Prostanoid signaling, localization, and expression of IP receptors in rat thick ascending limb cells.

It is widely held that only one prostacyclin (IP) receptor exists that can couple to guanine stimulatory nucleotide binding proteins (Gs) leading to activation of adenyl cyclase. Although IP receptor mRNA is expressed in vascular arterial smooth muscle cells and platelets, with lower level expression in mature thymocytes, splenic lymphocytes, and megakaryocytes, there is no molecular evidence for IP receptor expression in renal epithelial cells. The purpose of the present study was to obtain molecular evidence for the expression and localization of the IP receptor and to study the signaling pathways of IP receptor in rat medullary thick ascending limb (MTAL). Biochemical studies showed that IP prostanoids do not increase cAMP in rat MTAL. However, in the presence of vasopressin, inhibition of cAMP formation by prostacyclin (PGI2) analogs is pertussis toxin sensitive and does not activate protein kinase C. In situ hybridization studies localized IP receptor mRNA expression to MTAL in the rat kidney outer medulla. The results of RT-PCR of freshly isolated RNA from MTAL, with primers specific for the mouse IP receptor cDNA, produced an amplification product of the correct predicted size that contained an expected Nco I endonuclease restriction site. We conclude that rat renal epithelial cells express the IP receptor, coupled to inhibition of cAMP production.

Localization of the ROMK protein on apical membranes of rat kidney nephron segments.

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 (KIR 1.2) 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. Immunofluoresence 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.

Hypercalcemia stimulates expression of intrarenal phospholipase A2 and prostaglandin H synthase-2 in rats. Role of angiotensin II AT1 receptors.

In chronic hypercalcemia, inhibition of thick ascending limb sodium chloride reabsorption is mediated by elevated intrarenal PGE2. The mechanisms and source of elevated PGE2 in hypercalcemia are not known. We determined the effect of hypercalcemia on intrarenal expression of cytosolic phospholipase A2 (cPLA2), prostaglandin H synthase-1 (PGHS-1), and prostaglandin H synthase-2 (PGHS-2), enzymes important in prostaglandin production. In rats fed dihydrotachysterol to induce hypercalcemia, Western blot analysis revealed significant upregulation of both cPLA2 and PGHS-2 in the kidney cortex and the inner and outer medulla. Immunofluorescence localized intrarenal cPLA2 and PGHS-2 to interstitial cells of the inner and outer medulla, and to macula densa and cortical thick ascending limbs in both control and hypercalcemic rats. Hypercalcemia had no effect on intrarenal expression of PGHS-1. To determine if AT1 angiotensin II receptor activation was involved in the stimulation of cPLA2 and PGHS-2 in hypercalcemia, we treated rats with the AT1 receptor antagonist, losartan. Losartan abolished the polydipsia associated with hypercalcemia, prevented the increase in cPLA2 protein in all regions of the kidney, and diminished PGHS-2 expression in the inner medulla. In addition, losartan completely prevented the increase in urinary PGE2 excretion in hypercalcemic rats. Intrarenal levels of angiotensin II were unchanged in hypercalcemia. These data indicate that hypercalcemia stimulates intrarenal cPLA2 and PGHS-2 protein expression. Our results further support a role for angiotensin II, acting on AT1 receptors, in mediating this stimulation.

Expression of the Na(+)-K(+)-2Cl- cotransporter BSC2 in the nervous system.

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.

Rabbit cortical collecting ducts express a novel prostacyclin receptor.

Prostaglandin E2 (PGE2) inhibits vasopressin-stimulated water conductivity (AVP-Lp) and inhibits Na+ reabsorption in the rabbit cortical collecting duct (CCD). Inhibition of Na+ reabsorption is mediated by increased intracellular calcium ion concentration ([Ca2+]i). Prostacyclin (PGI2) has also been shown to inhibit Na+ reabsorption in the CCD. The present studies were designed to examine the effect of the PGI2 agonist, Iloprost (ILP), on AVP-Lp and [Ca2+ in the isolated perfused rabbit CCD and to determine whether ILP activates different receptors than PGE2. ILP and PGE2 each maximally inhibited AVP-Lp equipotently at 10(-7) M. When CCDs were exposed to PGE2 and ILP simultaneously, or if PGE2 was added in the presence of ILP, inhibition of AVP-Lp was additive. Additivity was not observed if the PGI2 agonist, carbaprostacyclin (c-PGI2), was added with ILP, or if the PGE2 agonist, sulprostone, was added with PGE2, or if ILP was added to CCDs preexposed to PGE2. In fura 2-loaded CCD, ILP and PGE2 added separately increased [Ca2+]i. The response to c-PGI2 could be desensitized by prior exposure to ILP. ILP did not cause desensitization to PGE2, but PGE2 could desensitize the CCD to ILP. We conclude that PGI2 inhibits AVP-Lp by activation of a novel IP3 prostacyclin receptor and increases [Ca2+]i by activation of an IP1 prostacyclin receptor in the rabbit CCD. Functional evidence is presented that PGI2 cannot occupy PGE2 receptors and that PGE2 can occupy but cannot activate PGI2 receptors linked to inhibition of AVP-Lp.

