We used in situ hybridization and immunocytochemistry with polyclonal antibodies against the mouse bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter (mBSC2) to determine the location of this cotransporter in rat brain. Northern blots and in situ hybridization showed the presence of cotransporter mRNA in the brain, with an especially high level of expression in the choroid plexus (CP). Affinity-purified anti-BSC2 antibody identified proteins of 145-155 kDa on Western blot analysis and immunoprecipitation of brain and CP membrane protein. Indirect immunofluorescence demonstrated that BSC2 protein is located on the apical surface of the CP and is heterogeneously distributed in cell bodies and dendrites of neurons in the central and peripheral nervous system. The apical localization of BSC2 in the CP was confirmed by 86Rb+ uptakes in primary cultures of CP cells grown on permeable filters and confocal immunofluorescence microscopy. The apical localization of the cotransporter in CP epithelium suggests a role for the cotransporter in cerebrospinal fluid K+ homeostasis. In neurons, the cotransporter may help regulate intracellular Cl- concentration and thereby affect neuronal response to gamma-aminobutyric acid.

The ATP-sensitive, inwardly rectifying K+ channel, ROMK, has been suggested to be the low-conductance ATP-sensitive K+ channel identified in apical membranes of mammalian renal thick ascending limb (TAL) and cortical collecting duct (CCD). Mutations in the human ROMK gene (KIR1.2) have been identified in kindreds with neonatal Bartter’s syndrome. In the present study, we generated polyclonal antibodies raised against both a COOH-terminal (amino acids 252–391) ROMK-maltose binding protein (MBP) fusion protein and an NH2-terminal (amino acids 34–49) ROMK peptide. Affinity-purified anti-ROMK COOH-terminal antibody detected the 45-kDa ROMK protein in kidney tissues and HEK-293 cells transfected with ROMK1 cDNA. The antibody also recognized 85- to 90-kDa proteins in kidney tissue; these higher molecular weight proteins were abolished by immunoabsorption with ROMK-MBP fusion protein and were also detected on Western blots using anti-ROMK NH2-terminal antibody. Immunofluorescence studies using anti-ROMK COOH-terminal antibody showed intense apical staining along the loop of Henle and distal nephron; staining with preimmune and immunoabsorbed serum was negative. When colocalized with distal nephron markers [the thiazide-sensitive cotransporter (rTSC1), the bumetanide-sensitive cotransporter (rBSC1), the vacuolar type H+-ATPase, and neuronal nitric oxide synthase (NOS I)], the ROMK protein was found primarily at the apical border of cells in the TAL, macula densa, distal convoluted tubule, and connecting tubule. Within the CCD, the ROMK protein was expressed in principal cells and was absent from intercalated cells. The tubule localization and polarity of ROMK staining are consistent with the distribution of ROMK mRNA and provide more support for ROMK being the low-conductance K+secretory channel in the rat distal nephron.

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The pathogenetic factors involved in norepinephrine- (NE) induced reversible acute renal failure (ARF) were examined in untreated (U) and mannitol-treated (M) animals. At 3 and 24 h after NE infusion renal blood flow (RBF) was significantly higher in M compared to U animals (174 vs. 138 and 191 vs. 148 ml/min, respectively, both P less than 0.05). At 3 h, glomerular filtration rate (GFR) was higher in M animals (8 vs. 4 ml/min, P less than 0.01), while at 24 h protection was even greater (18 vs. 3 ml/min, P less than 0.01). In U animals proximal tubule pressure (Pt) was lower at 1 h than before NE (13 vs. 23 mmHg, P less than 0.01); from 1 to 3 h Pt increased to elevated levels in parallel with restoration of RBF (r = 0.62, P less than 0.01). At 3 h in U animals stop-flow pressure (SFP), as an index of glomerular capillary pressure, was below normal (35 vs. 44 mmHg, P less than 0.05) yet Pt was increased (35 vs. 23 mmHg, P less than 0.05). Thus little transglomerular pressure gradient was present for ultrafiltration. Further evidence of tubular obstruction was obtained by microperfusion at 6 nl/min, which increased Pt from 30 to 45 mmHg (P less than 0.001), a finding not present in unobstructed tubules. Delayed excretion (approximately 20 min) of microinjected [3H]inulin also was compatible with renal ischemia and tubule obstruction. Microinjection studies provided no evidence for backleak of tubular fluid. At 1 h, Pt was higher in M vs. U animals (31 vs. 13 mmHg, P less than 0.05). In M animals at 3 h SFP was normal (50 vs. 44 mmHg) and Pt was below SFP (32 vs. 50 mmHg, P less than 0.01), thus preserving a substantial transglomerular pressure gradient for ultrafiltration. In summary, reduced GFR in U animals is characterized by a combination of reduced glomerular capillary pressure and tubule obstruction. In contrast, animals receiving mannitol were protected against ARF through maintenance of glomerular capillary pressure and prevention of tubular obstruction, perhaps by increasing Pt within the first hour of the NE insult.

CQI can enhance educational program quality and outcomes, if the process is designed to collect relevant information and the results are used for program improvement.