Intravital two-photon microscopy of SuperClomeleon in live mice detected a 13.1±2.0% decrease in the Cl-dependent FRET ratio following hypertonic saline and bumetanide infusion (p<0.01).
Intravital two-photon microscopy of SuperClomeleon can successfully measure dynamic changes in intracellular chloride concentration in the distal convoluted tubules of live mice.
p-value: p=< 0.01
Distal tubule-mediated NaCl reabsorption plays an essential role in salt homeostasis and blood pressure regulation. Cl reabsorption in the DCT occurs via the apical Na/Cl cotransporter NCC (Slc12A3) and is regulated by exit pathways via the Cl channel CLC-K2 and K/Cl cotransporter (KCC). Changes in intracellular Cl concentration (Cli) in the DCT play an essential role in regulating NCC. Despite its importance, few methods have been developed to measure NaCl transport or Cli in DCT in live animals, with the last methods relying on in vivo micropuncture. Previous measurements in the 1990s using microelectrodes or fluorescence in isolated DCT showed that baseline cytoplasmic Cl is 7-10 mM. Mulitphoton (MP) microscopy is a powerful tool for measuring kidney function in live mice with intact regulatory systems. SuperClomeleon is a FRET-based fluorescent protein consisting of a Cl-sensitive Topaz/YFP coupled to a Cl-insensitive Cerulean/CFP. It has been used to measure Cli in hippocampal neurons with 2P-microcopy and has a reported Kd ranging from 9-24 mM Cl. We hypothesize that intravital 2P microscopy of SuperClomeleon, expressed specifically in DCT, can be used to perform dynamic measurements of cytoplasmic Cl and NCC activity in the distal tubule in vivo. To generate SuperClomeleon expression in the distal tubule, we crossed a flox-stop-flox SuperClomeleon mouse line with Slc12a3-Cre-ERT2 mice to produce iDistal-SuperClomeleon mice. Male iDistal-SuperClomeleon mice were fed tamoxifen diet for 3 weeks, then rested for one week, anesthetized with isoflurane and canulated through the left carotid artery. The left kidney was exposed and imaged by 2P microscopy. To increase distal NaCl delivery, we infused a bolus of hypertonic saline and loop diuretic furosemide in 1 minute. Using the Na-sensitive CoroNa Green fluorescent indicator, we found this caused a large increase in Na delivery to the distal nephron (ΔFi = 53.8±15.8%, n = 5 mice, p< 0.05). To image SuperClomeleon and measure changes in FRET in DCT, we used 830 nm excitation while acquiring emission at 535nm (YFP) and 485 nm (CFP). We found that it was very easy to visualize DCTs expressing SuperClomeleon in the cortex, at a depth of 50-70 µm from the capsule, and 3-6 DCT were observed per field. When we increased distal NaCl delivery (hypertonic saline plus 20mg/kg bumetanide), we observed a rapid decrease in the Cl-dependent FRET ratio (YFP/CFP) within 2 min, which plateaued by 10 min, indicative of an increase in Cli concentration in DCT. The maximal decrease in YFP/CFP ratio averaged 13.1±2.0% from baseline at 4±1 min (n = 13 tubules in 4 mice, p< 0.01). Our data support that SuperClomeleon can be used to measure dynamic changes in Cli in DCT in vivo. We continue working on a protocol to measure NCC activity in DCT by quantifying the initial rate of Cl entry. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Wu et al. (Fri,) conducted a other in Intracellular chloride measurement in distal convoluted tubules (n=9). Hypertonic saline plus bumetanide vs. Baseline was evaluated on Maximal decrease in YFP/CFP FRET ratio from baseline (p=< 0.01). Intravital two-photon microscopy of SuperClomeleon in live mice detected a 13.1±2.0% decrease in the Cl-dependent FRET ratio following hypertonic saline and bumetanide infusion (p<0.01).