Endogenous prostaglandin E2 mediates inhibition of rat thick ascending limb Cl reabsorption in chronic hypercalcemia.

The hypothesis that endogenous PGE2 mediates defective thick ascending limb (TAL) Cl reabsorption (percent delivered load: FRCl%) in rats with vitamin D-induced chronic hypercalcemia (HC) was tested by measuring FRCl% in loop segments microperfused in vivo in HC and control rats treated acutely with indomethacin (Indo) or its vehicle, and obtaining the corresponding outer medullary [PGE2]. Microperfusion conditions were developed in which FRCl% was exclusively furosemide sensitive. To determine the cellular mechanism, tubules were perfused acutely with forskolin (FSK), cAMP, or the protein kinase C inhibitor staurosporine (SSP). Outer medullary [PGE2] in HC rats was 9 to 10 times greater than control and could be normalized by Indo. FRCl% was 20% lower in HC rats infused with vehicle, and Indo, FSK, and cAMP returned FRCl% to normal despite sustained HC. Indo or FSK had no effect on FRCl% in control rats and Indo did not prevent inhibition of FRCl% by luminal PGE2 (1 microM). Luminal SSP (10(-7), 10(-8) M) in HC did not return FRCl% to control values. We conclude that impaired TAL FRCl% in HC occurs at a pre-cAMP site and is due to endogenous PGE2 and not to HC.

K infusion corrects thick ascending limb Cl reabsorption in K-depleted rats by an aldosterone-independent mechanism.

Others have provided evidence that thick ascending limb (TAL) NaCl reabsorption is aldosterone dependent in adrenalectomized animals. In rats fed a K-free diet, plasma K concentration ([K]) is reduced and plasma aldosterone concentration [Aldo] is decreased. Because aldosterone release is regulated by extracellular fluid (ECF) [K], the purpose of the present study was to determine whether aldosterone deficiency mediates inhibition of TAL NaCl transport in K-depleted rats (K-Dep). Cl reabsorption was measured in functionally isolated loop segments microperfused in vivo (22 nl/min) using a modified perfusate that minimizes proximal nephron reabsorption. The results of our studies show that the defect in TAL Cl reabsorption in K-Dep rats is quantitatively significant and can be rapidly reversed by the acute systemic infusion of K. However, acute administration of aldosterone, in the presence of sustained K-Dep, failed to reverse the impairment in TAL Cl reabsorption in K-Dep rats. Furthermore, acute infusion of K, in the presence of an aldosterone antagonist, in K-Dep rats rapidly corrected the defect in TAL Cl reabsorption. Additional studies showed that despite normalization of ECF [K] by acute K infusion in K-Dep rats, plasma [Aldo] failed to increase. In contrast, acute infusion of KCl in control rats increased plasma [Aldo] by 46%, but Cl reabsorption was unchanged. In summary, these results provide conclusive evidence that the correction of defective TAL Cl reabsorption in response to the acute administration of K in K-Dep rats occurs via an aldosterone-independent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Reset of the osmotic threshold for vasopressin in rats fed a low NaCl, K-free diet.

