Monitoring Hydrogen Peroxide (H2O2) in live kidneys by Multiphoton Microscopy and genetically encoded probes

Abstract

Hydrogen Peroxide (H2O2) is an important physiological factor in the control of renal function. In the kidney, H2O2 is enhanced in some forms of hypertension and in diabetes, where it may play a role in renal damage. We previously found that H2O2 potently stimulates renin release from mouse juxtaglomerular (JG) cells. While all cells have the capacity to produce H2O2, the relative production levels in the different cell types of the kidney cortex (glomeruli vs proximal tubules vs JG cells), is not clear. Most H2O2 measurements have been performed ex‐vivo or in isolated renal cells, but few measurements of H2O2 have been performed in vivo. To begin addressing this, we used a genetically encoded H2O2 sensor named HyPer and multi‐photon (MP) microscopy imaging of the live mouse kidney. Before using HyPer in vivo, experiments were conducted in cultured cells expressing HyPer to determine the best wavelength for MP excitation and emission, by monitoring the increase in fluorescence after addition of H2O2. Then, HyPer was transduced into the renal cortex by subcapsular infusion of a plasmid, for 7 days. After transduction, mice were anesthetized with isoflurane, the left kidney exteriorized and a carotid cannula for infusion was placed. The renal cortical vasculature and glomeruli were visualized by using Rhodmaine‐R18 simultaneously with HyPer Imaging. We observed strong HyPer fluorescence primarily in proximal tubules, and in a few cases inside glomeruli. We then tested the ability of PEG‐Catalase plus the H2O2 scavenger Ebselen to decrease the HyPer signal in proximal tubules. After finding a region to image, baseline measurements were obtained for 5 minutes at 15 sec intervals, and then Catalase/Ebselen was infused into the carotid artery. Fluorescence was monitored for another 25 minutes. We observed a steady decrease in fluorescence over time that reached 35±5% after 20 minutes (p<0.05, n=3), suggesting a decrease in H2O2. Monitoring H2O2 in specific cell types can be performed by using cell‐selective promoters to drive expression of HyPer. To monitor H2O2 in JG cells we generated adenoviruses encoding HyPer under control of the Ren1 (renin 1) promoter. Renal subcapsular catheters were used to infuse adenoviruses for 7 days. MP imaging was performed to locate glomeruli expressing Hyper in the afferent arteriole or glomerular pole. After finding this, baseline measurements were performed for five minutes and the Catalase/Ebselen was infused. We observed a steady decrease in fluorescence over time that reached 33±7% after 20 minutes (p<0.05, n=3), suggesting a decrease in H2O2 in JG cells expressing HyPer. We conclude that HyPer can be used to monitor and compare H2O2 levels in different cell types in the kidney cortex by using cell‐type specific promoters and MP microscopy. This method should allow the comparison of H2O2 levels in renal cortical cell types between different strains or during pathological conditions in live animals.Support or Funding InformationNIH 5R03DK105300‐03 to MMThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.