Kidney Week

Abstract: PO1419

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

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Basic
  October 22, 2020 | Location: On-Demand
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid, Electrolyte, and Acid-Base Disorders

• 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

• Cornelius, Ryan J., Oregon Health & Science University, Portland, Oregon, United States

Ryan J. Cornelius,

• Sharma, Avika, Oregon Health & Science University, Portland, Oregon, United States

Avika Sharma,

• Su, Xiao-Tong, Oregon Health & Science University, Portland, Oregon, United States

Xiao-Tong Su,

• Ellison, David H., Oregon Health & Science University, Portland, Oregon, United States

David H. Ellison,

• McMahon, Andrew P., Keck School of Medicine of the University of Southern California, Los Angeles, California, United States

Andrew P. McMahon,

• McCormick, James A., Oregon Health & Science University, Portland, Oregon, United States

James A. McCormick,

Background

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 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.

Methods

CRISPR/Cas9 was used to introduce Cre-ERT2 into the 3' UTR near the stop codon of the Slc12a3 gene encoding NCC (Slc12a3-Cre-ERT2 mice). Here, we crossed Slc12a3-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’.

Results

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. Crossing to TdTomato mice revealed higher leakiness (64.5%), suggesting differential sensitivity of the floxed site. Western blotting revealed no differences in abundances of total or the active-phosphorylated form of NCC in Slc12a3-Cre-ERT2 mice of either sex compared to controls. Furthermore, functional analysis of NCC showed no effects on NCC activity in Slc12a3-Cre-ERT2 mice. Plasma K⁺ and Mg²⁺ concentrations, and thiazide-sensitive Na⁺ and K⁺ excretion did not differ in Slc12a3-Cre-ERT2 mice compared to controls.

Conclusion

Thus, the Slc12a3-Cre-ERT2 mice have high recombination efficiency and complete fidelity in cell-specificity. Our data show that Cre expression is entirely localized to the DCT and the genetic modification has no effect on NCC expression and renal function. The Slc12a3-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.

Funding

• NIDDK Support