Anat Cell Biol.  2021 Dec;54(4):501-517. 10.5115/acb.21.124.

Protective effect of vitamin C against ivermectin induced nephrotoxicity in different age groups of male wistar rats: bio-histopathological study

• Tawfeek SE
• Domouky AM
• Abdel-Kareem RH

• KMID: 2523580
• DOI: http://doi.org/10.5115/acb.21.124

Abstract

Ivermectin (Ive) has exceedingly efficient against several microorganisms including viruses; therefore, it could help as a potential treatment of COVID-19. Because of increasing consumption of ivermectin and vitamin C (Vit.C) in hope to treat COVID-19, and because of ivermectin nephrotoxic effects have not been fully clarified especially in juvenile age, it was conducted to examine the histopathological and biochemical effects of ivermectin on adult and juvenile kidneys, and to assess the possible protective role of Vit.C against this potential toxicity. Rats were divided to 4 subgroups (Control subgroup, Vit.C subgroup, Ive subgroup, and Vit.C+Ive subgroup), 1 week after 4 doses of ivermectin (0.4 mg/kg Ive±1.25 mg/kg Vit.C), blood samples obtained for assessment of kidney function test, part of kidneys prepared for determination of matrix metalloproteinase-9 and antioxidant enzymes essay. Other parts prepared for histopathological and ultrastructural examination. Results showed that administration of ivermectin led to attenuation in kidney function and in activities of the antioxidant enzymes and increase in matrix metalloproteinase-9 activity. In addition, there were histological damages (shrunken glomeruli, widened urinary space, cytoplasmic vacuolation and pyknotic nuclei with epithelial exfoliation, extravasated blood, and mononuclear cell infiltration) and immunohistochemistry revealed increase in percentage of Bax proapoptotic protein expression. Also, ultrastructure examination showed alteration in cell architecture. All these changes were more obvious in juvenile group while co-administration of Vit.C led to significant protection more in adult group. In conclusion, Ivermectin should be used cautiously especially in juvenile age, and co-administration of Vit.C is highly recommended.

Keyword

Ivermectin, Renal toxicity; Vitamin C; COVID-19

Figure

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  Fig. 1 Anatomical parameters examination in different study groups showing that Juvenile (Jv) rats treated with ivermectin (Ive subgroup) have a significant decrease in body weight (A) and kidney weight (B) compared with control and vitamin C (Vit.C) subgroups, while kidney weight/body weight % (C) remain insignificant difference. Adult (Ad) rats reveal significant difference in body weight (D) and no significant difference neither in kidney weight (E) nor kidney weight/body weight % (F). The percentage of change happen in corresponding Ive subgroups, the juvenile rats exhibit more body weight loss (G), also more kidney weight loss (H, I). Values are presented as mean±SD.

• 

  Fig. 2 Biochemical parameters analysis in different study groups showing that rats treated with ivermectin (Ive subgroup) have a highly significant increase in the kidney function marker; Serum creatinine concentrations (A), Blood urea nitrogen (B). Measuring the activities of the TAC (C), antioxidant enzymes catalase assay (CAT) (D), superoxide dismutase assay (SOD) (E), glutathione peroxidase assay (GPx) (F), and reactive oxygen species (G) showing that administration of vitamin C (Vit.C) alone leads to increase in antioxidant enzymes activities, on the other hand, rats treated with ivermectin (Ive subgroups) display a highly significant decrease in the activities of the antioxidant enzymes, comparing juvenile (Jv) and adult (Ad) groups showing that Jv rats have more decline in oxidants activity in treated group and less protected effect of Vit.C. Matrix metalloproteinase 9 (MMP9) is statistically significantly higher in Ive treated subgroups (H). Values are presented as mean±SD. TAC, total antioxidant capacity.

• 

  Fig. 3 H&E (×400) stained sections of renal tissues in different experimental groups: control and vitamin C (Vit.C) subgroups (A–D) showing; normal shaped glomeruli (g), normal renal space (S) lined by parietal layer of Bowman’s capsules (B) and renal tubules (dt, distal tubule; pt, proximal tubule) lined by normal cells with vesicular nuclei (short arrow). Ivermectin treated sub groups (Ive subgroup) (E–H) showing; shrunken glomeruli (G), widened urinary space (*), dilated tubules (dt & pt) lined by vacuolated cells with pyknotic nuclei (V), some desquamated cells in the lumen (arrow head), inflammatory cell infiltration (curved arrows) and extravasated blood (H). Vit.C+Ive subgroups (I, J) showing; normal shaped glomeruli (g) and tubules (dt, distal tubule; pt, proximal tubule), some shrunken glomeruli (G), relatively wide renal space (*) and some extravasated blood (H). In (I), few tubules exhibit pyknotic nuclei with cytoplasmic vacuolation (V) and epithelial cell exfoliation (arrow head). Morphometrically, in the Ive subgroups, cortical surface area (L) and glomerular area (K) showing significant decrease with significant increase in urinary space cross sectional (M), these changes are more obvious in juvenile (Jv) group (N, O) than adult (Ad) group.

