Three‐dimensional analysis of potassium deprivation‐induced tubular remodeling using optical clearing

Abstract

Tubular remodeling can be induced by various stimuli and plays a critical role in functional adaptation. For instance, hypokalemia is associated with increased kidney mass caused by cellular hypertrophy and hyperplasia. However, accurate quantitative analysis of tubular remodeling is still lacking since most kidney morphometry was performed with two‐dimensional techniques. Here, we combined optical clearing and advanced light microscopy as a new approach for three‐dimensional analysis of tubular remodeling in mouse kidneys, upon dietary potassium (K+) restriction.Adult C57BL/6J mice were fed for 3 days with K+‐deficient diet (approx. 0.002% K+) or normal K+ diet (1.2% K+) and were injected with Bromodeoxyuridine (BrdU) intraperitoneally before kidneys were perfusion‐fixed 4 h later. Kidneys were cut into 1.0–1.5 mm thick slices and stained for BrdU then optically cleared with Ethyl Cinnamate. Confocal microscopy was used to identify the total number of BrdU positive cells per unit volume. In addition, 4 μm sections were used for co‐staining of BrdU with segment‐specific antibodies to localize proliferating cells. Whole blood was collected via terminal cardiac puncture and analyzed using i‐STAT analyzer (Abbot Point of Care Inc.).As expected, serum K+ levels were significantly lower in mice on K+ ‐deficient diet than in mice on normal diet (3.7 ± 0.1 mmol/L vs. 4.2 ± 0.1 mmol/L, p<0.005, n=6–7). After optical clearing, confocal microscopy allowed high‐resolution imaging of BrdU positive cells within the whole thick kidney slice. Quantification of BrdU positive cells revealed that the cell proliferation rate was low in control mice, but that K+ restriction strongly induced cell proliferation (3748 ± 392 cells/mm3 vs. 1089 ± 341 cells/mm3 p<0.05, n=3). Co‐localization using thin sections and segment‐specific markers revealed that this increase in proliferation was restricted to the S3 segment of the proximal tubule and outer medullary collecting duct intercalated cells.In this study, we validated a combination of optical clearing and advanced light microscopy as a new toolbox for imaging and quantification of tubular remodeling, which will decrease our reliance on biased two‐dimensional morphometric techniques and time‐consuming stereological approaches. Our analysis revealed that dietary K+ restriction caused preferential hyperplasia in S3 segments and in intercalated cells. The former observation might reflect a functional adaptation after low‐K+ induced injury of proximal tubules, whereas the latter likely represent a compensatory response to increase renal potassium uptake.Support or Funding InformationDFG German Research Foundation Fellowship (332853055) and Else Kröner‐Fresenius Stiftung (2015_A197) to TS, Humboldt Foundation and National Health and Medical Research Council of Australia Research Fellowships to VGP, R01DK098141 to JAM, and R01DK054983 to DHE.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.