Urolithiasis and hepatotoxicity are linked to the anion transporter Sat1 in mice

Abstract

Urolithiasis, a condition in which stones are present in the urinary system, including the kidneys and bladder, is a poorly understood yet common disorder worldwide that leads to significant health care costs, morbidity, and work loss. Acetaminophen-induced liver damage is a major cause of death in patients with acute liver failure. Kidney and urinary stones and liver toxicity are disturbances linked to alterations in oxalate and sulfate homeostasis, respectively. The sulfate anion transporter-1 (Sat1; also known as Slc26a1) mediates epithelial transport of oxalate and sulfate, and its localization in the kidney, liver, and intestine suggests that it may play a role in oxalate and sulfate homeostasis. To determine the physiological roles of Sat1, we created Sat1-/- mice by gene disruption. These mice exhibited hyperoxaluria with hyperoxalemia, nephrocalcinosis, and calcium oxalate stones in their renal tubules and bladder. Sat1-/- mice also displayed hypersulfaturia, hyposulfatemia, and enhanced acetaminophen-induced liver toxicity. These data suggest that Sat1 regulates both oxalate and sulfate homeostasis and may be critical to the development of calcium oxalate urolithiasis and hepatotoxicity.

Figure 1. Targeted disruption of Sat1.

(A) Sat1 targeting strategy. Exons (boxes 1–4), restriction sites, probes, and primers P1–P5 are shown. Neo^R, neomycin resistance sequence; TK, thymidine kinase sequence. (B) Southern analysis of Hind III– and Not I–digested DNA from Sat1^+/+, Sat1^+/–, and Sat1^–/– mice. Probe A detected 9.9-kb wild-type and 5.2-kb targeted allele fragments. (C) Southern analysis of Bcl I–digested DNA from Sat1^+/+, Sat1^+/–, and Sat1^–/– mice. The neo^R probe detected a single 4.7-kb fragment in Sat1^+/– and Sat1^–/– mice. (D) Forward (P4) and reverse (P5) primers amplified an 8.9-kb product in DNA samples from Sat1^+/– and Sat1^–/– mice. M, molecular mass ladder; Neg, negative control. (E) Primers P1 and P2 amplified a 0.5-kb wild-type fragment; P1 and P3 amplified a 0.4-kb targeted allele fragment. (F) Selective disruption of the Sat1 gene located on the opposite strand within the Idua gene. Sat1 exons (white boxes 1–4), Idua exons (black boxes 1–14), primers P6 and P7, and neomycin resistance sequence are shown.

Figure 2. Analysis of Sat1 protein and mRNA.

(A) Schematic of Sat1 cDNA and the predicted knockout cDNA lacking exon 3. Exons 1–4 (boxes), protein coding sequences (shading) of wild-type (3,821 nucleotides) and Sat1 knockout (3,201 nucleotides) cDNA, and primers P8 and P9 are shown. (B) Northern analysis of RNA from liver (L), kidney (K), and ileum (I) of Sat1^+/+, Sat1^+/–, and Sat1^–/– mice. RNA was hybridized with a ³²P-labeled Sat1 cDNA (top), showing the 3.8-kb transcript, and GAPDH cDNA (bottom). (C) Total RNA from kidney was RT-PCR amplified. A 1,716-bp wild-type product and a 1,096-bp knockout Sat1 cDNA product (top) were amplified using primers P8 and P9. A 983-bp product (bottom) was amplified using GAPDH primers. (D) Total RNA from Sat1^+/+ intestinal segments was RT-PCR amplified. A 1,716-bp product was amplified using primers P8 and P9. A 983-bp product (bottom) was amplified using GAPDH primers. (E) Western analysis of protein from intestinal BLMVs and apical BBMVs. The Sat1 antibody detected a 75.4-kDa protein in Sat1^+/+ BLMVs, but not in Sat1^+/+ BBMVs or in Sat1^–/– mice.

Figure 3. Urolithiasis in Sat1^–/– mice.

(A and B) Representative H&E-stained kidney sections showing infiltration of leukocytes (arrow) around the renal cortical vessels in 100% of Sat1^–/– mice (n = 8), absent in Sat1^+/+ mice. (C–F) Representative Yasue-stained kidney (C and D) and bladder (E and F) sections showing calcium oxalate stones (dark staining) in kidney tubules of 47% Sat1^–/– mice (n = 15) and bladders of 26% Sat1^–/– mice (n = 19), but not in any Sat1^+/+ mice (n = 10). Gross histological analyses of additional tissues in which Sat1 is expressed (liver and brain) showed no structural differences between Sat1^–/– and Sat1^+/+ mice (not shown). Scale bars: 100 μm (A and B); 500 μm (C and D); 2 mm (E and F).

Figure 4. Energy dispersive X-ray spectrometry analysis of kidney stones in Sat1^–/– mice.

(A and B) Representative electron micrograph (A) and energy dispersive X-ray micrograph (B) showing kidney stones (arrowheads in B) in the renal cortex and outer medullary region of Sat1^–/– mice. Higher-magnification view of the boxed region in B is shown in the inset. Scale bar: 1 mm; 100 μm (inset). (C) Elemental profile of kidney stones showing abundance of calcium (11.01% ± 0.03%), oxygen (36.53% ± 0.22%), and carbon (51.46% ± 0.08%), as well as trace levels (≤0.30%) of sodium, magnesium, aluminium, sulfur, chromium, and iron. CPS, counts per second; keV, kiloelectron volts.

Figure 5. Increased hepatotoxicity in Sat1^–/– mice.

(A and B) Serum ALT and liver GSH levels in Sat1^+/+ (white bars) and Sat1^–/– (black bars) mice 0, 2, and 12 hours after administration of 250 mg/kg APAP. Results are mean ± SEM (n = 4–8 per group). *P < 0.05 versus time 0 Sat1^+/+; ^#P < 0.05 versus time 0 Sat1^–/–. (C and D) Representative H&E-stained liver sections of Sat1^+/+ and Sat1^–/– mice (n = 6–7 per group) 12 hours after administration of 250 mg/kg APAP, showing liver necrosis in Sat1^–/– mice. Scale bars: 100 μm.