Influence of parasite load on renal function in mice acutely infected with Trypanosoma cruzi

Abstract

Background: Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi. Despite the vast number of studies evaluating the pathophysiological mechanisms of the disease, the influence of parasite burden on kidney lesions remains unclear. Thus, the main goal of this work was to evaluate the effect of T. cruzi infection on renal function and determine whether there was a correlation between parasite load and renal injury using an acute experimental model of the disease.

Methodology/principal findings: Low, medium and high parasite loads were generated by infecting C57BL/6 mice with 300 (low), 3,000 (medium) or 30,000 (high) numbers of "Y" strain trypomastigotes. We found that mice infected with T. cruzi trypomastigotes show increased renal injury. The infection resulted in reduced urinary excretion and creatinine clearance. We also observed a marked elevation in the ratio of urine volume to kidney and body weight, blood urea nitrogen, chloride ion, nitric oxide, pro- and anti-inflammatory cytokines and the number of leukocytes in the blood and/or renal tissues of infected mice. Additionally, we observed the presence of the parasite in the cortical/medullary and peri-renal region, an increase of inflammatory infiltrate and of vascular permeability of the kidney. Overall, most renal changes occurred mainly in animals infected with high parasitic loads.

Conclusions/significance: These data demonstrate that T. cruzi impairs kidney function, and this impairment is more evident in mice infected with high parasitic loads. Moreover, these data suggest that, in addition to the extensively studied cardiovascular effects, renal injury should be regarded as an important indicator for better understanding the pan-infectivity of the parasite and consequently for understanding the disease in experimental models.

Figure 1. Parasitemia and survival of mice in the acute stage of T. cruzi infection.

C57BL/6 mice were challenged with 3×10² (low dose), 3×10³ (medium dose) or 3×10⁴ (high dose) blood trypomastigotes. Parasitemia (A) was determined by counting the number of parasites in 5 µL of blood collected from tail snips at the indicated time points. Each point represents the mean of individual values from 10 mice. In the survival curve (B), 10 animals were individually monitored for 30 days of infection. ^δ0p≤0.05 indicates a significant difference when the mice infected with medium-inoculum were compared to the mice infected with high inoculum, ^δ1p≤0.05 indicates a significant difference when the mice from the low-inoculum group were compared to the mice from the high-inoculum group, ^δ2p≤0.05 indicates a significant difference when mice from the low-inoculum group were compared to mice from the medium-inoculum group, and *p≤0.05 indicates a significant difference when animals from the infected groups were compared to the uninfected control mice.

Figure 2. Determination of the urine excretion (24 hours) and the index between the kidney and body weight.

The index between the kidney and body weight (A–D), urine excretion (E–H) and the correlation between the index and urine excretion (I–L) were evaluated as indicators of renal lesions. The bodies and kidneys of infected and uninfected mice were weighed at the indicated time points (6, 9, 12 and 18 days p.i.) to calculate the index. At the same time points, the animals were placed in metabolic cages for 24 hours to quantify the urine volume. *p≤0.05 indicates a significant difference between the animals that received a high inoculum and the uninfected animals. ^δp≤0.05 indicates a significant difference between the animals that received a high inoculum and the animals that received the low inoculum.

Figure 3. Effect of T. cruzi parasite loads on plasma urea concentration, BUN/creatinine ratio, creatinine clearance and plasma chloride ion levels.

C57BL/6 mice were challenged with 3×10² (low dose), 3×10³ (medium dose) or 3×10⁴ (high dose) blood trypomastigotes, and 6, 9, 12 and 18 days post-infection, the plasma and urine (24 hours) of these animals were collected. The plasma urea (A–D) and creatinine levels were measured, and the ratios between blood urea nitrogen (BUN) and creatinine (E–H) were calculated. To determine the creatinine clearance, the urine creatinine levels were measured over a 24-hour period (I–L). The concentration of chloride ions (mEq/L) was measured in the plasma from the same mice (M–P). We used commercial kits for these analyses, as described in Materials and Methods. Each bar represents the mean ± standard deviation of individual values from 10 mice. *p≤0.05 indicates a significant difference when animals from the highly infected group were compared to the uninfected control animals.

Figure 4. Analysis of the presence of T.cruzi amastigotes and inflammatory infiltrates in the renal tissues.

C57BL/6 mice were challenged with low, medium and high loads of trypomastigotes, and at 9 and 18 days post-infection, the inflammatory infiltrate and the presence and location of T. cruzi amastigotes in the renal tissues were evaluated. T. cruzi amastigotes were found in both cortical/medullary (A) and peri-renal (B) tissues. The inflammatory infiltrate was evidenced in the tubular region (C) and in the Bowman’s capsule (D). After demonstrating the presence of nests of T. cruzi amastigotes and the inflammatory infiltrates, we evaluated the comparative percentage of positive antigen labeling for T. cruzi in 5 different slides collected from the different inocula at 9 and 18 days post-infection (E).

Figure 5. Increased circulating cells in mice infected with T. cruzi.

C57BL/6 mice were infected with increasing doses of trypomastigotes, and at 6, 9, 12 and 18 days post-infection the number of cells/mm³ in the blood was determined. At each time point, the total leukocytes (A), neutrophils (B), lymphocytes (C), and monocytes (D) were measured. Total cells were counted using a Neubauer chamber, and the differential cell counts (100 cells total) were obtained using stained blood smear slides. The data are reported as the means ± SEM of 10 mice. *p<0.05 versus the uninfected group.

Figure 6. Effect of T. cruzi parasite loads on cytokine and nitric oxide production in kidney tissues.

C57BL/6 mice were challenged with low, medium and high loads of blood trypomastigotes. At 6, 9, 12 and 18 days post-infection they were euthanized and their kidneys were removed to measure the concentrations of cytokines and nitric oxide. The cytokines TNF-α (A–D), IFN-γ (E–H) and IL-10 (I–L) were measured according to the manufacturer’s instructions, using commercially available ELISA kits. For measurement of nitric oxide, the Griess reaction was used. The absorbance was read at 570 nm. *p≤0.05 indicates a significant difference when animals from the medium and highly infected groups were compared to the uninfected control mice.

Figure 7. Effect of T. cruzi parasite loads on vascular permeability in the kidney tissue.

C57BL/6 mice were challenged with low, medium and high loads of trypomastigotes and at 9 day post-infection, the accumulation of Evans Blue in the renal tissues was assessed. In A–D, a representative image of Evans Blue accumulation in the kidney from each group is demonstrated. E shows the mean percentage ± SEM of Evans Blue accumulation in the renal parenchyma. *p≤0.05 indicates a significant difference when mice from the medium and highly infected groups were compared to the uninfected control mice.