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 been identified to have diuretic activity and might 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 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, and urine osmolality was significantly lower. 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. These results revealed that functional deficiency of all UTs caused urea selective urine concentrating defect with little physiological abnormality in extrarenal organs.

Noninvasive imaging of renal urea handling by CEST‐MRI

“…After 5 minutes of dynamic scanning, a bolus of either 150 μL of 2M urea or 150 μL of 0.9% saline was administered through the intraperitoneal infusion line. The amount of urea administered was determined from previous mouse renal physiology studies . After 20 minutes of dynamic scanning, an additional full Z‐spectrum set of CEST‐weighted images was acquired (both water saturation shift reference and CEST).…”

GRHL2 Is Required for Collecting Duct Epithelial Barrier Function and Renal Osmoregulation

“…[56][57][58][59] In addition, urinary concentrating defects were observed in mice with genetic defects that perturbed the accumulation of sodium chloride (via functions of the thick ascending loop of Henle) [60][61][62] or urea (via specific urea transporters). 7,63 We examined these different components of the urinary concentration machinery in Grhl2 CD2/2 mice. AVP levels were normal, water reabsorption was functionally unaltered as shown by swelling/deswelling experiments in isolated CDs, and AQP2 membrane translocation in response to cAMP stimulation in isolated CD cells occurred normally.…”

“…IMCD urea transporters contribute to the establishment of medullary hypertonicity. 7 To rule out that Grhl2 CD2/2 kidneys exhibit a defect of urea transporter expression, we analyzed UT-A1 or UT-A3 expression in medullas of control and Grhl2 CD2/2 kidneys using Western blot and immunofluorescence, revealing similar expression levels and unchanged cellular localization (Supplemental Figure 16). Together, these data indicated that CD water transport systems, AVP signaling, and medullary urea transporter expression were normal in Grhl2 CD2/2 mice.…”

“…tip, resulting in a urea gradient between the urine and the interstitium. In case of water restriction, specialized urea transporters are inserted into the cell membrane, [6][7][8] and urea follows its gradient into the medulla. 9 Compared with other segments of the nephron, the CD epithelium forms particularly tight barriers between the urinary space and the interstitial environment.…”

Emerging Targets of Diuretic Therapy

“…The function of UT‐A2 is not as clear as other UTs. A recent all‐UT‐knockout mouse model exhibited a severe urine concentrating defect due to defective urea accumulation in the inner medulla, slightly lower blood pressure, but no disturbance of renal Na + transporters activities and internal electrolyte balance . The lower blood pressure likely resulted from water diuresis.…”