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. 2015 Sep;3(9):e12558.
doi: 10.14814/phy2.12558.

TLR4 Mutant Mice Are Protected From Renal Fibrosis and Chronic Kidney Disease Progression

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Free PMC article

TLR4 Mutant Mice Are Protected From Renal Fibrosis and Chronic Kidney Disease Progression

Ana C P Souza et al. Physiol Rep. .
Free PMC article

Abstract

Chronic kidney disease (CKD) is associated with persistent low-grade inflammation and immunosuppression. In this study we tested the role of Toll-like receptor 4, the main receptor for endotoxin (LPS), in a mouse model of renal fibrosis and in a model of progressive CKD that better resembles the human disease. C3HeJ (TLR4 mutant) mice have a missense point mutation in the TLR4 gene, rendering the receptor nonfunctional. In a model of renal fibrosis after folic acid injection, TLR4 mutant mice developed less interstititial fibrosis in comparison to wild-type (WT) mice. Furthermore, 4 weeks after 5/6 nephrectomy with continuous low-dose angiotensin II infusion, C3HeOuJ (TLR4 WT) mice developed progressive CKD with albuminuria, increased serum levels of BUN and creatinine, glomerulosclerosis, and interstitial fibrosis, whereas TLR4 mutant mice were significantly protected from CKD progression. TLR4 WT mice also developed low-grade systemic inflammation, splenocyte apoptosis and increased expression of the immune inhibitory receptor PD-1 in the spleen, which were not observed in TLR4 mutant mice. In vitro, endotoxin (LPS) directly upregulated NLRP3 inflammasome expression in renal epithelial cells via TLR4. In summary, TLR4 contributes to renal fibrosis and CKD progression, at least in part, via inflammasome activation in renal epithelial cells, and may also participate in the dysregulated immune response that is associated with CKD.

Keywords: Albuminuria; endotoxemia; inflammasome; inflammation; spleen apoptosis.

Figures

Figure 1
Figure 1
Effect of TLR4 deficiency on renal fibrosis following folic acid injection. (A) Schematic view of the folic acid model. (B) Survival curves after folic acid injection during the length of study. (C) Relationship between severity of acute kidney injury, as indicated by day 2 BUN (log-transformed), and severity of interstitial fibrosis at day 14. TLR4 mutant mice exhibit protection from more severe kidney fibrosis relative to the TLR4 wild type among complete cases, adjusting for postinjury (day 2) BUN: the mean decrease in fibrosis score was –1.01 ± 0.359; P = 0.014). (D) Masson trichrome staining: TLR4 wild-type (top panel) and TLR4 mutant (bottom panel), comparing mice that were matched (see arrows in panel C) by log-transformed BUN values at day 2.
Figure 2
Figure 2
Effect of TLR4 deficiency on albuminuria following 5/6 nephrectomy with angiotensin II infusion. (A) Schematic view of progressive CKD model: mice were subjected to 2/3 left nephrectomy (Nx) on week –1, after baseline urine collection; a week later (zero) mice were subjected to right Nx and a minipump with or without angiotensin II was implanted subcutaneously. Urine was collected weekly and mice were euthanized after 4 weeks. (B) Albuminuria excretion over time (N = 8–10/group). ACR, albumin-to-creatinine ratio.
Figure 3
Figure 3
Effect of TLR4 deficiency on kidney function in a progressive CKD model. (A) Serum BUN and (B) serum creatinine at 4 weeks: Control (mice not subjected to any procedure), 5/6Nx alone, and 5/6Nx with angiotensin II infusion (5/6Nx+AngII) (N = 6–10/group).
Figure 4
Figure 4
Effect of TLR4 deficiency on renal histological scores in a progressive CKD model. Glomerulosclerosis (PAS stain, A) and interstitial fibrosis (Masson Trichrome stain, B) scores 4 weeks after 5/6Nx+AngII (N = 6–10/group).
Figure 5
Figure 5
Effect of TLR4 deficiency on splenocyte apoptosis, splenic PD-1 expression, and serum HMGB-1. (A) Spleen apoptosis measured by caspase-3 positive staining (N = 6–8/group). (B) Splenic PD-1 cell staining (N = 4/group). (C) Serum HMGB-1 levels (N = 7–8/group) in mice 4 weeks after 5/6Nx+AngII.
Figure 6
Figure 6
Effect of TLR4 deficiency on systemic inflammation in a progressive CKD model. (A) Serum IL-6 and (B) serum TNF-α levels expressed as fold changes over nonprogressive CKD (5/6Nx alone) model (N = 7/group).
Figure 7
Figure 7
Effect of TLR4 deficiency on primary renal epithelial cell culture. TLR4 WT- and mutant-derived renal epithelial cells were challenged with different concentrations of LPS for 20 h. IL-6 levels in the supernatant (A), and cellular mRNA expression of IL-6 (B), NLRP3 (C) and IL-1β (D).

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