Hydroxyproline metabolism in mouse models of primary hyperoxaluria

Abstract

Primary hyperoxaluria type 1 (PH1) and type 2 (PH2) are rare genetic diseases that result from deficiencies in glyoxylate metabolism. The increased oxalate synthesis that occurs can lead to kidney stone formation, deposition of calcium oxalate in the kidney and other tissues, and renal failure. Hydroxyproline (Hyp) catabolism, which occurs mainly in the liver and kidney, is a prominent source of glyoxylate and could account for a significant portion of the oxalate produced in PH. To determine the sensitivity of mouse models of PH1 and PH2 to Hyp-derived oxalate, animals were fed diets containing 1% Hyp. Urinary excretions of glycolate and oxalate were used to monitor Hyp catabolism and the kidneys were examined to assess pathological changes. Both strains of knockout (KO) mice excreted more oxalate than wild-type (WT) animals with Hyp feeding. After 4 wk of Hyp feeding, all mice deficient in glyoxylate reductase/hydroxypyruvate reductase (GRHPR KO) developed severe nephrocalcinosis in contrast to animals deficient in alanine-glyoxylate aminotransferase (AGXT KO) where nephrocalcinosis was milder and with a lower frequency. Plasma cystatin C measurements over 4-wk Hyp feeding indicated no significant loss of renal function in WT and AGXT KO animals, and significant and severe loss of renal function in GRHPR KO animals after 2 and 4 wk, respectively. These data suggest that GRHPR activity may be vital in the kidney for limiting the conversion of Hyp-derived glyoxylate to oxalate. As Hyp catabolism may make a major contribution to the oxalate produced in PH patients, Hyp feeding in these mouse models should be useful in understanding the mechanisms associated with calcium oxalate deposition in the kidney.

Fig. 1.

Western blot of mouse tissues incubated with polyclonal rabbit antibodies against mouse glyoxylate reductase and hydroxypyruvate reductase (GRHPR) and mouse GAPDH. WT, wild-type; KO, knockout.

Fig. 2.

Plasma hydroxyproline (Hyp) levels with and without 1% Hyp feeding for 7 days in WT (open bar), AGXTKO (hatched bar), and GRHPR KO (filled bar) mice (n = 6 per group). Data are expressed as means ± SD.

Fig. 3.

Urinary oxalate 24-h excretion with and without 1% Hyp feeding for 7 days in WT (open bar), AGXT KO (hatched bar), and GRHPR KO (filled bar) mice (n = 6 per group). Data are expressed as means ± SD.

Fig. 4.

Urinary glycolate 24-h excretion with and without 1% Hyp feeding for 7 days in WT (open bar), AGXT KO (hatched bar), and GRHPR KO (filled bar) mice (n = 6 per group). Data are expressed as means ± SD.

Fig. 5.

Representative histology after Yasue staining in a GRHPR KO mouse after 4 wk of 1% Hyp feeding. Black deposits represent calcium crystal deposits. The bulk of calcium crystal deposits was found intraluminally, with some smaller interstitial deposits as well.

Fig. 6.

Renal oxalate levels in AGTKO, GRHPR KO, and WT mice after feeding with 1% Hyp for 1 wk (open bar) or 4 wk (filled bar). Data expressed as means ± SE, n = 6.

Fig. 7.

Plasma cystatin C levels over time with 1% Hyp feeding in WT (open bar), AGXT KO (hatched bar), and GRHPR KO (filled bar) mice (n = 5 per group). Data expressed as means ± SE. *Significant increase (P ≤ 0.05) compared with 0 wk.