NALP3-mediated inflammation is a principal cause of progressive renal failure in oxalate nephropathy

Abstract

Oxalate nephropathy with renal failure is caused by multiple disorders leading to hyperoxaluria due to either overproduction of oxalate (primary hyperoxaluria) or excessive absorption of dietary oxalate (enteric hyperoxaluria). To study the etiology of renal failure in crystal-induced kidney disease, we created a model of progressive oxalate nephropathy by feeding mice a diet high in soluble oxalate (high oxalate in the absence of dietary calcium). Renal histology was characterized by intratubular calcium-oxalate crystal deposition with an inflammatory response in the surrounding interstitium. Oxalate nephropathy was not found in mice fed a high oxalate diet that also contained calcium. NALP3, also known as cryopyrin, has been implicated in crystal-associated diseases such as gout and silicosis. Mice fed the diet high in soluble oxalate demonstrated increased NALP3 expression in the kidney. Nalp3-null mice were completely protected from the progressive renal failure and death that occurred in wild-type mice fed the diet high in soluble oxalate. NALP3 deficiency did not affect oxalate homeostasis, thereby excluding differences in intestinal oxalate handling to explain the observed phenotype. Thus, progressive renal failure in oxalate nephropathy results primarily from NALP3-mediated inflammation.

Figures

Figure 1. Wild-type mice fed a high oxalate diet in the absence of calcium develop renal failure

(a) BUN and (b) creatinine were measured in wild-type mice placed for 14 days on a diet high in soluble oxalate (50 μmoles sodium oxalate added per gram of virtually calcium-free diet) or fed the same amount of oxalate in a calcium-containing diet (50 μmoles sodium oxalate added per gram of diet containing 150 μmoles/g calcium carbonate). Data expressed as the means ± SEM; n=3 per group; p< 0.05.

Figure 2. Wild-type mice fed a diet high in soluble oxalate develop oxalate nephropathy and inflammation

(a) Polarization microscopy of whole mouse kidneys of mice fed a high oxalate diet for 14 days in the absence of calcium demonstrates crystal deposition in the cortex and cortico-medullary junction while (b) animals placed on a high oxalate diet in the presence of calcium show no crystal deposition. (c) Hematoxylin and eosin (HE) staining reveals crystals (arrow) lining the lumen of dilated and flattened tubules with surrounding inflammation noted by the presence of mononuclear cells in mice fed an oxalate diet in the absence of dietary calcium. 200x. (d) Normal renal parenchyma in mice fed a high oxalate diet in the presence of calcium. 200x. (e) F4/80 staining for macrophages/monocytes is positive (dark color) in mice fed a high oxalate diet in the absence of calcium. 200x. (f) F4/80 staining is negative in mice fed an oxalate diet in the presence of calcium. 200x. n=3 per group, representative images are shown.

Figure 3. Nalp3 and IL-1β mRNA levels are increased in kidneys of mice fed a high oxalate diet

(a) Nalp3 and (b) IL-1β mRNA was measured in wild-type mice maintained on a control diet (no oxalate) versus high soluble oxalate diet (50 μmoles sodium oxalate added per gram) for 6 days. Data expressed as the means ± SEM; n=3 for control diet, n=4 for oxalate diet; p< 0.05.

Figure 4. Nalp3-deficiency or dietary calcium prevents progressive oxalate nephropathy

(a) BUN and (b) creatinine were measured in age- and gender-matched wild-type and Nalp3-null mice on an oxalate and calcium free diet to determine baseline BUN and creatinine. Animals were then switched to a diet high in soluble oxalate. In addition, a separate group of wild-type mice were fed a high oxalate diet with calcium. Data expressed as means ± SEM; n=12 (6 male/6 female) Nalp3-null and wild-type mice fed the diet high in soluble oxalate; n=6 (3 male/3 female) wild-type mice fed high oxalate with calcium. There was no statistically significant difference at baseline for BUN and creatinine (p=0.70). Mean serum BUN was significantly higher in the wild-type mice fed a high oxalate diet than either other group at days 12 and 18, and for creatinine at days 6, 12 and 18 via Duncan tests (p-values <0.0001 by ANOVA).

Figure 5. Nalp3-deficiency does not affect oxalate homeostasis

(a) Urinary oxalate/creatinine ratio and (b) Plasma oxalate increased about 15-fold and 30-fold respectively from baseline following a switch from an oxalate-free to a diet high in soluble oxalate. Urinary oxalate excretion was identical in age and gender-matched wild-type and Nalp3-null mice. Data expressed as means ± SEM; n= 2 at baseline, n=4 at day 1, n=5 at days 3 and 6; p>0.05 each time point.

Figure 6. Nalp3-deficiency does not affect initial renal crystal deposition

(a) Polarization microscopy of whole mouse kidneys of wild-type mice fed a high oxalate diet in the absence of calcium demonstrated equal amounts of crystal deposition compared with (b)Nalp3-null mice at 3 days. (c) Crystal deposition was increased in wild-type mice compared with (d)Nalp3-null mice after 6 days. (e) Crystal quantification expressed per kidney surface area in wild-type and Nalp3-null mice. Data expressed as the means ± SEM; n=3 male mice per group; p< 0.05. Representative images are shown.

Figure 7. Nalp3-deficiency reduces but does not eliminate renal inflammation

(a) F4/80 staining of whole mouse kidneys (dark staining as indicated by white arrows) of wild-type mice fed a high oxalate diet in the absence of calcium demonstrated increased F4/80 staining in wild-type as compared with (b)Nalp3-null mice at 6 days. Higher magnification (200x) confirmed more abundant F4/80 staining for macrophages/monocytes in wild-type mice fed a high oxalate diet in the absence of calcium (c) as compared with (d)Nalp3-null mice at 6 days (e) Inflammation quantification expressed per kidney surface area in wild-type and Nalp3-null mice. Data expressed as the means ± SEM; n=4 male mice per group; p< 0.05. Representative images are shown.

Figure 8. Nalp3-deficiency protects from oxalate diet induced mortality

(a) Wild-type mice placed on a diet high in soluble oxalate demonstrated 100% mortality at 30 days. In contrast, Nalp3-deficiency or the addition of dietary calcium results in 100% survival at 30 days. Data expressed as Kaplan-Meier Survival Curve; n=12 (6 male/6 female) Nalp3-null and wild-type mice fed the diet high in soluble oxalate; n=6 (3 male/3 female) wild-type mice fed high oxalate with calcium (P < 0.0001 for WT vs. NALP3 −/− and WT with added dietary calcium). (b) Dietary calcium protects from hyperoxalemia in wild-type mice while Nalp3-null mice fed a diet high in soluble oxalate had persistent hyperoxalemia at 30 days. (c) BUN and (d) creatinine was compared between wild-type mice placed on a high oxalate diet in the presence of dietary calcium and Nalp3-null mice fed a diet high in soluble oxalate for 30 days. Data b–d expressed as the means ± SEM; n=6 (3 male/3 female) for wild-type mice with added dietary calcium and n=12 (6 male/6 female) for Nalp3-null mice fed a diet high in soluble oxalate; p < 0.05.