Measuring urine concentration capacity and luminal Na in live mice by intravital multi-photon microscopy
Authors: Wu Andrew , Liao Tang-Dong , Ortiz Pablo 
Published: May 2023

Multiphoton (MP) microscopy of live animals is a useful tool for visualizing the structure and function of the kidney cortex with intact regulatory systems. It has also been applied to monitor ion fluxes (chloride and pH) in the live mouse brain. However, measuring and quantifying dynamic changes in urine concentration capacity and luminal Na in distal tubules have not been done by intravital MP microscopy. We hypothesize that intravital MP microscopy of the kidney can be used to perform dynamic measurements of sodium and urine concentrating capacity in the nephron in vivo. Male C57BL/6 mice were anesthetized with isoflurane and canulated through the left carotid artery. Tetramethylrhodamine-Dextran (TMR-Dextran 10,000 M.W., which filters freely and is not reabsorbed) and CoroNa Green, a Na ion indicator, were injected as a bolus. We exposed the left kidney and imaged proximal and distal tubules using MP microscopy at 850 nm excitation while acquiring emission at >580 nm (TMR-Dextran) and 525/25 nm (CoroNa Green). As expected, dextran concentration was higher in distal tubules after concentration in the loop of Henle and are easily visualized. After obtaining baseline measurements, we injected a timed bolus of furosemide (0.5 mg/kg) to inhibit the Na-K-2Cl cotransporter (NKCC2) and rapidly decrease urine concentration. Given that NKCC2-mediated salt reabsorption is necessary to establish the interstitial concentration gradient, we predicted that furosemide would cause a rapid dilution of dextran and increase sodium concentration in distal tubules. 5 minutes after furosemide treatment, we observed a 33±7% (p<0.05) decrease in TMR-Dextran fluorescence intensity (FI) in distal tubules, which further decreased by 55±19% and reached a plateau after 20 min. There were no significant changes in dextran FI in proximal tubules (n=10 mice). Changes in flow rate and concentrating capacity affect the CoroNa Green FI in distal tubules, independently of Na concentration. Therefore, we ratioed changes in CoroNa Green FI over those of TMR-Dextran. We found that furosemide increased the CoroNa Green/dextran ratio FI in the distal tubules by 42±18% at 5 minutes, which further increased to 131±25% after 20 minutes (p<0.05). There was a minor but significant increase in the CoroNa Green/dextran ratio 3 minutes after furosemide in proximal tubules (n=8), which later returned to baseline. Time controls after saline injection showed no effect on TMR-dextran nor CoroNa Green/dextran FI (n=5). These results indicate that we were able to measure the changes in sodium and urine concentration capacity after furosemide treatment. Our data show that intravital MP microscopy is an effective tool for dynamically measuring urine concentrating capacity in vivo. Future experiments will focus on the effect of other diuretics, salt diets, and hydration status on renal tubular dynamics. NIH-NIDDK, AHA This is the full abstract presented at the American Physiology Summit 2023 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.