• SLC22A12 is an organic anion transporter, also known as the urate transporter (URAT1), which plays a critical role in the kidneys. Uric acid, a byproduct of purine metabolism, is normally filtered by the kidneys and excreted in urine. URAT1 is located on the luminal membrane of the proximal tubules and is responsible for reabsorbing uric acid from the tubular fluid (urine) into the proximal tubule cells. The reabsorbed uric acid is then transported back into the bloodstream via another transporter, GLUT9, thereby regulating uric acid levels and maintaining its balance in the body. Dysfunction of URAT1 can lead to abnormal accumulation of uric acid, increasing the risk of gout and uric acid stone diseases. As a result, URAT1 has become an important target for the development of uric acid-lowering drugs.

Targeting strategy:

• The exons 1-10 of mouse Urat1 gene that encode signal peptide, extracellular domain, cytoplasmic portion and transmembrane domain is replaced by human counterparts in B-hURAT1 mice. The promoter, 5’UTR and 3’UTR region of the mouse gene are also replaced by human counterparts. The URAT1 expression is driven by human URAT1 promoter, while mouse Urat1 gene transcription and translation will be disrupted.

Protein expression analysis:

• URAT1 was detected in heart, liver, kidney and colon of homozygous mice and wild-type mice, and weakly detected in brain, as anti-URAT1 antibody is cross reactive between human and mouse URAT1 protein.

mRNA expression analysis:

• Mouse Urat1 mRNA was only detected in kidney of wild-type mice. Human URAT1 mRNA was exclusively detectable in homozygous B-hURAT1 mice but not in wild-type mice.

Gene targeting strategy for B-hURAT1 mice. The exons 1-10 of mouse Urat1 gene that encode signal peptide, extracellular domain, cytoplasmic portion and transmembrane domain is replaced by human counterparts in B-hURAT1 mice. The promoter, 5’UTR and 3’UTR region of the mouse gene are also replaced by human counterparts. The URAT1 expression is driven by human URAT1 promoter, while mouse Urat1 gene transcription and translation will be disrupted.

Strain specific analysis of URAT1 mRNA expression in wild-type C57BL/6JNifdc and B-hURAT1 mice by RT-PCR. Kidney RNA were isolated from wild-type C57BL/6JNifdc (+/+) and homozygous B-hURAT1 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human URAT1 primers. Mouse Urat1 mRNA was only detected in wild-type mice. Human URAT1 mRNA was exclusively detectable in homozygous B-hURAT1 mice but not in wild-type mice.

Strain specific analysis of URAT1 gene expression in wild-type C57BL/6JNifdc mice and B-hURAT1 mice by RT-qPCR. Tissue RNA were isolated from homozygous B-hURAT1 mice (H/H) (male, 7w, n=3). Human URAT1 mRNA was exclusively detectable in kidneys of homozygous B-hURAT1 mice. Values are expressed as mean ± SEM. Significance was determined by unpaired t-test.  *P < 0.05, **P < 0.01, ***P < 0.001.

Species specific analysis of URAT1 mRNA expression in heterozygous B-hURAT1 mice by RT-qPCR. Kidney was collected from heterozygous B-hURAT1 mice (H/+) (male or female, n=3, 7-week-old). The URAT1 mRNA expression level in male mice was higher than female mice. (A) The analysis was used mouse Urat1 primer. (B) The analysis was used human URAT1 primer. Values are expressed as mean ± SEM.