Metabolic consequences of cystinuria

Abstract

Background: Cystinuria is an inherited disorder of renal amino acid transport that causes recurrent nephrolithiasis and significant morbidity in humans. It has an incidence of 1 in 7000 worldwide making it one of the most common genetic disorders in man. We phenotypically characterized a mouse model of cystinuria type A resultant from knockout of Slc3a1.

Methods: Knockout of Slc3a1 at RNA and protein levels was evaluated using real-time quantitative PCR and immunofluorescence. Slc3a1 knockout mice were placed on normal or breeder chow diets and evaluated for cystine stone formation over time suing x-ray analysis, and the development of kidney injury by measuring injury biomarkers. Kidney injury was also evaluated via histologic analysis. Amino acid levels were measured in the blood of mice using high performance liquid chromatography. Liver glutathione levels were measured using a luminescent-based assay.

Results: We confirmed knockout of Slc3a1 at the RNA level, while Slc7a9 RNA representing the co-transporter was preserved. As expected, we observed bladder stone formation in Slc3a1^-/- mice. Male Slc3a1^-/- mice exhibited lower weights compared to Slc3a1^+/+. Slc3a1^-/- mice on a regular diet demonstrated elevated blood urea nitrogen (BUN) without elevation of serum creatinine. However, placing the knockout animals on a breeder chow diet, containing a higher cystine concentration, resulted in the development of elevation of both BUN and creatinine indicative of more severe chronic kidney disease. Histological examination revealed that these dietary effects resulted in worsened kidney tubular obstruction and interstitial inflammation as well as worsened bladder inflammation. Cystine is a precursor for the antioxidant molecule glutathione, so we evaluated glutathione levels in the livers of Slc3a1^-/- mice. We found significantly lowered levels of both reduced and total glutathione in the knockout animals.

Conclusions: Our results suggest that that diet can affect the development and progression of chronic kidney disease in an animal model of cystinuria, which may have important implications for patients with this disease. Additionally, reduced glutathione may predispose those with cystinuria to injury caused by oxidative stress. Word count: 327.

Keywords: Chronic kidney disease; Cystine; Cystinuria; Kidney stones; Nephrolithiasis.

Fig. 1

Slc3a1 transcripts and rBAT protein expression are lost in male Slc3a1 knockout mice. Slc3a1^−/− mice demonstrate loss of Slc3a1 RNA (a) but retention of Slc7a9 RNA (b) in kidney and small intestine. Bladder and liver are provided as negative controls. Shown are the averages of two independent experiments done in triplicate (mean ± SD). (c) Representative Western blot of rBAT from wild type and Slc3a1^−/− mice. Immunofluorescence was used to evaluate for rBAT expression in wild type (d) and male Slc3a1^−/− mice (e). Knockout animals demonstrate loss of rBAT expression in the proximal tubule

Fig. 2

Male Slc3a1^−/− mice demonstrate differing rates of bladder stone formation dependent upon diet. a X-rays of mice with typical bladder stone formation. b 42% of mice on normal chow had bladder stones by 28 weeks, whereas 100% of mice had bladder stones by 28 weeks on breeder chow (N = 12). c X-ray of Slc3a1^−/− animals revealed the development bladder stone formation on breeder chow as depicted by a Kaplan-Meier plot (N = 8)

Fig. 3

Male Slc3a1 −/− mice exhibited lower weight compared to wild type mice of the same age. a Comparison of weight of 10–12 week old wild type (WT) and Slc3a1^−/− (KO) animals on breeder chow (*, p < 0.05 Mann Whitney test, N = 5–6 ± SD). b Picture of age matched (12 week old) WT (left) and KO (right) male mice. c All mice exhibited the same nose-to-tail length (N = 5–6 ± SD)

Fig. 4

Male Slc3a1 −/− mice have higher blood urea nitrogen (BUN) and creatinine indicative of the development of CKD. a Comparison of BUN in wild type (WT) and Slc3a1^−/− (KO) animals on a regular or breeder chow diet. KO animals exhibited elevated BUN on both regular and breeder chow when compared to WT animals on the same diet (*, p < 0.05 Mann Whitney test, N = 6–19 ± SEM). b Comparison of serum creatinine in WT and KO animals on a regular or breeder chow diet. KO animals exhibited elevated creatinine on the breeder chow diet but not the regular chow diet (*, p < 0.05, Mann Whitney test, N = 6–19 ± SEM). Each dot on the graph represents an individual measurement from an individual mouse

Fig. 5

Histological analysis of wild-type and male Slc3a1^−/− kidneys and bladders at 10–12 months of age. Kidneys of mice with elevated creatinine (b and d) demonstrate more tubular dilatation and fibrosis when compared to mice with a normal creatinine (a and c) whether on regular (a and b) or breeder chow (c and d). Bladders of wild-type (e), knockout with normal creatinine (f), and knockout with elevated creatinine (g) mice are also shown. The knockouts demonstrate inflammatory infiltrate and cystine crystals. Panel (g) demonstrates cystine crystals embedded within the bladder wall (arrows). Shown are representative H&E stains from each group of mice

Fig. 6

Glutathione levels in male Slc3a1^−/− mice at 8 weeks of age. Reduced (GSH) and oxidized (GSSG) glutathione levels were measured in the livers of wild-type and Slc3a1^−/− animals. GSH, GSSG, total and the ratio of reduced to oxidized forms (GSH:GSSG) were all lowered in Slc3a1^−/− mice (N = 6 ± SEM)