Pivotal Role of Toll-Like Receptors 2 and 4, Its Adaptor Molecule MyD88, and Inflammasome Complex in Experimental Tubule-Interstitial Nephritis

Tubule-interstitial nephritis (TIN) results in decreased renal function and interstitial inflammation, which ultimately leads to fibrosis. Excessive adenine intake can cause TIN because xanthine dehydrogenase (XDH) can convert this purine into an insoluble compound, which precipitates in the tubuli. Innate immune sensors, such as Toll-like receptors (TLR) and inflammasome complex, play a crucial role in the initiation of inflammation. The aim of this study was to evaluate the roles of TLR-2 and -4, Myd88 and inflammasome complex in an experimental model of TIN. Here, we show that wild-type (WT) mice fed adenine-enriched food exhibited significant renal dysfunction and enhanced cellular infiltration accompanied by collagen deposition. They also presented higher gene and protein expression of pro-inflammatory cytokines. In contrast, TLR-2, -4, MyD88, ASC and Caspase-1 KO mice showed renoprotection associated with expression of inflammatory molecules at levels comparable to controls. Furthermore, treatment of WT animals with allopurinol, an XDH inhibitor, led to reduced levels of uric acid, oxidative stress, collagen deposition and a downregulation of the NF-kB signaling pathway. We concluded that MyD88 signaling and inflammasome participate in the development of TIN. Furthermore, inhibition of XDH seems to be a promising way to therapeutically target the developing inflammatory process

Oxidative Stress and Modification of Renal Vascular Permeability Are Associated with Acute Kidney Injury during P. berghei ANKA Infection

Malaria associated-acute kidney injury (AKI) is associated with 45% of mortality in adult patients hospitalized with severe form of the disease. However, the causes that lead to a framework of malaria-associated AKI are still poorly characterized. Some clinical studies speculate that oxidative stress products, a characteristic of Plasmodium infection, as well as proinflammatory response induced by the parasite are involved in its pathophysiology. Therefore, we aimed to investigate the development of malaria-associated AKI during infection by P. berghei ANKA, with special attention to the role played by the inflammatory response and the involvement of oxidative stress. For that, we took advantage of an experimental model of severe malaria that showed significant changes in the renal pathophysiology to investigate the role of malaria infection in the renal microvascular permeability and tissue injury. Therefore, BALB/c mice were infected with P. berghei ANKA. To assess renal function, creatinine, blood urea nitrogen, and ratio of proteinuria and creatininuria were evaluated. The products of oxidative stress, as well as cytokine profile were quantified in plasma and renal tissue. The change of renal microvascular permeability, tissue hypoxia and cellular apoptosis were also evaluated. Parasite infection resulted in renal dysfunction. Furthermore, we observed increased expression of adhesion molecule, proinflammatory cytokines and products of oxidative stress, associated with a decrease mRNA expression of HO-1 in kidney tissue of infected mice. The measurement of lipoprotein oxidizability also showed a significant increase in plasma of infected animals. Together, our findings support the idea that products of oxidative stress, as well as the immune response against the parasite are crucial to changes in kidney architecture and microvascular endothelial permeability of BALB/c mice infected with P. berghei ANKA.State of Sao Paulo Foundation for Research Support (FAPESP)State of Sao Paulo Foundation for Research Support (FAPESP) [07/07139-3, 10/52180-4, 12/02270-2]CAPESCAPESBrazilian Council of Scientific and Technologic Development (International Associated Laboratory of Renal Immunopathology, CNPq/Inserm)Brazilian Council of Scientific and Technologic Development (International Associated Laboratory of Renal Immunopathology, CNPq/Inserm)Complex Fluids INCT (FAPESP/CNPq)Complex Fluids INCT (FAPESP/CNPq

Analysis of sequestration of infected red blood cell in renal tissue.

<p>(A) Representative figure of presence of malaria pigment hemozoin in renal tissue sections visualized by hematoxylin and eosin, or under polarized light. Detection of hemozoin at glomeruli (<b>i</b>) and vascular endothelium (<b>ii</b>). (B) Hemozoin quantification in histological section of renal tissue. (C) <i>P. berghei</i> ANKA mRNA quantified by qPCR in renal tissue of BALB/c infected mice. Each graph represents the mean of 5–10 animals per group ± standard deviation. One-way ANOVA with Bonferroni post-test was performed using GraphPad Prism. *** p<0.001 vs. the control group – day 0, # p<0.01 vs. day 12.</p

<i>Ex vivo</i> adherence of <i>P. berghei</i> ANKA^GFP iRBC to renal tissue.

