Congestive heart failure in rats is associated with increased expression and targeting of aquaporin-2 water channel in collecting duct

We tested whether severe congestive heart failure (CHF), a condition associated with excess free-water retention, is accompanied by altered regulation of the vasopressin-regulated water channel, aquaporin-2 (AQP2), in the renal collecting duct. CHF was induced by left coronary artery ligation. Compared with sham-operated animals, rats with CHF had severe heart failure with elevated left ventricular end-diastolic pressures (LVEDP): 26.9 ± 3.4 vs. 4.1 ± 0.3 mmHg, and reduced plasma sodium concentrations (142.2 ± 1.6 vs. 149.1 ± 1.1 mEq/liter). Quantitative immunoblotting of total kidney membrane fractions revealed a significant increase in AQP2 expression in animals with CHF (267 ± 53%, n = 12) relative to sham-operated controls (100 ± 13%, n = 14). In contrast, immunoblotting demonstrated a lack of an increase in expression of AQP1 and AQP3 water channel expression, indicating that the effect on AQP2 was selective. Furthermore, postinfarction animals without LVEDP elevation or plasma Na reduction showed no increase in AQP2 expression (121 ± 28% of sham levels, n = 6). Immunocytochemistry and immunoelectron microscopy demonstrated very abundant labeling of the apical plasma membrane and relatively little labeling of intracellular vesicles in collecting duct cells from rats with severe CHF, consistent with enhanced trafficking of AQP2 to the apical plasma membrane. The selective increase in AQP2 expression and enhanced plasma membrane targeting provide an explanation for the development of water retention and hyponatremia in severe CHF.

Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells.

The aquaporins (AQPs) are a family of homologous water-channel proteins that can be inserted into epithelial cell plasma membranes either constitutively (AQP1) or by regulated exocytosis following vasopressin stimulation (AQP2). LLC-PK1 porcine renal epithelial cells were stably transfected with cDNA encoding AQP2 (tagged with a C-terminal c-Myc epitope) or rat kidney AQP1 cDNA in an expression vector containing a cytomegalovirus promoter. Immunofluorescence staining revealed that AQP1 was mainly localized to the plasma membrane, whereas AQP2 was predominantly located on intracellular vesicles. After treatment with vasopressin or forskolin for 10 min, AQP2 was relocated to the plasma membrane, indicating that this relocation was induced by cAMP. The location of AQP1 did not change. The basal water permeability of AQP1-transfected cells was 2-fold greater than that of nontransfected cells, whereas the permeability of AQP2-transfected cells increased significantly only after vasopressin treatment. Endocytotic uptake of fluorescein isothiocyanate-coupled dextran was stimulated 6-fold by vasopressin in AQP2-transfected cells but was only slightly increased in wild-type or AQP1-transfected cells. This vasopressin-induced endocytosis was inhibited in low-K+ medium, which selectively affects clathrin-mediated endocytosis. These water channel-transfected cells represent an in vitro system that will allow the detailed dissection of mechanisms involved in the processing, targeting, and trafficking of proteins via constitutive versus regulated intracellular transport pathways.

Serial microanalysis of renal transcriptomes

Large-scale gene expression studies can now be routinely performed on macroamounts of cells, but it is unclear to which extent current methods are valuable for analyzing complex tissues. In the present study, we used the method of serial analysis of gene expression (SAGE) for quantitative mRNA profiling in the mouse kidney. We first performed SAGE at the whole-kidney level by sequencing 12,000 mRNA tags. Most abundant tags corresponded to transcripts widely distributed or enriched in the predominant kidney epithelial cells (proximal tubular cells), whereas transcripts specific for minor cell types were barely evidenced. To better explore such cells, we set up a SAGE adaptation for downsized extracts, enabling a 1,000-fold reduction of the amount of starting material. The potential of this approach was evaluated by studying gene expression in microdissected kidney tubules (50,000 cells). Specific gene expression profiles were obtained, and known markers (e.g., uromodulin in the thick ascending limb of Henle's loop and aquaporin-2 in the collecting duct) were found appropriately enriched. In addition, several enriched tags had no databank match, suggesting that they correspond to unknown or poorly characterized transcripts with specific tissue distribution. It is concluded that SAGE adaptation for downsized extracts makes possible large-scale quantitative gene expression measurements in small biological samples and will help to study the tissue expression and function of genes not evidenced with other high-throughput methods.

