Volume 84, Issue 2, Pages 277-284 (August 2013) The calcium sensing receptor modulates fluid reabsorption and acid secretion in the proximal tubule  Giovambattista Capasso, Peter J. Geibel, Sara Damiano, Philippe Jaeger, William G. Richards, John P. Geibel  Kidney International  Volume 84, Issue 2, Pages 277-284 (August 2013) DOI: 10.1038/ki.2013.137 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Luminal Ca2+ dose curve on fluid reabsorption (Jv) in rat proximal tubule. Bath Ca2+ was maintained at a constant 1.2mmol/l. Data are from three tubules per concentration, with three collections made for each concentration and five rats per data set. N.S., not significant. Kidney International 2013 84, 277-284DOI: (10.1038/ki.2013.137) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Increasing luminal Ca2+ concentration enhances net fluid reabsorption (Jv, nl/mm/min) of proximal tubule in the Casr+/+::Gcm2+/+ mouse. All data are the means±s.d. from three sequential collections per tubule, with two tubules per mouse used and five animals used for each group. P-values were calculated using GraphPad and a paired Student’s t-test. Control Jv was a solution that contained 1.2mmol/l CaCl. This solution did not differ from the control perfusate; 1.2mmol/l CaCl solutions were identical to the initial control, except that they were added at a different randomized perfusion time point. The order of solution exchanges in the lumen was randomized; to prevent contamination from the previous solution after the exchange, fluid generated during the first 5min following the switch was collected and discarded. N.S., not significant. Kidney International 2013 84, 277-284DOI: (10.1038/ki.2013.137) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Variations of luminal Ca2+ concentration in the Casr−/−::Gcm2−/− mouse and the effects on fluid reabsorption (Jv). Note that lack of a functional receptor prevents Ca2+ from having an effect on Jv. The data represent three sequential collections under each experimental condition. There were three tubules per mouse, and five mice were used for the study. All data are the means±s.d. from three sequential collections per tubule, with three tubules per mouse used and five animals for each group. P-values were calculated using GraphPad and a paired Student’s t-test. Control Jv was a solution that contained 1.2mmol/l CaCl. This solution did not differ from the control perfusate. This was the Jv that was recorded following perfusion with either a low (0.1mmol/l) or high (2.0mmol/l) CaCl solution. The order of solution exchanges in the lumen was randomized; to prevent contamination from the previous solution after the exchange, fluid generated during the first 5min following the switch was collected and discarded. N.S., not significant. Kidney International 2013 84, 277-284DOI: (10.1038/ki.2013.137) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 The effect of changes in luminal Ca2+ concentration on the rate of fluid absorption in single in vivo microperfused rat proximal tubules. Data shown are pooled from 12 tubules (5 rats). Each tubule was sequentially perfused with the perfusion solutions containing different Ca2+ concentrations. N.S., not significant. Kidney International 2013 84, 277-284DOI: (10.1038/ki.2013.137) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Effects of calcium-sensing receptor activator R-568 on Jv in Casr+/+::Gcm2+/+ mice perfused in vitro. Please note that addition of R-568 to the luminal perfusate in the continued presence of low Ca2+ increased fluid reabsoprtion (Jv) to levels comparable to control. N.S., not significant. Kidney International 2013 84, 277-284DOI: (10.1038/ki.2013.137) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 Effects of the calcium-sensing receptor activator R-568 on fluid reabsorption (Jv) in Casr−/−::Gcm2−/− mice perfused in vitro. Note that addition of R-568 to the luminal perfusate in the continued presence of low Ca2+ did not modify Jv from initial low levels. N.S., not significant. Kidney International 2013 84, 277-284DOI: (10.1038/ki.2013.137) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 7 Effect of calcium-sensing receptor activator R-568 (100nmol/l) on absolute fluid absorption (Jv) in proximal tubule of rats. The addition of R-568 to the luminal perfusate increased Jv to values comparable to those obtained with 2mmol/l Ca2+ in the perfusion solution. N.S., not significant. Kidney International 2013 84, 277-284DOI: (10.1038/ki.2013.137) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 8 Effects of luminal perfusion of R-568 on Na+-dependent acid extrusion from Casr+/+::Gcm2+/+ and Casr−/−::Gcm2−/− mice. Addition of 400nmol/l of the calcimimetic R-568 leads to a significant increase in Na+-dependent proton extrusion from mouse proximal tubules. The lack of a functional calcium-sensing receptor (CaSR) prevents an enhanced sodium–hydrogen exchanger (NHE) activity in the tubule. Kidney International 2013 84, 277-284DOI: (10.1038/ki.2013.137) Copyright © 2013 International Society of Nephrology Terms and Conditions