<p>(<b>A</b>) Representative microscopic image of the <i>ex vivo</i> adherence assays showing iRBC adhering to renal tissue sections from control (right) and infected (left) mice (200X magnification). (<b>B</b>) Adhesion of iRBC treated or not with proteinase K prior incubation with the frozen kidney sections. All data represent the number of bound iRBC per area. (mean±s.e.m). Two-way ANOVA with Bonferroni post-test was performed using GraphPad Prism. *P<0.05; **P<0.01. (n.d.: not-detected).</p

Impairment of renal function during <i>P. berghei</i> ANKA malaria infection.

<p>(A) Parasitemia, and (B) survival of BALB/c mice infected with 10⁶ parasitized erythrocytes by <i>P. berghei</i> ANKA. Renal function was assessed by (C) plasma creatinine, (D) blood urea nitrogen (BUN) and (E) quantification of erythrocyte protoporphyrin estimated on different days after infection. Results represent the mean of 5–10 animals per group ± standard deviation. One-way ANOVA with Bonferroni post-test was performed to renal assessment using GraphPad Prism. * P<0.05 vs the control group – day 0, ** p<0.01 vs the control group – 0 days.</p

Assessment of products of oxidative stress during malaria-associated AKI.

<p>(A) Plasmatic quantification of toxic heme (B), detection of conjugated dienes by Cooper and (C) quantification of plasma levels of oxidized low density lipoprotein in BALB/c mice infected with 10⁶ parasitized erythrocytes by <i>P. berghei</i> ANKA. (D) mRNA expression of LOX-1 in renal tissue during infection in BALB/c mice. The results represent the average of 5–10 animals per group ± standard deviation. One-way ANOVA with Bonferroni post-test was performed using GraphPad Prism. * P<0.05 vs the control group – day 0, ** p<0.01 vs the control group – 0 days, *** p <0.001 vs the control group – 0 days.</p

Evaluation of hypoxia, HIF-1α in renal tissue and apoptosis during malaria-associated AKI.

<p>(A) Representative immunohistochemistry, and (B) quantification of renal hypoxia in control and <i>P. berghei</i> ANKA infected mice. (C) mRNA expression of HIF-1α in renal tissue. (D) Evaluation of apoptosis in kidney section of control and <i>P. berghei</i> ANKA infected mice. Each graph represents the mean of 5–10 animals per group ± standard deviation. One-way ANOVA with Bonferroni post-test was performed using GraphPad Prism. * P<0.05 vs control group – day 0, ** p<0.01 vs control group – day 0.</p

<i>P. berghei</i> ANKA malaria infection induces endothelium injury and changes in renal architecture.

<p>(A) Renal microvascular permeability change assessed by Evans blue dye and (B) representative pathophysiology of renal tissue stained with hematoxylin-eosin (HE) and examined in light microscopy (Leica DM LB2, Leica Microsystems). Each graph represents the mean of 5–10 animals per group ± standard deviation. One-way ANOVA with Bonferroni post-test was performed using GraphPad Prism. * P<0.05 vs. the control group – day 0, ** p<0.01 vs. the control group – day 0, *** p<0.001 vs. the control group – day 0.</p

Effect of <i>P. berghei</i> ANKA malaria infection in renal pro-inflammatory response.

<p>mRNA expression of (A) IFN-γ, (B) ICAM-1 and (C) iNOS in renal tissue of BALB/c mice infected with 10⁶ parasitized erythrocytes by <i>P. berghei</i> ANKA. Renal tissue protein expression of (D) TNF-α, (E) IL-1β and (F) IL-6. (G) Representative image and graphic quantification of bands expressed of an IKK western blot. The graphs of A to F represent the average of 3–5 animals per group ± standard deviation. One-way ANOVA with Bonferroni post-test was performed to mRNA expression and quantification of IKK using GraphPad Prism. Unpaired Student-t test was performed to renal tissue protein expression using GraphPad Prism. * P<0.05 vs the control group – day 0, ** p<0.01 vs the control group – 0 days.</p