Graduation Date

Spring 5-5-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Cellular & Integrative Physiology

First Advisor

Erika I. Boesen

Abstract

In the kidney, endocytic recycling regulates the abundance of channels and transporters in the membrane of the tubular epithelium, and thereby controls the kidney’s ability to regulate water homeostasis. In recent years, a family of proteins called Eps15 homology domain-containing (EHD) proteins has emerged as important regulators of the endocytic recycling pathway. Mammals express four paralogs of EHD proteins, EHD1-4, that are expressed in different tissues. Although EHD4 is expressed in the kidney, the specific physiological role of EHD4 in the kidney remains unknown. Therefore, this dissertation was focused to elucidate the physiological role of EHD4 in the kidney. In the mouse kidney, EHD4 was found to be expressed most abundantly in the inner medullary collecting duct (IMCD), a site for fine-tuning final urine concentration. Studying the renal parameters of Ehd4-/- (EHD4-KO) mice showed that EHD4-KO mice had a higher urine flow and lower urine osmolality than wild-type (WT) mice. EHD4 was found to be expressed in the hypothalamus, but its deletion did not affect the plasma osmolality or arginine vasopressin (AVP) excretion. EHD4-KO mice were able to exhibit anti-diuretic responses to 24-h water restriction similar to the WT mice, suggesting that the diuretic phenotype of EHD4-KO mice is due to defective renal water and solute handling. Formation of a concentrated urine requires the presence of the renal medullary osmotic gradient, partially contributed by renal urea handling, and fine-tuning of water reabsorption via aquaporin (AQP) 2 in the IMCD. Given the abundant expression of EHD4 in the IMCD, it was hypothesized that EHD4 regulates AQP2 trafficking in the principal cells and renal urea handling. Both in vivo and in vitro analysis showed that in the absence of EHD4, the apical membrane abundance of AQP2 was significantly attenuated. EHD4 was also found to regulate basolateral membrane abundance of AQP4 in principal cells. Although EHD4 was found to co-localize with AQP2, immuno-complex containing AQP2 did not show the presence of EHD4, suggesting a lack of direct physical interaction between the two proteins. Additionally, EHD4-KO mice had higher urea excretion than WT mice, suggesting a dysfunctional urea handling in these mice. To delineate the exact role of EHD4 in renal urea handling, mice were subjected to a series of experiments involving intake of modified protein diets, which confirmed a role of EHD4 in the regulation of renal urea handling. Overall, this dissertation describes a novel role for EHD4 in the regulation of AQP2, AQP4, and renal urea handling in the kidney.

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