Mouse model for Lowe syndrome/Dent Disease 2 renal tubulopathy

Abstract

The Lowe oculocerebrorenal syndrome is an X-linked disorder characterized by congenital cataracts, cognitive disability, and proximal tubular dysfunction. Both this syndrome and Dent Disease 2 result from loss-of-function mutations in the OCRL gene, which encodes a type II phosphatidylinositol bisphosphate 5-phosphatase. Ocrl-deficient mice are unaffected, however, which we believe reflects a difference in how humans and mice cope with the enzyme deficiency. Inpp5b and INPP5B, paralogous autosomal genes that encode another type II phosphoinositide 5-phosphatase in mice and humans, respectively, might explain the distinct phenotype in the two species because they are the closest paralogs to Ocrl and OCRL in their respective genomes yet differ between the two species with regard to expression and splicing. Here, we generated Ocrl(-/-) mice that express INPP5B but not Inpp5b. Similar to the human syndromes, all showed reduced postnatal growth, low molecular weight proteinuria, and aminoaciduria. Thus, we created an animal model for OCRL and Dent Disease 2 tubulopathy by humanizing a modifier paralog in mice already carrying the mutant disease gene.

Figure 1.

Early growth retardation in 1H mice. Mouse weights (mean + SEM) for 1H (n = 14), 2H (n = 10), and 1M (n = 10) mice established from transgenic line C. Mice were weighed weekly from 4 to 16 weeks. Growth curves were fitted empirically with cubic equations centered at 10 weeks and the null hypothesis that the curve for 1M could be described with the same four coefficients as either 1H or 2H could be rejected at P < 0.0001, whereas the null hypothesis that 1H and 2H could be fitted with a curve with the same four coefficients could not be rejected (P = 0.1335). 2M mice were published previously and had no growth deficit compared with wild-type mice.

Figure 2.

Low molecular weight proteinuria. Coomassie stain of urine from 2-month-old mice with genotypes as follows: Lane 1, male Inpp5b^+/−;Ocrl^+/y;BAC⁺. Lanes 2 and 5, female Inpp5b^−/−;Ocrl^+/+;BAC⁻. Lane 3, male 1M. Lane 4, male 2H. Lanes 6 and 7, 1H. Lane 8, FVB wild-type mouse. Solid line rectangle outlines the LMW proteinuria in two independent 1H mice, whereas the dotted line rectangle indicates the absence of LMW proteinuria in a 1M and a 2H mouse. Equal amounts of total urine protein were loaded per lane.

Figure 3.

High urinary levels of two proteins characteristic of low molecular weight proteinuria. (A) Western blot analysis of urine with anti-secretoglobin (CC16) from four mice of each genotype: 1H mice (lanes 1, 2, 9, and 10), 2H mice (lanes 3, 4, 11, and 12), 1M mice (lanes 5, 6, 13, and 14), and 2M mice (lanes 7, 8, 15, and 16). Also shown is the signal from the Mup1 protein due to nonspecific antibody binding caused by the massive amount of Mup1 normally present in the urine of male mice. (B) Western blot analysis of urine with anti–vitamin D binding protein of the same urine samples, loaded in the same order, as was used in (A).

Figure 4.

Aminoaciduria. Amino acid concentrations (mM) relative to creatinine (mM) from 4-month-old 2M (n = 8), 1M (n = 6), 2H (n = 5), and 1H (n = 10) mice. (A) Amino acids showing highly significant differences (P < 0.001) between 2M and 1H mice and a strong correlation of aminoaciduria versus genotype (increasing from 2M to 1M to 2H to 1H). (B) Amino acids showing significant differences (P < 0.05) between 2M and 1H mice and a strong correlation of aminoaciduria versus genotype. (C) Amino acids showing little or no significant differences between 2M and 1H mice but with 7 of 10 demonstrating significant (P < 0.05) correlation of aminoaciduria versus genotype.