Optical clearing and 3D‐Imaging Reveals a Sexual Dimorphism in the Structure and Remodeling Response of the Distal Convoluted Tubule

Abstract

IntroductionThe abundance of the distal convoluted tubule (DCT) thiazide‐sensitive sodium chloride cotransporter (NCC) is well appreciated to be greater in females than males. Because structural remodeling of the DCT is dependent on NCC activity, it has been generally assumed that there is a corresponding sexual dimorphism in DCT morphology. Until now, this has never been directly examined. Here, combining new optical clearing techniques with staining for markers of DCT segments, state‐of‐the‐art high‐speed volumetric imaging and analysis tools we quantitatively assess DCT morphology in male and female mice, and study DCT remodeling response to furosemide.MethodsMale and Female (3‐month‐old) mice were treated with vehicle or Furosemide administered in the food (100 mg/kg per day) for seven days. Total NCC and phospho‐active NCC abundance (pNCC) was evaluated by Western Blot in one kidney, the other kidney was perfused and fixed for imaging. Kidneys were cleared using the Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC) pipeline, co‐stained with antibodies that label the early DCT (DCT1, parvalbumin) and the entire DCT (DCT1 & 2; NCC), imaged using a high‐speed spinning disc confocal microscope, and processed with 3D rendering and analysis software (IMARIS).ResultsWe confirmed previous studies that females have greater NCC expression in the basal state. Surprisingly, the length of the DCT was longer in male (~620 μm) than female mice (~560 μm). Furosemide treatment significantly increased the abundance of NCC and pNCC in both sexes, and this was paralleled by an expansion of the DCT. The remodeling response to furosemide was more profound in females (~20% increase in DCT length) than males (~8%). The DCT elongation effect of furosemide treatment in females stemmed largely from an increase in DCT1 length rather than a change in DCT2. By contrast, furosemide expanded the DCT1 and DCT2 to the same extent in males.SummaryOur data indicate that sexual dimorphism in NCC does not originate from differences in the DCT length. In fact, male have a longer DCT than female mice, which suggests that the density of NCC per unit of length is higher in females. Altogether, the differences in the remodeling response suggests that females have more “plastic” DCTs compared to males.Support or Funding InformationThis study was supported by NIH and Ben J. Lipps postdoctoral Fellowship award.