Mitochondria ROS in podocytes of freshly isolated glomeruli during salt‐sensitive hypertension
Authors: Domondon Mark , Sultanova Regina , Polina Iuliia , Kurashkina Elizaveta , Heslop Kareem , Maldonado Eduardo N. , Ilatovskaya Daria 
Published: June 2021

The hallmark of salt‐sensitive (SS) hypertension is an early onset of proteinuria, which results from loss of epithelial cells of the glomerular filtration barrier – podocytes. Several cell culture studies show that mitochondria dysfunction can contribute to podocyte apoptosis, thus leading to proteinuria. During hypertension, mitochondria in the kidney cells are known to lose cristae membranes, reduce their size and have reduced matrix density, leading to decreased ATP levels and increased reactive oxygen species (ROS). The goal of this study was to develop the method to monitor mitochondrial damage in the podocytes and test the role of mitochondria in podocyte dysfunction during SS.Hypertension was induced in Dahl SS rats by a high salt diet challenge (HS, 4% NaCl, 21 days); control group was maintained on the low salt diet (0.4% NaCl, LS). At the end of the protocol blood pressure was measured using the tail cuff, kidney flush via abdominal aorta was performed, tissues were collected, glomeruli were isolated by differential sieving and then loaded with TMRM (mitochondria membrane potential marker) to visualize podocytic mitochondria, or MitoPY1 (a mitochondria‐targeting ROS indicator), and imaged with a Zeiss 880 confocal microscope. Blood electrolyte levels were measured using a CareLyte Electrolyte ISE Analyzer. Kidney samples were stained using Masson's trichrome in order to characterize damage. Using a BD CARV II Confocal Imager, fifty glomeruli from each sample were blindly scored on a scale of 0–4.Systolic blood pressure was elevated in the HS diet fed rats (171 ± 9 vs 148 ± 6 mmHg in HS vs LS diet fed groups, respectively, n=11 per group); a trend for renal hypertrophy was noted in the HS group compared to controls. No differences in plasma Na+, Cl− or K+ were observed. Histological scoring revealed that HS diet fed rats had exacerbated glomeruli damage compared to the LS group (3.1 ± 0.2 vs 1.9 ± 0.1, p = 0.006). Using an uncoupling agent FCCP we confirmed in the TMRM‐loaded samples that the signals we are recording are generated specifically by mitochondria since upon FCCP application we observed fast (within 40 ± 12 s) quenching of TMRM fluorescence. In MitoPY1‐stained glomeruli we detected higher baseline mitochondrial ROS level, as well as more fragmented mitochondria in the podocytes of the HS diet, fed group. In addition, we revealed a differential response to an acute application of 20 μM H2O2 between the LS vs HS groups. Future directions of the study involve assessing mitochondria biogenesis, performing a detailed structural analysis of mitochondria using electron microscopy, and testing a panel of mitochondrial damage markers in qPCR in the setting of SS hypertension. Further work will be devoted to discovering molecular pathways contributing to altered mitochondria structure and function in podocytes in SS hypertension.Support or Funding InformationSupported by NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.