A Novel Distal Convoluted Tubule‐Specific Tamoxifen‐Inducible Cre‐Recombinase Driven by the NaCl Cotransporter Gene

Abstract

The use of knockout and transgenic mouse models coupled with Cre‐lox technologies has revolutionized research in kidney transport physiology by allowing site‐specific genetic recombination in individual nephron segments. Although several groups have tried to generate a distal convoluted tubule (DCT)‐specific mouse Cre‐recombinase driven by the thiazide‐sensitive NaCl cotransporter (NCC) promoter, this goal has remained elusive. The DCT is a major site for the regulation of Na+ and K+ transport. The segment consists of two distinct segments the early (DCT1) and late (DCT2) DCT. The only previously recognized mouse model available allowing targeted gene modification in the DCT is the DCT1‐specific mouse with Cre‐recombinase under control of the Pvalb gene encoding parvalbumin. The model, however, has limitations including activity in neurons that prevent comprehensive characterization of transport pathways in the DCT. Recently, a novel mouse line expressing estrogen‐inducible DCT‐specific Cre was generated and donated to Jackson Laboratory. CRISPR/Cas9 was used to introduce Cre‐ERT2 into the 3' UTR near the stop codon of the Slc12a3 gene encoding NCC (NCC‐Cre‐ERT2 mice). Here, we crossed NCC‐Cre‐ERT2 mice with YFP floxed mice to test whether the Cre expression would mimic that of NCC, and to determine whether the construct is ‘leaky’. Without tamoxifen, approximately 6% of NCC positive cells expressed YFP, indicating minimal leakiness. After five days of tamoxifen injection, mice showed YFP expression in almost all NCC positive cells and there was complete overlap of YFP expression in NCC positive cells. Western blotting showed lower NCC abundance in NCC‐Cre‐ERT2 males compared to controls, but female NCC‐Cre‐ERT2 mice NCC abundance was not significantly different from the abundance in control littermates. Despite the difference in NCC abundance in males, abundance of the active phosphorylated form of NCC did not differ. Furthermore, functional analysis of NCC showed no effects on NCC activity in NCC‐Cre‐ERT2 mice. Plasma K+ and Mg2+ concentrations, and thiazide‐sensitive Na+ and K+ excretion did not differ in NCC‐Cre‐ERT2 mice compared to controls. Thus, the NCC‐Cre‐ERT2 mice have high recombination efficiency, minimal leakiness, and complete fidelity in cell‐specificity. Our data show that Cre expression is entirely localized to the DCT and the genetic modification has very mild or no effect on NCC expression and renal function. The NCC‐Cre‐ERT2 mice are the first mice generated with Cre recombinase activity along the entire DCT, and will be a powerful tool to study DCT function.Support or Funding InformationNIH grants DK098141 and DK117903 to JAM. NIH grant DK51496 and VA grant 1I01BX002228‐01A1 to DHE.