Mice rescued from severe malaria are protected against renal injury during a second kidney insult

Abstract

Malaria is a worldwide disease that leads to 1 million deaths per year. Plasmodium falciparum is the species responsible for the most severe form of malaria leading to different complications. Beyond the development of cerebral malaria, impairment of renal function is a mortality indicator in infected patients. Treatment with antimalarial drugs can increase survival, however the long-term effects of malaria on renal disease, even after treatment with antimalarials, are unknown. The aim of this study was to evaluate the effect of antimalarial drug treatment on renal function in a murine model of severe malaria and then evaluate kidney susceptibility to a second renal insult. Initially, mice infected with Plasmodium berghei ANKA achieved 20% parasitemia on day 5 post infection, which was completely abolished after treatment with 25 mg/kg artesunate and 40 mg/kg mefloquine. The treatment also decreased plasma creatinine levels by 43% and partially reversed the reduction in the glomerular filtration rate induced by infection. The urinary protein/creatinine ratio, collagen deposition, and size of the interstitial space decreased by 75%, 40%, and 20%, respectively, with drugs compared with untreated infected animals. In infected-treated mice that underwent a second renal insult, the plasma creatinine level decreased by 60% and the glomerular filtration rate increased compared with infected animals treated only with antimalarials. The number of glomerular cells, collagen deposition and the size of the interstitial space decreased by 20%, 39.4%, and 41.3%, respectively, in the infected group that underwent a second renal insult compared with the infected-treated groups. These functional and structural data show that renal injury observed in a murine model of severe malaria is partially reversed after antimalarial drug treatment, making the kidney less susceptible to a second renal insult.

Figure 1. Experimental design.

(A) C57BL/6 mice were separated into four experimental groups that were euthanized on day 5 (5d) or day 21 (21d) post infection (p.i.): noninfected group (control_5d); mice infected with 10⁶ infected red blood cells euthanized on day 5 p.i. (infected_5d); noninfected mice that received treatment with antimalarials (control+treated_21d); and the Plasmodium berghei ANKA (PbA)-infected group treated with antimalarials and euthanized on day 21 p.i. (infected+treated_21d). (B) Additional experimental groups that were euthanized on day 21 underwent or not a second kidney insult induced with intraperitoneal injections of bovine serum albumin (BSA): noninfected group (control_21d); noninfected mice with BSA-induced renal injury (control+BSA_21d); noninfected mice that received treatment with antimalarials and subsequent BSA injections (control+treated+BSA_21d); PbA-infected mice treated with antimalarials (infected+treated_21d); and PbA-infected mice that were treated and subsequently underwent a second kidney insult (infected+treated+BSA_21d). (C) Parasitemia was determined from blood smears stained with Diff-Quick in all infected groups on day 1 (A), day 5 (B), day 8 (C), and day 21 (D) p.i.

Figure 2. Effect of antimalarial treatment on malaria-induced renal damage.

Animals were euthanized at day 5 or 21 post infection and plasma and urine samples were collected for analysis of renal function. Analysis of (A) urinary flow, (B) serum creatinine, (C) blood urea nitrogen (BUN), (D) creatinine clearance (CCr), (E) BUN/serum creatinine ratio, and (F) urinary protein/creatinine (UPC) ratio in different experimental groups as depicted in the figure. *Statistically significant compared with control_5d or control+treated_21d. #Statistically significant compared with control_5d or infected_5d (P<0.05).

Figure 3. Effect of antimalarial treatment on the number of glomerular cells.

Animals were euthanized on day 5 or day 21 p.i., perfused, and the kidneys were collected for histologic analysis. The number of glomerular cells in the renal cortex was visualized using periodic acid−Schiff. Representative photomicrography of noninfected mice (control_5d) (A), 5 days p.i. (infected_5d) (B), noninfected mice that received treatment with antimalarial drugs (control+treated_21d) (C), and infected mice that received treatment with antimalarial drugs (infected+treated_21d) (D) (n = 6 per group). Bar = 40 µm. The number of glomerular cells is quantified in (E). The results are expressed as means ± standard error. *Statistically significant compared with control_5d or control+treated_21d. #Statistically significant compared with control_5d or infected_5d (P<0.05).

Figure 4. Effect of antimalarial treatment on collagen deposition in the renal cortex of mice.

