Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice

Abstract

To investigate the role of aquaporin-1 (AQP1) water channels in proximal tubule function, in vitro proximal tubule microperfusion and in vivo micropuncture measurements were done on AQP1 knockout mice. The knockout mice were generated by targeted gene disruption and found previously to be unable to concentrate their urine in response to water deprivation. Unanesthetized knockout mice consumed 2.8-fold more fluid than wild-type mice and had lower urine osmolality (505 +/- 40 vs. 1081 +/- 68 milliosmolar). Transepithelial osmotic water permeability (Pf) in isolated microperfused S2 segments of proximal tubule from AQP1 knockout [-/-] mice was 0.033 +/- 0.005 cm/s (SE, n = 6 mice, 37 degreesC), much lower than that of 0.15 +/- 0.03 cm/s (n = 8) in tubules from wild-type [+/+] mice (P < 0.01). In the presence of isosmolar luminal perfusate and bath solutions, spontaneous fluid absorption rates (nl/min/mm tubule length) were 0.31 +/- 0.12 (-/-, n = 5) and 0.64 +/- 0.15 (+/+, n = 8). As determined by free-flow micropuncture, the ratios of tubular fluid-to-plasma concentrations of an impermeant marker TF/P in end proximal tubule fluid were 1.36 +/- 0. 05 (-/-, n = 8 mice [53 tubules]) and 1.95 +/- 0.09 (+/+, n = 7 mice [40 tubules]) (P < 0.001), corresponding to 26 +/- 3% [-/-] and 48 +/- 2% [+/+] absorption of the filtered fluid load. In collections of distal tubule fluid, TF/P were 2.8 +/- 0.3 [-/-] and 4.4 +/- 0.5 [+/+], corresponding to 62 +/- 4% [-/-] and 76 +/- 3% [+/+] absorption (P < 0.02). These data indicate that AQP1 deletion in mice results in decreased transepithelial proximal tubule water permeability and defective fluid absorption. Thus, the high water permeability in proximal tubule of wild-type mice is primarily transcellular, mediated by AQP1 water channels, and required for efficient near-isosmolar fluid absorption.

Figure 1

P_f and J_v in isolated microperfused proximal tubules. S2 segments of proximal tubule were dissected from wild-type [+/+] and AQP1 knockout [−/−] mice and microperfused in vitro at 37°C as described in Methods. (A) Osmotic water permeability. Tubules were perfused with 295 mosM buffer and bathed in a 345 mosM buffer to give a 50 mosM bath-to-lumen osmotic gradient. P_f was computed from the increase in concentration of a membrane-impermeant marker at the distal end of the tubule. (B) Near-isosmolar fluid reabsorption. Tubules were perfused and bathed in an isosmolar buffer. J_v was computed from the increase in luminal marker concentration. For A and B, each point is the averaged data from 1 or 2 tubules from one mouse. Averaged data with SEs (n = number of different mice) is shown at the right of each data set.

Figure 2

Blood pressure and urinary parameters in anesthetized mice prepared for micropuncture. (A) Mean arterial blood pressure (MAP). (B) Urine flow. (C) Urine osmolality. (D) Glomerular filtration rate. Each point represents averaged measurements from one mouse. Averaged data with SEs is shown at the right of each data set. Significance values are given in Table 1.

Figure 3

Free-flow micropuncture data. Averaged data shown for each mouse that underwent micropuncture with end proximal tubule fluid collection (A) and distal tubule fluid collection (B). See Methods for details and Table 1 for significance values. TF/P, ratio of marker in collected tubule fluid vs. plasma; SNGFR, single nephron GFR; % abs, percentage fluid absorption.