Effect of <i>T. cruzi</i> parasite loads on cytokine and nitric oxide production in kidney tissues.

<p>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.</p

Effect of <i>T. cruzi</i> parasite loads on vascular permeability in the kidney tissue.

<p>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.</p

Analysis of the presence of <i>T.cruzi</i> amastigotes and inflammatory infiltrates in the renal tissues.

<p>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 <i>T. cruzi</i> amastigotes in the renal tissues were evaluated. <i>T. cruzi</i> 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 <i>T. cruzi</i> amastigotes and the inflammatory infiltrates, we evaluated the comparative percentage of positive antigen labeling for <i>T. cruzi</i> in 5 different slides collected from the different inocula at 9 and 18 days post-infection (E).</p

Parasitemia and survival of mice in the acute stage of <i>T. cruzi</i> infection.

<p>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.</p

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

<p>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.</p