Nlrp3 prevents early renal interstitial edema and vascular permeability in unilateral ureteral obstruction

Abstract

Progressive renal disease is characterized by tubulo-interstitial injury with ongoing inflammation and fibrosis. The Nlrp3 inflammasome contributes to these pathophysiological processes through its canonical effects in cytokine maturation. Nlrp3 may additionally exert inflammasome-independent effects following tissue injury. Hence, in this study we investigated potential non-canonical effects of Nlrp3 following progressive renal injury by subjecting WT and Nlrp3-deficient (-/-) mice to unilateral ureter obstruction (UUO). Our results revealed a progressive increase of renal Nlrp3 mRNA in WT mice following UUO. The absence of Nlrp3 resulted in enhanced tubular injury and dilatation and an elevated expression of injury biomarker NGAL after UUO. Moreover, interstitial edema was significantly elevated in Nlrp3-/- mice. This could be explained by increased intratubular pressure and an enhanced tubular and vascular permeability. In accordance, renal vascular leakage was elevated in Nlrp3-/- mice that associated with reduced mRNA expression of intercellular junction components. The decreased epithelial barrier function in Nlrp3-/- mice was not associated with increased apoptosis and/or proliferation of renal epithelial cells. Nlrp3 deficiency did not affect renal fibrosis or inflammation. Together, our data reveal a novel non-canonical effect of Nlrp3 in preserving renal integrity and protection against early tubular injury and interstitial edema following progressive renal injury.

Figure 1. Progressive renal injury enhances renal Nlrp3, but not ASC mRNA expression.

Relative expression levels of Nlrp3 (A) and ASC (B) mRNA in total kidney specimen following progressive renal injury. Nlrp3 mRNA expression was significantly enhanced 1, 3, 7 and 14 days post-UUO compared with contralateral kidneys, whereas ASC mRNA remained constitutively expressed. Data are mean±SEM of 6 mice per group. *:p<0.05.

Figure 2. Nlrp3 deficiency increases renal injury following UUO.

(A) Semi-quantitative tubular injury score in wild type (white bars) and Nlrp3−/− (black bars) mice following UUO. Nlrp3−/− mice developed significantly more tubular injury when compared to their respective wild type mice 1, 3, and 7 days post-UUO. (B) Representative PASD microphotographs of renal tissue subjected to 1 day of UUO, with apparent interstitial edema in the Nlrp3−/− kidneys (magnification x200). (C) Nlrp3−/− mice displayed enhanced mRNA expression of the renal injury biomarker NGAL compared to their wild type mice following UUO. Data are mean±SEM of 7 mice per group. Contral.  =  contralateral kidneys of mice subjected to 14 days of UUO. *:p<0.05.

Figure 3. Nlrp3 preserves early expression of renal epithelial- and endothelial-associated Claudins following UUO.

Renal mRNA expression of epithelium-associated Claudin-1 (A) and Claudin-2 (B) and vascular endothelium-associated Claudin-5 (C) and VE-Cadherin (D) was reduced in obstructed kidneys of Nlrp3−/− mice (black bars) when compared to wild type mice (white bars) 1 day post-UUO. Whereas VE-Cadherin remained lower, Nlrp3−/− mice displayed a subtle increase in Claudin-1 and -2 expression 14 days post-UUO compared to wild type mice. Contral.  =  contralateral kidneys of mice subjected to 14 days of UUO. Data are mean±SEM of 6 mice per group. *:p<0.05.

Figure 4. Nlrp3 deficiency elevates vascular leakage in the ligated and non-ligated kidney following UUO.

(A) The accumulation of interstitial FITC-Dextran was significantly elevated in both obstructed (UUO) and contralateral (Ctrl) kidneys of Nlrp3−/− mice compared to wild type mice 1 day post-UUO. (B) Representative microphotographs of renal tissue subjected to 1 day UUO, demonstrating elevated interstitial FITC-Dextran accumulation in Nlrp3−/− obstructed kidneys (magnification 200x). Data are mean±SEM of 4 mice per group. *:p<0.05.

Figure 5. Nlrp3 deficiency mildly reduces number of apoptotic but not proliferating tubular epithelial cells following UUO.

The number of (A) apoptotic (active caspase 3⁺) TECs was mild but significantly lower in contralateral and 3-day obstructed kidneys of Nlrp3−/− (black bars) mice compared to wild type mice (white bars), whereas at other time points no differences were observed. (B) The number of proliferating TECs was comparable between wild type and Nlrp3−/− mice at all time points. Data are mean±SEM of 7 mice per group. Contral.  =  contralateral kidneys of mice subjected to 14 days of UUO. *:p<0.05. The panels show representative microphotographs of renal tissue sections, magnification x200.

Figure 6. Nlrp3 deficiency reduces early renal IL1-β levels but does not affect macrophage accumulation following UUO.

(A) Macrophages progressively accumulated in obstructed kidneys upon UUO and were comparable between wild type (white bars) and Nlrp3−/− (black bars) mice following UUO. (B) Representative microphotographs of macrophage accumulation in renal tissue sections of mice subjected to 7 days of UUO, magnification x200. (C) Nlrp3−/− mice displayed less renal IL1-β in their contralateral and obstructed kidneys after 1 day of UUO, whereas levels were comparable to wild type mice at later time points. Data are mean±SEM of 7 mice per group and indicated as pg IL1-β per mg protein. Contral.  =  contralateral kidneys of mice subjected to 14 days of UUO. *:p<0.05.

Figure 7. Nlrp3 deficiency does not affect myofibroblast accumulation and collagen deposition following UUO.

Renal myofibroblast accumulation (A) and collagen deposition (B) in obstructed and contralateral kidneys of wild type (white bars) and Nlrp3−/− (black bars) mice following UUO. Nlrp3 deficiency did not affect the accumulation of myofibroblasts in kidneys following UUO (A). Except for a difference observed at 3 days post-UUO Nlrp3 deficiency did not influence renal collagen deposition following UUO (B). Data are mean±SEM of 7 mice per group. Contral.  =  contralateral kidneys of mice subjected to 14 days of UUO. *:p<0.05. The panels show representative microphotographs of renal tissue of mice subjected to 14 days of UUO, magnification x200.