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  Fig. 4 PAS stained (×400) sections of renal tissues in different experimental groups (A–D) (control & Vit.C) showing; well-formed tubular basement membranes (arrow heads), Basement membranes of Bowman’s capsules appear as thin regular PAS+Ive (thick arrows) and well developed brush borders of proximal convoluted tubule (arrows). Ive treated sub groups, (E, F) showing; strong positive PAS reaction of thickened basement membrane of both Bowman’s capsule (thick arrows) and renal tubules (arrow heads) and discontinuity PAS reaction at the brush borders of proximal tubular cells (arrows). Vit.C+Ive subgroups (G, H) showing; PAS positive reaction at the thin Bowman’s capsule (thick arrows) and tubular basement membrane (arrow heads) and continued brush border of proximal tubules (arrows). Vit.C, vitamin C; Ive, ivermectin; Jv, juvenile; Ad, adult.

• 

  Fig. 5 Bax immune-stained (×400) sections of renal tissues in different experimental groups showing; positive proapoptotic protein in the cytoplasm of renal tubules (arrow heads) is normally expressed in control and Vit.C subgroups (A–D), while Ive treated subgroups (E, F) display patchy expression. Vit.C+Ive subgroups (G, H) showing improvement in the Bax proapoptotic protein expression. Morphometrical analysis of percentages of Bax positivity showing high significant increase in Ive subgroups and significant increase in Vit.C+Ive subgroups (I, J). Vit.C, vitamin C; Ive, ivermectin; Jv, juvenile; Ad, adult.

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  Fig. 6 Electron micrographs of kidney tubules of control subgroup; juvenile (Jv) (A: proximal tubule, ×12,000; C: distal tubule, ×6,000) and adult (Ad) (B: proximal tubule, ×12,000; D: distal tubule, ×12,000) showing the following features: N, normal nucleus; n, nucleolus; m, mitochondria; arrow head, nuclear envelop; arrow, basal lamina; curved arrow, apical microvilli; Lu, lumen; L, lysosomes; RER, rough endoplasmic reticulum and the electron micrographs of glomerulus in both Jv (E: ×30,000) and Ad (F: ×30,000) of the control subgroup showing; P, podocyte nucleus; BC, blood capillary; arrow, basal lamina; arrow head, foot process of podocytes; zigzag arrow, septum between foot process. Electron micrographs of proximal tubule of Vit.C subgroup of Jv (G: ×6,000), and Ad (H: ×6,000) showing; N, normal nucleus; n, nucleolus; m, mitochondria; L, lysosomes; MV, microvilli; arrow, basement membrane. Vit.C, vitamin C; Ive, ivermectin.

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  Fig. 7 Electron micrographs of kidney tubules of ivermectin (Ive) treated sub groups; juvenile (Jv) (A: proximal tubule, ×5,000; C: distal tubule, ×6,000) and adult (Ad) (B: proximal tubule, ×6,000; D: distal tubule, ×12,000) showing the following features: N, normal nucleus; aN, apoptotic nucleus; m, ballooned mitochondrial; R, cytoplasmic rarefaction; C, intraluminal casts; L, secondery lysosomes; RER, degenerated rough endoplasmic reticulum; HG, luminal hemorrhage; arrowhead, irregular nuclear envelop; arrow, thick basement membrane. The region of glomerulus in both Jv (E: ×30,000) and Ad (F: ×30,000) of the same subgroup with the following features: P, podosyt; arrow, thick basement membrane; arrowhead, fused foot process of podocytes; zigzag arrow, fused septum between foot process; E, endothelial cells. Vit.C+Ive subgroups of Jv (G: ×6,000; I: ×12,000), and Ad (H: ×6,000; J: ×6,000) rats showing N, normal nucleus; n, nucleolus; aN, apoptotic nucleus m, mitochondria; MV, microvilli; BL, blood capillary; L, lysosomes; arrow, basement membrane; arrow head, regular nuclear envelop in (H) and relatively normal foot process in (I). Moreover, glomerulus in (I) showing P, intact podocyte and E, endothelial cells.

Reference

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