Kidney Week

Abstract: FR-PO578

Renal Kallikrein-1 Contributes to Cleavage of Gamma-Epithelial Sodium Channel (ENaC) in Response to a Short-Term, Low-Sodium, High-Potassium Diet

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Basic
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid, Electrolytes, and Acid-Base Disorders

• 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

• Curry, Joshua N., Oregon Health & Science University, Portland, Oregon, United States

Joshua N. Curry, MD, PhD

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

Xiao-Tong Su, PhD

• Maeoka, Yujiro, Hiroshima Daigaku Byoin, Hiroshima, Hiroshima, Japan

Yujiro Maeoka, MD, PhD

• Yang, Chao-Ling, Oregon Health & Science University, Portland, Oregon, United States

Chao-Ling Yang, MD

• Delpire, Eric J., Vanderbilt University Medical Center, Nashville, Tennessee, United States

Eric J. Delpire, PhD

• Welling, Paul A., Johns Hopkins Medicine, Baltimore, Maryland, United States

Paul A. Welling, MD

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

David H. Ellison, MD, FASN

Background

Tissue kallikrein, encoded in part by Klk1, is a serine protease that has been proposed to cleave and activate Na⁺ channels (ENaC). In the kidney, kallikrein-1 is found primarily in the aldosterone sensitive distal nephron, and its transcript abundance is markedly reduced by deletion of the mineralocorticoid receptor. While global deletion of kallikrein-1 leads to defective renal K⁺ handling and reduced gamma-ENaC cleavage, it is not clear if these findings result from loss of renal kallikrein-1 or from systemic abnormalities observed with global kallikrein-1 loss.

Methods

In order to determine the direct effects of renal kallikrein-1 on ENaC, we generated mice with conditional deletion of kallikrein-1 in the distal nephron: Klk1^fl/ffl Calbindin-Cre (KS-Klk1 KO). Mice were treated with either control (Ctrl, 0.49% NaCl, 1%. KCl) or low Na⁺, high K⁺ (LNHK, 0.03% NaCl, 5% KCl) diet for 5 days. On Day 4, urine was collected during 24 hours. Urine electrolytes were measured by flame photometry or colorimetric assay, and kallikrein activity was measured using amidolytic assay. Western blotting was used to quantify gamma-ENaC, with or without Peptide -N-Glycosidase F (PNGase F).

Results

Deletion of kallikrein-1 was confirmed by western blotting; it resulted in striking, but incomplete, reduction in urinary kallikrein excretion. Serum [K⁺] was similar across genotypes on both Ctrl and LNHK diets. Urine [Na⁺] and [K⁺] excretion rates were also similar between genotypes. Cleaved gamma-ENaC was higher on LNHK than Ctrl diet in both groups, but the ratio of cleaved:uncleaved was not as high in KS-Klk1 KO (WT 3.3+/-0.04 vs KO 2.05+/-0.55). To improve ENaC resolution, we treated kidney samples with PNGase F. Under LNHK conditions the 52 kDa (distally cleaved) gamma-ENaC band was 52% lower in KS-Klk1 KO (WT 100% vs KO 51.5%, p=0.006). There was also a trend towards lower phosphorylated-NCC in the KS-Klk1 KO mice on LNHK diet, suggesting a compensatory response.

Conclusion

These results suggest that kidney kallikrein-1 contributes to, but is not the only protease, that cleaves gamma-ENaC at its distal site in response to a short-term low Na⁺ and high K⁺ diet. Further studies are needed to better understand how renal kallikreins are produced and their role in gamma-ENaC cleavage and ENaC function.

Funding

• NIDDK Support