Graduation Year

2021

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Timo Rieg, M.D., Ph.D.

Committee Member

Craig Doupnik, Ph.D.

Committee Member

Ruisheng Liu, M.D., Ph.D.

Committee Member

Niketa Patel, Ph.D.

Committee Member

Kay-Pong Daniel Yip, Ph.D.

Keywords

epithelial transport, acid-base balance, electrolyte and water homeostasis, blood pressure

Abstract

The sodium-hydrogen exchanger isoform 3 (NHE3, SLC9A3) mediates most Na+/H+ exchange in the gastrointestinal tract and the kidney, where it plays crucial roles in Na+ and fluid absorption as well as acid-base homeostasis. Whole body NHE3 knockout (NHE3-/-) mice show overt absorptive defects in both the intestine and kidneys. Genetic mutations of NHE3 gene in humans are associated with congenital sodium diarrhea (CSD). In the kidneys, NHE3 is expressed in the proximal tubule (PT) and the thick ascending limb (TAL). Whole kidney-specific (PT and TAL) NHE3 knockout (NHE3Pax8cre) mice and PT-specific NHE3 knockout (PT-NHE3-/-) mice have shown the importance of renal NHE3 in Na+ homeostasis and blood pressure regulation. To date, the relative contribution of NHE3 in the intestine versus the kidneys and the role of NHE3 in the TAL for Na+, fluid and acid-base homeostasis not been selectively investigated due to a lack of suitable animal models. To fill these two knowledge gaps, two series of experiment were conducted by using novel tamoxifen inducible intestinal epithelial cell specific (NHE3IEC-KO) and TAL-specific NHE3 knockout (NHE3TAL-KO) mouse models. 1) No difference was found in blood parameters or other phenotypes between NHE3IEC-KO and control (NHE3loxlox) mice before tamoxifen administration. After tamoxifen administration, NHE3 was completely deleted in the intestine of NHE3IEC-KO mice. Loss of NHE3 causes defective intestinal absorption, indicated by watery, alkaline diarrhea and dilated intestine associated with a compensatory higher fluid intake in HE3IEC-KO mice. Three weeks after tamoxifen, ~25% mortality rate is found in NHE3IEC-KO mice. NHE3IEC-KO mice also show metabolic acidosis, lower blood bicarbonate levels, hyponatremia and hyperkalemia in combination with drastically elevated plasma aldosterone levels. These results indicate that intestinal NHE3 plays a significant role in acid-base, Na+ and volume homeostasis. This NHE3IEC-KO mouse model provides a suitable tool for studying the role of intestinal NHE3 and explains the phenotype of individuals with CSD carrying SLC9A3 mutations. 2) After tamoxifen administration, deletion of NHE3 in the TAL was confirmed by mRNA expression and immunofluorescent staining. Two weeks after tamoxifen administration, NHE3TAL-KO mice showed a ~25% lower urine osmolality associated with ~20% higher water intake compared to Con mice. No differences were found in blood pH, urinary pH, urinary Na+ or Cl-/creatinine ratios or glomerular filtration rate. Of note, NHE3TAL-KO mice have a higher K+/creatinine ratio compared to control mice. To test for a role of the TAL in urinary acidification we used furosemide which stimulates urinary acidification. In response to furosemide no dose-dependent differences were observed in Cl- excretion or urinary flow rate. Of note, NHE3TAL-KO mice showed an acute decrease in urinary pH similar to control mice followed by an immediate recovery of urinary pH, the latter completely absent in control mice. Taken together, the results indicate that NHE3 in the TAL contributes to urinary concentrating ability and the sustained response of the TAL to furosemide-induced urinary acidification. The new NHE3TAL-KO mouse model provides a useful tool to delineate the specific role of NHE3 in the TAL for renal functions. This thesis provides unprecedent insights into the functions of NHE3 in the intestine and TAL of the kidneys.

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