EHD4 deletion results in a urine concentrating defect in mice
Authors: Rahman Shamma Shakila , Moffitt Alexandra Elizabeth Jane , Storck Mathew , Band Hamid , Boesen Erika Ingrid 
Published: November 2023

C‐terminal Eps15 Homology Domain‐containing protein 4 (EHD4) regulates endocytic recycling but its function in the kidney is unknown. Previously we found that, despite similar water intake, Ehd4−/− (EHD4‐KO) mice produced a higher volume of osmotically dilute urine compared to wild type (WT) mice, suggesting defective water handling in EHD4‐KO mice. Formation of a concentrated urine requires the presence of an appropriate interstitial osmotic gradient, generated in part by urea accumulation in the renal medulla, and fine regulation of water reabsorption via aquaporin‐2 (AQP2). Given that EHD4 is expressed in the inner medullary collecting duct (IMCD), we hypothesized that deletion of EHD4 disrupts urea transport in the IMCD and also decreases AQP2 apical membrane accumulation, resulting in pronounced diuresis in EHD4‐KO mice. To test the first part of our hypothesis, we analyzed 24‐h urinary urea excretion and found that EHD4‐KO mice had higher urea excretion than WT mice (13.5 ± 1.7 vs. 5.2 ± 1.1 mg/d in male EHD4‐KO vs. WT mice, n = 6–8, P = 0.001; 11.5 ± 1.0 vs. 6.1 ± 1.1 mg/d in female EHD4‐KO vs. WT mice, n = 4–5, P = 0.008), a finding compatible with impaired interstitial accumulation of urea. Next, we performed immunofluorescence staining of renal inner medulla (IM) and found more dispersed intracellular localization of AQP2 in principal cells of EHD4‐KO mice compared to a more clear apical localization of AQP2 in WT mice. To test if the dispersed AQP2 localization in EHD4‐KO mice was due to decreased circulating arginine vasopressin (AVP), a key regulator of AQP2 trafficking, we analyzed 24‐h urinary AVP excretion as an indirect indicator of plasma AVP. AVP excretion was comparable between male EHD4‐KO and WT mice (3.1 ± 0.4 and 3.6 ± 0.3 pg/d respectively, n = 6 per group, P = 0.39); however, Western blot of IM revealed similar levels of phospho‐S256‐AQP2 in both genotypes, indicating that events leading to phosphorylation of AQP2 were functional in EHD4‐KO mice, and suggesting that EHD4 may regulate subsequent intracellular trafficking of AQP2 to the apical membrane. To further test the role of EHD4 in urinary concentrating ability, female WT and EHD4‐KO mice were subjected to 24‐h water restriction. Urine volumes of the two groups after dehydration were comparable (0.3 ± 0.1 versus 0.8 ± 0.2 ml/d in WT and EHD4‐KO mice, n = 3 per group, P = 0.16); however, urine osmolality of EHD4‐KO mice was lower than WT mice (3820 ± 115 versus 2677 ± 201 mOsmol/kg H20 for WT and EHD4‐KO mice, n = 3 per group, P = 0.008), indicating that EHD4‐KO mice have a mild defect in the urinary concentrating mechanism. Together these data suggest that EHD4 plays an important role in the urinary concentrating mechanism, potentially through effects on urea transport and membrane trafficking of AQP2. Future studies will be focused on identifying the exact molecular mechanisms of how EHD4 regulates urea transport and AQP2 membrane trafficking in the IMCD, and therefore allow us to better understand water homeostasis.Support or Funding InformationPre‐doctoral fellowship, American Heart Association (15PRE25580003) to SR; NIH grants CA105489, CA87986 and CA99163 to HB