Requirement of aquaporin-1 for NaCl-driven water transport across descending vasa recta

Abstract

Deletion of AQP1 in mice results in diminished urinary concentrating ability, possibly related to reduced NaCl- and urea gradient-driven water transport across the outer medullary descending vasa recta (OMDVR). To quantify the role of AQP1 in OMDVR water transport, we measured osmotically driven water permeability in vitro in microperfused OMDVR from wild-type, AQP1 heterozygous, and AQP1 knockout mice. OMDVR diameters in AQP1(-/-) mice were 1.9-fold greater than in AQP1(+/+) mice. Osmotic water permeability (P(f)) in response to a 200 mM NaCl gradient (bath > lumen) was reduced about 2-fold in AQP1(+/-) mice and by more than 50-fold in AQP1(-/-) mice. P(f) increased from 1015 to 2527 microm/s in AQP1(+/+) mice and from 22 to 1104 microm/s in AQP1(-/-) mice when a raffinose rather than an NaCl gradient was used. This information, together with p-chloromercuribenzenesulfonate inhibition measurements, suggests that nearly all NaCl-driven water transport occurs by a transcellular route through AQP1, whereas raffinose-driven water transport also involves a parallel, AQP1-independent, mercurial-insensitive pathway. Interestingly, urea was also able to drive water movement across the AQP1-independent pathway. Diffusional permeabilities to small hydrophilic solutes were comparable in AQP1(+/+) and AQP1(-/-) mice but higher than those previously measured in rats. In a mathematical model of the medullary microcirculation, deletion of AQP1 resulted in diminished concentrating ability due to enhancement of medullary blood flow, partially accounting for the observed urine-concentrating defect.

Figure 1

Light micrographs of OMDVR from AQP1^+/+ mice and AQP1^–/– mice. Four OMDVR from AQP1^+/+ mice are shown in the left panel, and 3 OMDVR from AQP1^–/– mice are shown at right. Deletion of AQP1 leads to an increase in OMDVR diameter.

Figure 2

P_f of OMDVR from AQP1^+/+, AQP1^+/–, and AQP1^–/– mice. P_f was measured in microperfused AQP1^+/+ (n = 11), AQP1^+/– (n = 8), and AQP1^–/– (n = 11) OMDVR by driving water flux across the walls with a transmural gradient of NaCl (bath, 350 mM; lumen, 150 mM). Osmotic equilibration of NaCl along the axis of the microperfused vessel was measured with microchloride assay of the bath perfusate and collectate. Compared with wild-type mice, P_f was lower in heterozygotes and was nearly zero in homozygote AQP1 null mice (P < 0.05, all comparisons).

Figure 3

Osmotic water transport across AQP1^+/+ and AQP1^–/– OMDVR wall. (a) Left: Collectate fluorescence reversibly rose as raffinose or NaCl was added to and then removed from the bath of a microperfused AQP1^+/+ OMDVR. Despite the much larger transmural NaCl gradient (400 mOsm/L), raffinose (200 mM) concentrated the FITCDx volume marker with equal effectiveness. After 30 minutes of incubation in 2 mM pCMBS, raffinose continued to drive water efflux, but NaCl was ineffective. Right: Collectate fluorescence reversibly rose as raffinose was added to and then removed from the bath, but NaCl was ineffective at driving water movement across the wall of a microperfused AQP1^–/– OMDVR. Raffinose-driven water efflux was not reduced by pCMBS. Raf, raffinose. (b) The effect of pCMBS on P_f of AQP1^+/+ OMDVR was tested. Paired measurements of P_f were obtained using transmural raffinose and NaCl gradients in random order. In all vessels, raffinose was more effective than was NaCl at inducing osmotic water movement (P < 0.05). After 30 minutes of incubation with pCMBS (2 mM), P_f measured with NaCl was reduced to nearly zero, but P_f measured with raffinose was only partly inhibited (P < 0.05 for both comparisons). A 5-minute treatment with DTT (5 mM) reversed the pCMBS effects. (c) The effect of pCMBS of inhibiting water flux across the AQP1^–/– OMDVR wall was tested. P_f was measured in OMDVR from AQP1^–/– mice as water efflux was driven by the addition of raffinose (200 mM) to the bath. Vessels were incubated for 30 minutes in pCMBS (2 mM, n = 7) or vehicle (n = 4). The pathway across which raffinose drives water efflux in AQP1^–/– OMDVR wall is insensitive to mercurials.

Figure 4

Summary of measurements of P_f. All P_f measurements are summarized (ordinate, mean ± SEM) for AQP1^–/– mice (left) and AQP1^+/+ mice (right). The solute used to drive water movement is shown on the abscissa. GA-fixed vessels were used except for 8 AQP1^–/– vessels. P_f was the same when measured with raffinose whether or not the GA fixation step was included. For purposes of calculating P_f, the influx of the solute used to drive water movement was most often calculated by measuring lumen-to-bath efflux of the equivalent isotope and simulating the experiment with a mathematical model (see Methods). In AQP1^+/+ OMDVR, osmolar equilibration was monitored in 2 ways: by measuring efflux of ²²Na (n = 14) or measuring influx of NaCl by microassay of collectate chloride (n = 11; see Figure 2). These approaches yielded similar values. Note that the sum of P_f measured with NaCl in AQP1^+/+ vessels (AQP1 only) and P_f measured with raffinose in AQP1^–/– vessels (non-AQP1 only) is equal to P_f measured with raffinose in AQP1^+/+ OMDVR (AQP1 and non-AQP1 pathways). The number of vessels in each group is shown on the figure beside the corresponding error bar.

Figure 5

Summary of diffusive permeabilities of mouse OMDVR. All measurements of diffusional permeabilities to the tracers shown on the abscissa are summarized (mean ± SEM). In both AQP1^+/+ OMDVR and AQP1^–/– OMDVR, permeabilities were uniformly high. The number of vessels in each group is shown on the figure beside the corresponding error bar.

Figure 6

Molecular sieving of [³H]raffinose and [¹⁴C]inulin by AQP1^–/– OMDVR. To demonstrate molecular sieving across the non-AQP1 pathway, water efflux was driven by adding raffinose (200 mM) to the bath of AQP1^–/– vessels perfused with ²²Na, [³H]raffinose, or [¹⁴C]inulin. (a and b) Water efflux occurred in response to the raffinose gradient as documented by a fall in collection rate (Q_c) and a rise in R_Dx. (c–e) Collectate-to-perfusate activity ratios of the tracers (R_Na, R_raf, and R_IN) were measured during zero volume flux (J_v = 0) and raffinose-driven volume flux (J_v > 0). Compared with J_v = 0, R_raf and R_IN increased when J_v = + (P < 0.05), demonstrating molecular sieving (ς_raf and ς_in > 0).

Figure 7

Effect of AQP1 deletion on predicted renal medullary interstitial osmolality. The predicted interstitial osmolality is shown as a function of corticomedullary axis (corticomedullary junction: x/L = 0; papillary tip: x/L = 1) for various values of DVR P_f. AQP1 expression in OMDVR is predicted to enhance concentrating ability by mediating water efflux from DVR to AVR, secondarily reducing blood flow to the papillary tip.