Identification of a renal-specific oxido-reductase in newborn diabetic mice

Aldose reductase (ALR2), a NADPH-dependent aldo-keto reductase (AKR), is widely distributed in mammalian tissues and has been implicated in complications of diabetes, including diabetic nephropathy. To identify a renal-specific reductase belonging to the AKR family, representational difference analyses of cDNA from diabetic mouse kidney were performed. A full-length cDNA with an ORF of 855 nt and yielding a ≈1.5-kb mRNA transcript was isolated from a mouse kidney library. Human and rat homologues also were isolated, and they had ≈91% and ≈97% amino acid identity with mouse protein. In vitro translation of the cDNA yielded a protein product of ≈33 kDa. Northern and Western blot analyses, using the cDNA and antirecombinant protein antibody, revealed its expression exclusively confined to the kidney. Like ALR2, the expression was up-regulated in diabetic kidneys. Its mRNA and protein expression was restricted to renal proximal tubules. The gene neither codistributed with Tamm–Horsfall protein nor aquaporin-2. The deduced protein sequence revealed an AKR-3 motif located near the N terminus, unlike the other AKR family members where it is confined to the C terminus. Fluorescence quenching and reactive blue agarose chromatography studies revealed that it binds to NADPH with high affinity (KdNADPH = 66.9 ± 2.3 nM). This binding domain is a tetrapeptide (Met-Ala-Lys-Ser) located within the AKR-3 motif that is similar to the other AKR members. The identified protein is designated as RSOR because it is renal-specific with properties of an oxido-reductase, and like ALR2 it may be relevant in the renal complications of diabetes mellitus.

Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion

Pendrin is an anion transporter encoded by the PDS/Pds gene. In humans, mutations in PDS cause the genetic disorder Pendred syndrome, which is associated with deafness and goiter. Previous studies have shown that this gene has a relatively restricted pattern of expression, with PDS/Pds mRNA detected only in the thyroid, inner ear, and kidney. The present study examined the distribution and function of pendrin in the mammalian kidney. Immunolocalization studies were performed using anti-pendrin polyclonal and monoclonal antibodies. Labeling was detected on the apical surface of a subpopulation of cells within the cortical collecting ducts (CCDs) that also express the H+-ATPase but not aquaporin-2, indicating that pendrin is present in intercalated cells of the CCD. Furthermore, pendrin was detected exclusively within the subpopulation of intercalated cells that express the H+-ATPase but not the anion exchanger 1 (AE1) and that are thought to mediate bicarbonate secretion. The same distribution of pendrin was observed in mouse, rat, and human kidney. However, pendrin was not detected in kidneys from a Pds-knockout mouse. Perfused CCD tubules isolated from alkali-loaded wild-type mice secreted bicarbonate, whereas tubules from alkali-loaded Pds-knockout mice failed to secrete bicarbonate. Together, these studies indicate that pendrin is an apical anion transporter in intercalated cells of CCDs and has an essential role in renal bicarbonate secretion.

Cellular and subcellular localization of the vasopressin- regulated urea transporter in rat kidney.

The renal urea transporter (RUT) is responsible for urea accumulation in the renal medulla, and consequently plays a central role in the urinary concentrating mechanism. To study its cellular and subcellular localization, we prepared affinity-purified, peptide-derived polyclonal antibodies against rat RUT based on the cloned cDNA sequence. Immunoblots using membrane fractions from rat renal inner medulla revealed a solitary 97-kDa band. Immunocytochemistry demonstrated RUT labeling of the apical and subapical regions of inner medullary collecting duct (IMCD) cells, with no labeling of outer medullary or cortical collecting ducts. Immunoelectron microscopy directly demonstrated labeling of the apical plasma membrane and of subapical intracellular vesicles of IMCD cells, but no labeling of the basolateral plasma membrane. Immunoblots demonstrated RUT labeling in both plasma membrane and intracellular vesicle-enriched membrane fractions from inner medulla, a subcellular distribution similar to that of the vasopressin-regulated water channel, aquaporin-2. In the outer medulla, RUT labeling was seen in terminal portions of short-loop descending thin limbs. Aside from IMCD and descending thin limbs, no other structures were labeled in the kidney. These results suggest that: (i) the RUT provides the apical pathway for rapid, vasopressin-regulated urea transport in the IMCD, (ii) collecting duct urea transport may be increased by vasopressin by stimulation of trafficking of RUT-containing vesicles to the apical plasma membrane, and (iii) the rat urea transporter may provide a pathway for urea entry into the descending limbs of short-loop nephrons.