Animals were euthanized on day 5 or 21 post infection (p.i.), perfused, and the kidneys were collected for histologic analysis. Collagen deposition in the renal cortex was visualized using Picrosirius Red stain. Representative photomicrography of noninfected mice (control_5d) (A), 5 days p.i. (infected_5d) (B), noninfected mice that received treatment with antimalarial drugs (control+treated_21d) (C), and infected mice that received treatment with antimalarials (infected+treated_21d) (D) (n = 6 per group). Bar = 20 µm. Collagen deposition is quantified in (E). Values are expressed as a percentage of collagen deposition per area (means ± standard error). *Statistically significant compared with control_5d or control+treated_21d. #Statistically significant compared with control_5d or infected_5d (P<0.05).

Figure 5. Effect of antimalarial treatment on the size of the interstitial space.

Animals were euthanized on day 5 or 21 post infection (p.i.), perfused, and the kidneys were collected for histological analysis. The size of the interstitial space in the renal cortex was visualized using periodic acid−Schiff. Representative photomicrography of noninfected mice (control_5d) (A), 5 days p.i. (infected_5d) (B), noninfected mice that received treatment with antimalarial drugs (control+treated_21d) (C), and infected mice that received treatment with antimalarial drugs (infected+treated_21d) (D) (n = 6 per group). Bar = 20 µm. The size of the interstitial space is quantified in (E). Values are expressed as a percentage of interstitial space per area (means ± standard error). *Statistically significant compared with control_5d or control+treated_21d. #Statistically significant compared with control_5d or infected_5d (P<0.05).

Figure 6. Malaria-surviving mice show less susceptibility to a second kidney insult.

Animals were euthanized on day 21 post infection (p.i.) and plasma and urine samples were collected for analysis of renal function. Analysis of control_21d, control+BSA _21d, control+treated+BSA_21d, infected+treated_21d, and infected+treated+BSA_21d: (A) serum creatinine, (B) blood urea nitrogen (BUN); (C) BUN/serum creatinine ratio; (D) creatinine clearance (CCr); and (E) urinary protein/urinary creatinine (UPC) ratio. *Statistically significant compared with control_21d. #Statistically significant compared with infected+treated _21d (P<0.05).

Figure 7. Glomerular cell number decreases in mice subjected to a second kidney insult.

Animals were euthanized on day 21 post infection (p.i.), perfused, and the kidneys were collected for histologic analysis. The number of glomerular cells in the renal cortex was visualized using periodic acid−Schiff. Representative photomicrographs of (A) control_21d, (B) control+BSA _21d, (C) control+treated+BSA _21d, (D) infected+treated _21d, and (E) infected+treated+BSA _21d (n = 6 per group). Bar = 40 µm. The number of glomerular cells is quantified in (F). The results are expressed as means ± standard error. *Statistically significant compared with control_21d. #Statistically significant compared with infected+treated _21d (P<0.05).

Figure 8. Renal collagen deposition decreases in mice subjected to a second kidney insult.

Animals were euthanized on day 21 post infection (p.i.), perfused, and the kidneys were collected for histologic analysis. Collagen deposition in the renal cortex was visualized using Picrosirius Red. Representative photomicrographs of (A) control_21d, (B) control+BSA_21d, (C) control+treated+BSA_21d, (D) infected+treated_21d, and (E) infected+treated+BSA_21d (n = 6 per group). Bar = 20 µm. Collagen deposition is quantified in (F). Values are expressed as a percentage of collagen deposition per area (means ± standard error). *Statistically significant compared with control_21d. #Statistically significant compared with infected+treated _21d (P<0.05).

Figure 9. The size of the interstitial space is decreased in mice subjected to a second kidney insult.

Animals were euthanized on day 21 post infection (p.i.), perfused, and the kidneys were collected for histologic analysis. The size if the interstitial space in the renal cortex was visualized using periodic acid−Schiff. Representative photomicrographs of (A) control_21d, (B) control+BSA_21d, (C) control+treated+ BSA_21d, (D) infected+treated_21d, and (E) infected+treated+BSA_21d (n = 6 per group). Bar = 20 µm. The size of the interstitial space is quantified in (E). Values are expressed as a percentage of interstitial space per area (means ± standard error) *Statistically significant compared with control_21d. #Statistically significant compared with infected+treated _21d (P<0.05).