Fluid reabsorption in proximal convoluted tubules of mice with gene deletions of claudin-2 and/or aquaporin1

Abstract

Schnermann J, Huang Y, Mizel D. Fluid reabsorption in proximal convoluted tubules of mice with gene deletions of claudin-2 and/or aquaporin1. Am J Physiol Renal Physiol 305: F1352-F1364, 2013. First published September 18, 2013 doi:10.1152/ajprenal.00342.2013.-Deletions of claudin-2 (Cldn2) and aquaporin1 (AQP1) reduce proximal fluid reabsorption (PFR) by about 30% and 50%, respectively. Experiments were done to replicate these observations and to determine in AQP1/claudin-2 double knockout mice (DKO) if the effects of deletions of these established water pores are additive. PFR was determined in inactin/ketamine-anesthetized mice by free-flow micropuncture using single-nephron I 125 -iothalamate (io) clearance. Animal means of PFR [% of glomerular filtration rate (GFR)] derived from TF/P iothalamate ratios in 12 mice in each of four groups [wild type (WT), Cldn2 Ϫ/Ϫ , AQP1 Ϫ/Ϫ , and DKO) were 45.8 Ϯ 0.85 (51 tubules), 35.4 Ϯ 1 (54 tubules; P Ͻ 0.01 vs. WT), 36.8 Ϯ 1 (63 tubules; P Ͻ 0.05 vs. WT), and 33.9 Ϯ 1.4 (69 tubules; P Ͻ 0.01 vs. WT). Kidney and single-nephron GFRs (SNGFR) were significantly reduced in all mutant strains. The direct relationship between PFR and SNGFR was maintained in mutant mice, but the slope of this relationship was reduced in the absence of Cldn2 and/or AQP1. Transtubular osmotic pressure differences were not different between WT and Cldn2 Ϫ/Ϫ mice, but markedly increased in DKO. In conclusion, the deletion of Cldn2, AQP1, or of both Cldn2 and AQP1 reduces PFR by 22.7%, 19.6%, and 26%, respectively. Our data are consistent with an up to 25% paracellular contribution to PFR. The reduced osmotic water permeability caused by absence of AQP1 augments luminal hypotonicity. Aided by a fall in filtered load, the capacity of non-AQP1-dependent transcellular reabsorption is sufficient to maintain PFR without AQP1 and claudin-2 at 75% of control. micropuncture; iothalamate; tubular fluid osmolarity; paracellular; transcellular SEEMINGLY EXCESSIVE AMOUNTS of water and solutes crossing the glomerular filtration barrier are reduced by the renal tubular epithelium to the small quantities that must be excreted in the urine to achieve fluid volume and blood pressure homeostasis. All segments of the nephron participate in this vital task of fluid and solute retrieval, but the greatest reabsorptive burden is carried by the proximal tubules of the nephron. The architecture of the proximal tubular epithelium suggests two principal pathways of transtubular movement: permeation through the epithelial cells (transcellular) or permeation between the epithelial cells (paracellular). There is agreement that solutes and water use both routes to gain access to the blood, but estimates of the magnitudes of transcellular vs. paracellular absorption rates have been difficult to establish. Nevertheless, measurements of transtubular and transmembrane fluxes by micropuncture, microperfusion, and membrane fractionation together with the subsequent identification and manipulation of the major solute transport proteins...

Significant water absorption goes paracellular in kidney proximal tubules

“…Thus, claudin-2 so far is the only claudin for which water permeability has been demonstrated. The present study by Schnermann et al (9) has convincingly proven and quantified that concept for kidney proximal tubules.…”

“…This was observed both in the absence and presence of claudin-2. In mice deficient of both AQP-1 and claudin-2, a reduction of fluid flow by 26% was found, which was not significantly different from values measured in singly deficient animals (9). This was an unexpected finding of the study, which has not be sufficiently explained yet.…”

“…This result corroborates the concept of a claudin-2-based paracellular water permeability and convincingly demonstrates a paracellular contribution to proximal tubule fluid reabsorption of 25%. Schnermann et al (9) performed a comprehensive analysis not only of proximal fluid reabsorption, but also on kidney and single-nephron filtration rate on a large number of mice. Furthermore, in a detailed expression analysis of wild-type mice and mice deficient for claudin-2, AQP1, and both, the authors demonstrated that the findings are due to the deletion of the specific protein and not to further alterations in gene expression.…”

Short-Term Functional Adaptation of Aquaporin-1 Surface Expression in the Proximal Tubule, a Component of Glomerulotubular Balance

“…22 Absence of the main proximal tubular sodium transporter, NHE3, or the principal water channel, AQP1, did not entirely abolish GTB but significantly reduced the slope of flow-dependent volume absorption. 16,18,23,24 We aimed to resolve whether the surface expression of AQP1 may be altered in the short term by inducing changes in luminal flow rate and FSS. The selected flow rates for the isolated, microperfused tubules reflected the end proximal physiologic range of 5-30 nl/min.…”

SIRT2 Regulates LPS-Induced Renal Tubular CXCL2 and CCL2 Expression

“…28 Anti-SIRT2 (Abcam, Inc., Cambridge, MA), anti-AQP1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-AQP 2 (Santa Cruz Biotechnology), anti-Tamm-Horsfall glycoprotein (MP Biomedicals, Verona, Italy), anti-Gr-1 (BD Pharmingen Bioscience, San Diego, CA), anti-ER-HR3 (BMA Biomedical, Augst, Switzerland), and FITC-labeled Lotus tetragonolobus lectin (Vector Laboratories, Burlingame, CA) were used for frozen sections of mouse kidney. 29,30 Secondary Alexa Fluor 488-or Alexa Fluor 555-conjugated antibodies against rat, rabbit, or mouse immunoglobulins (1:1000; Invitrogen, Carlsbad, CA) were used to visualize antigen-antibody complexes. Nuclei were stained with 49, 6-diamidino-2-phenylindole.…”