Diurnal rhythm of aquaporins expression and function in the kidney
Authors: Nguyen Phi Hung , Huynh Nha Van , Hyndman Kelly 
Published: May 2024

Aquaporins (AQPs) are a protein family that plays a major role in maintaining water homeostasis. Aquaporin 2 (AQP2) and 3 (AQP3) are expressed on the collecting duct and regulated by the antidiuretic hormone, arginine vasopressin (AVP). AVP binds to its receptor on the collecting duct leading to increased expression of AQP2 on the apical membrane and AQP3 on the basolateral membrane to drive water reabsorption and promote urine concentration. We know that there are biological circadian rhythms that are critical for maintaining homeostasis. Therefore, we aimed to determine AQP2 and AQP3 expression and urine concentrating ability during their active time and inactive time in male and female mice. We hypothesize that there is a diurnal pattern in AQP2 and AQP3 expression or abundance leading to the variation of urine concentrating ability. Male (n=12) and female (n=12) C57BL/6J mice (10-11 weeks old) were euthanized in the middle of their active period (midnight) or the middle of their inactive period (noon). Spot urine osmolality was determined using a freezing point osmometer. Plasma sodium, chloride, and blood urea nitrogen (BUN) plasma concentration using an i-STAT blood analyzer. We localized aquaporins in kidney sections by using a diaminobenzidine chromagen method with antibodies against AQP2, AQP2 pS256 (an activation site), AQP2 pS261(an inhibitory site), and AQP3. Then, using a blinded method, the program Fiji Image J was used to quantify aquaporins on collecting ducts by using the integrated density (IntDen) parameter after calibrating into optical density (OD). We found that urine osmolality of control mice during their active period (2449±442.3 mOsm/ kg) was higher than that in the inactive time (1684±705.6 mOsm/kg, p = 0.0043). The plasma sodium concentration was higher in the active time (145±1.782 mmol/ L) than in the inactive time (142.9±1.311 mmol/L) with p = 0.0026. The plasma chloride (Cl−) and plasma BUN were also higher in the active time (Cl− = 115.2±1.4 mmol/L; BUN = 23±3.6 mmol/L) than in the inactive time mice (Cl− = 111±2.0 mmol/L, p < 0.0001; BUN = 19.75±2.5 mmol/L, p = 0.0187). AQP2 was differentially expressed. During the active period, in both male and female mice, AQP2 was highly expressed in the collecting duct but was obviously reduced during the inactive period (active time = 4083±1006 OD; inactive time = 1243±608 OD, p < 0.001). Moreover, AQP2 activation as determined by phosphorylation S256, was also greater during the active period (1148±404 OD) than inactive (221±96 OD, p = 0.002). Interestingly, the inhibitory site, phosphorylation of S261, was also greater during the active period (592 ± 378 OD) than the inactive period (162 ± 36 OD, p = 0.039). We found no significant difference in AQP3 localization or abundance. In summary, we showed that there is diurnal variability in urine osmolality (higher in their active period) that is supported by increased AQP2 expression during the active period of the mice, regardless of sex. Future studies will aim to determine if antagonizing and stimulating the vasopressin 2 receptor affects urine concentration in a diurnal manner. Intramural UAB funds to KAH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.