Generation and phenotypic analysis of mice lacking all urea transporters

Abstract

Urea transporters (UT) are a family of transmembrane urea-selective channel proteins expressed in multiple tissues and play an important role in the urine concentrating mechanism of the mammalian kidney. UT inhibitors have diuretic activity and could be developed as novel diuretics. To determine if functional deficiency of all UTs in all tissues causes physiological abnormality, we established a novel mouse model in which all UTs were knocked out by deleting an 87 kb of DNA fragment containing most parts of Slc14a1 and Slc14a2 genes. Western blot analysis and immunofluorescence confirmed that there is no expression of urea transporter in these all-UT-knockout mice. Daily urine output was nearly 3.5-fold higher, with significantly lower urine osmolality in all-UT-knockout mice than that in wild-type mice. All-UT-knockout mice were not able to increase urinary urea concentration and osmolality after water deprivation, acute urea loading, or high protein intake. A computational model that simulated UT-knockout mouse models identified the individual contribution of each UT in urine concentrating mechanism. Knocking out all UTs also decreased the blood pressure and promoted the maturation of the male reproductive system. Thus, functional deficiency of all UTs caused a urea-selective urine-concentrating defect with little physiological abnormality in extrarenal organs.

Keywords: animal model; gene targeting; knockout; renal physiology; urine concentration.

Fig. 1. Gene targeting strategy for all-UT-knockout and identification of all-UT-knockout mice

A. top, organization and restriction map of the mouse Slc14a2 and Slc14a1 genes. Rectangles indicate exon segments that constitute coding sequences. Middle and bottom, targeting strategy for all UTs deletion. Homologous recombination results in the replacement of the indicated segments (thick line) of the Slc14a2 and Slc14a1 genes by a neo cassette selectable marker flanked by LoxP sites. DTA is used for negative selection. The constitutive knockout allele was obtained after Cre-mediated recombination. B. Western blot analysis of kidney using UT-A2, UT-A3 or UT-B antibody. C. UT-A1/A3, UT-A2 and UT-B immunofluorescence of kidney inner and outer medulla stained by UT-A3, UT-A2 or UT-B antibody. D. urea permeability measured in erythrocytes from wild-type and all-UT-knockout mice from the time course of erythrocyte volume as determined by light scattering in response to a 250 mM inwardly directed urea gradient (left). Water permeability measured in response to a 250 mM inwardly directed sucrose gradient (right, n=3).

Fig. 2. Renal phenotype of all-UT-knockout mice

A. body weight. B. 24 h urine output. C. urine osmolality measured in mice given free access to food and water (basal) and after a 24 h water deprivation. Means ± SEM, n = 6. D. representative images of H&E staining of kidney cortex, outer medulla and inner medulla of wild-type mice (upper) and all-UT-knockout mice (lower). Scale bar =100μm. E. urea concentration, sodium concentration, potassium concentration, and chloride concentration in inner medullary tissue. Data are presented as means ± SEM. n=6. **p < 0.01; *p < 0.05 vs. wild-type mice.

Fig. 3. Renal handling of acute urea loading

Three hundred micromoles of urea were injected (ip) just after the first 2-h urine collection (time 0). A. urinary osmolality (U_osm). B. urinary output. C. urinary urea concentration (U_urea). D. urea excretion (excr.). E. nonurea solute concentration (U_{non-urea solutes}). F. excretion of non-urea solutes. Data are presented as means ± SEM. n = 6. *p < 0.05 vs. wild-type mice.

Fig. 4. Renal handling of long term urea loading in mice fed diets containing 10, 20, or 40% protein

A. urine output. B. urinary osmolality (U_osm). C. urinary urea concentration (U_urea). D. urinary osmolar excretion (Osmolar excr.). E. urea excretion. F. excretion of non-urea solutes. Data are presented as means ± SEM. n = 6. *p < 0.05 vs. wild-type mice.

Fig. 5. Diagram of urea recycling in the kidney of wild-type (A) and all-UT-knockout mice (B)

Urea flows are indicated by blue arrows. See text for details.

Fig. 6. Determination of selective inhibition of PU-48 on UTs

Wild-type and UT-B null mice were observed in metabolic cages and subcutaneously injected with or without 100 mg/kg of PU-48 just after a 2-h urine collection (time 0). Urine samples were collected every 2 h. A. urine output. B. urinary osmolality (U_osm). C. urine urea concentration (U_urea). D. urine non-urea concentration (U_non-urea). E. urea excretion. F. excretion of non-urea solutes. Data are presented as means ± SEM. n = 6. *p < 0.05 vs. wild-type mice.

Fig. 7. Extra renal phenotypes of all-UT-knockout mice

A. left, forced swim test. Data are presented as means ± SEM. n = 10. *p < 0.05 vs. wild-type mice. right, sucrose preference test. Data are presented as means ± SEM. n = 10*. P < 0.05 vs. wild-type mice. B. blood pressure. DBP, Diastolic blood pressure; SBP, systolic blood pressure; MAP, mean arterial pressure. Data are presented as means ± SEM. n = 8. C. HE staining of testis tissue sections. D. breeding performance of maturing male mice. Male (M) mice at 4-week-old were housed with 8-week-old wild-type female (F) mice. Data are shown for 6 pairs of competing mates (left) and 6 pairs of wild-type controls (right) and are means ± SEM.

Fig. 8. Diagram of urea recycling in the kidney of wild-type and all-UT-knockout mice

Urea flows are indicated by blue arrows.