Control group showing positive stain in the glomeruli (G), Bowman's capsule (arrowhead), basement membrane of renal tubules (arrow) and the brush border of proximal tubules (dashed arrow). (B) Diabetic group showing faint reaction in glomeruli (G), Bowman's capsules (arrowheads), basement membrane (dashed arrows) and brush border of the proximal convoluted tubules (arrows). (C) Diabetic plus insulin treated group showing positive reaction in the glomeruli (G), Bowman's capsule (arrowheads), basement membrane of renal tubules (arrow) and the brush border of proximal tubules (dashed arrow). Notice the presence of areas of negative reaction (stars). (D) Diabetic plus metformin treated group showing areas of faint reaction in the tubular basement membrane (arrow) and glomerulus (dashed arrow) with the presence of positive reaction in other glomerulus (G) and Bowman's capsule (arrowhead). (E) Diabetic plus insulin and metformin treated group showing positive reaction in the glomerulus (G), Bowman's capsule (arrowhead), basement membrane of renal tubules (arrows) and the brush border of proximal tubules (dashed arrows) (periodic acid-Schiff, ×400).

Fig. 4. Photomicrographs of Toluidine blue stained sections in the fetal kidney. (A–C) Control group showing vesicle (V), S-shaped (S), commashaped (Co), and capillary loop (CL) stages of glomerular development. (D) Control group showing mature glomerulus (G) with the parietal layer of Bowman's capsule (BC), podocyte (Pd), mesangial cells (M), proximal convoluted tubules (P) and distal (D) convoluted tubules. (E) Diabetic group showing, loss in the normal architecture of the cortex, immature glomerulus (vesicular stage) (G) and marked vacuolations (V) within the glomerulus and tubules. (F) Diabetic plus insulin-treated group showing intact mature glomerulus (G), proximal (P) and distal (D) convoluted tubules. Notice small area of hemorrhage (H) and vacuolation (arrow). (G) Diabetic plus metformin-treated group showing shrunken glomerulus (G), wide Bowman's space (BS), and areas of hemorrhage (H). Notice degenerated tubules (T). (H) Diabetic plus insulin and metformin-treated group showing intact mature renal glomerulus (G), Bowman's capsule (BC), podocyte (Pd), proximal (P) and distal (D) convoluted tubules (toluidine blue, ×1,000).

Fig. 5. Electron photomicrographs in fetal rat glomerulus. (A) Control group showing glomerular capillary lumen (L) lined with endothelial cell with euchromatic nucleus (N) and forming a thin fenestrated layer of cytoplasm (EN) lining the basement membrane (BM) internally. The capillary surrounded with intact podocyte (P) with foot processes (FP). Uranyl acetate and lead citrate (×10,000). (B) Diabetic group showing glomerular capillary lumen (L) with thinned glomerular filtration barrier (BM) and red blood cell (RBC), absence of fenestrations of endothelial layer (EN), vacuolations (V), podocytes with loss of foot processes (P) and chromatin clumping (N). Uranyl acetate and lead citrate, ×5,000. (C) Diabetic group showing glomerular capillary lumen (L) with a thickened glomerular basement membrane (BM) and area of absence of EN and surrounded by podocyte (P) with short foot process effacement and widening (FP). Uranyl acetated and lead citrate, ×8,000. (D) Diabetic plus insulin-treated group showing capillary endothelial cell (E) with euchromatic nucleus (N), podocyte (P) with intact foot processes (arrowheads) and homogeneous glomerular basement membrane with normal thickness (white arrow). Other areas of thickened glomerular basement membrane (BM) and marked widening and effacement of foot processes (FP). Uranyl acetate and lead citrate, ×15,000. (E) Diabetic plus metformin-treated group showing area of thick glomerular basement membrane (BM) and chromatin condensation (Cn) in nuclei of capillary endothelial cell (EN) and podocytes (P). Notice the presence of ill-defined nuclear envelop (dashed arrow) and foot process with effacement and widening (FP). Uranyl acetate and lead citrate, ×8,000. (F) diabetic plus insulin and metformin-treated group showing glomerular capillary lumen (L), intact glomerular basement membrane (BM), podocytes (P) with narrow and short foot processes (FP), thin layer of cytoplasmic endothelium with characteristic interruptions (dashed arrow). Uranyl acetate and lead citrate, ×12,000.

Fig. 6. Electron photomicrographs in the proximal convoluted tubule epithelial cells of fetal rat kidney. (A) Control group showing basal euochromatic nucleus (N), apical brush border with closely packed microvilli (MV), pinocytic vesicles (V), lysosome (L), plenty of mitochondria (M) and basement membrane (BM). Uranyl acetate and lead citrate, ×8,000. (B) Diabetic group showing irregular shaped nucleus (N), ill-defined and irregular nuclear envelope (white arrow), chromatin condensation (Cn), apical sticky and profoundly thin microvilli (MV) with areas of total loss of brush border (dashed arrow), swollen mitochondria with disruption of cristae (M) and apoptotic body (star). Notice presence of pinocytotic vesicles (V), and the lumen filled with exudate (E). Uranyl acetate and lead citrate, ×6,000. (C) Diabetic plus insulin treated group showing closely packed apical long microvilli (MV), euochromatic nucleus (N) and plenty of mitochondria (M). Notice the presence of areas with microvilli loss (^*), vesicles (V), and nucleus with chromatin condensation (Cn). Uranyl acetate and lead citrate, ×5,000. (D) Diabetic plus metformin treated group showing plenty of lysosomes (L), edematous mitochondria (M), nucleus with chromatin condensation (Cn), apoptotic body (star) and apical profoundly thin and sloughed microvilli (MV). Uranyl acetate and lead citrate, ×8,000. (E) Diabetic plus insulin and metformin-treated group showing closely packed apical microvilli (MV), pinocytic vesicles (V), lysosomes (L), euchromatic nucleus (N), plenty of mitochondria (M) and intact basement membrane (BM). Uranyl acetate and lead citrate, ×8,000.

Fig. 7. Electron photomicrographs in the distal convoluted tubule epithelial cells of fetal rat kidney. (A) Control group showing with blunt microvilli (MV), rounded euchromatic nucleus (N) and plenty of mitochondria (M). Uranyl acetate and lead citrate, ×15,000. (B) Diabetic group showing lumen obliteration with exudate (E), few microvilli (MV), swollen and disrupted mitochondria (M), pinocytotic vesicles (V), apoptotic body (star) and nuclei with chromatin condensation (Cn). Uranyl acetate and lead citrate, ×8,000. (C) Diabetic plus insulin-treated group showing short apical microvilli (MV), euchromatic nucleus (N) while other nucleus showed chromatin condensation (Cn), swollen and disrupted mitochondria (M). Uranyl acetate and lead citrate, ×8,000. (D) Diabetic plus metformin-treated group showing ill-defined cell boundaries, swollen mitochondria (M) and destructed and ruptured nucleus (N). Uranyl acetate and lead citrate, ×8,000. (E) Diabetic plus insulin and metformin-treated group showing small microvilli (MV), euchromatic rounded nucleus (N) with chromatin condensation (Cn) and intact mitochondria (M). Uranyl acetate and lead citrate, ×8,000.