Activation of the renin-angiotensin system induced by feeding a low NaCl, K-free (LS) diet is associated with polydipsia and a chronic reduction in effective plasma osmolality (efPosm). We have recently shown that converting enzyme inhibition with enalapril (EP) abolishes polydipsia. The present study was designed to test the hypothesis that the osmotic threshold for vasopressin is reset in rats fed the LS diet and to examine the effect of EP on ambient and osmotically stimulated plasma vasopressin levels (PAVP). Animals were fed the LS diet or a control salt diet and treated with vehicle or the lowest dose of EP sufficient to prevent polydipsia (7.5 mg.kg-1.day-1) in rats fed the LS diet. PAVP and efPosm were measured under ambient conditions and after osmotic loading. Urine osmolality (Uosm) was measured under ambient conditions and after water loading. The chronic reduction in efPosm in LS rats was associated with the excretion of a Uosm 1-2 times greater than the corresponding Posm, PAVP similar to controls (LS, 2.27 +/- 1.08 vs. control, 1.19 +/- 0.22 pg/mL) and the ability to excrete a water load. Following osmotic loading, efPosm and PAVP increased significantly and similarly in both LS and control rats. EP administration had no effect on water intake, ambient efPosm and PAVP, and the AVP response to osmotic loading in rats fed the control diet. EP prevented polydipsia in LS rats, however it had no significant effect on ambient or osmotically stimulated PAVP or efPosm.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin D-induced chronic hypercalcemia inhibits thick ascending limb NaCl reabsorption in vivo.

Chronic hypercalcemia (HC) induced by dihydrotachysterol (DHT) is associated with a nephrogenic concentrating defect. The purpose of the present study is to assess the effects of HC on thick ascending limb (TAL) NaCl reabsorption in vivo by use of micro stop-flow technique. Feeding DHT (4.25 mg/kg diet) to rats was associated with an increase in plasma [Ca] within 24 h from 2.21 +/- 0.049 to 2.71 +/- 0.047 mM (P less than 0.001), which remained elevated during the 7-day period of study. Ambient plasma arginine vasopressin (AVP) in polydipsic HC rats was 3.10 +/- 0.605 pg/ml, a value not different from that measured in pair-fed control rats (1.82 +/- 0.260 pg/ml). A urine-concentrating defect developed after 3 days and occurred without nephrocalcinosis or reduced glomerular filtration rate (GFR). The estimated TAL [NaCl] after all stop-flow periods in HC rats did not decrease to the same extent as in controls. The rate constant of NaCl reabsorption derived from linear regression analysis of the ln[NaCl] vs. time for 0-15 s was significantly reduced by 25% in the HC nephrons. In addition, the minimum [NaCl] established after 30-60 s of contact time increased by 36% in HC nephrons. These data provide evidence that reduced TAL NaCl reabsorption, and not reduced GFR, nephrocalcinosis, or deficient AVP, contributes to the presence of a renal concentrating defect in DHT-induced chronic HC.

Inhibition of tobramycin reabsorption in nephron segments by metabolic alkalosis.

Chronic metabolic acidosis appears to potentiate aminoglycoside induced nephrotoxicity and renal cortical accumulation while some, but not all, studies show that bicarbonate loading reduces nephrotoxicity. The purpose of the present study was to determine if reabsorption of tobramycin in proximal and distal nephron segments in vivo is altered by systemic pH. To test this possibility on the single nephron level, 24 nl samples of [3H] tobramycin were micro-injected into proximal and distal nephron segments, and its recovery was compared to that of [14C] inulin in rats undergoing osmotic diuresis with NaHCO3 or Na2SO4 containing HCl. Results were obtained in 66 tubules in 20 rats. Although plasma and urine pH were altered as anticipated in bicarbonate-infused and acid-infused animals, urine and late proximal tubule flow rates were similar. When animals were acid infused, 24.8 +/- 1.90% of [3H] tobramycin injected into proximal nephrons was reabsorbed compared to 7.5 +/- 1.56% (P less than 0.001) when the animals were bicarbonate-infused. When [3H] tobramycin was injected into distal tubules, 5.9 +/- 0.75% was reabsorbed in acid-infused rats while virtually none of the injected tobramycin, 0.43 +/- 1.59%, was reabsorbed by the distal nephron of bicarbonate-loaded animals. Our results provide evidence that tobramycin reabsorption by both proximal and distal nephron segments is substantially reduced by bicarbonate-infusion.

Converting-enzyme inhibition abolishes polydipsia induced by dietary NaCl and K depletion.

The present studies were designed to test the hypothesis that angiotensin II (ANG II) mediates nonosmotic thirst in animals fed the low-NaCl K-free diet by preventing the increased generation of ANG II using the converting-enzyme inhibitor, enalapril. Animals were fed either a control salt or low-NaCl K-free diet and were treated with or without enalapril. Water intake in rats fed the low-NaCl K-free diet increased more than twofold on day 3 and remained elevated over the 10-day period of study. Treatment with enalapril (40 mg.kg-1.day-1) 1) prevented the striking rise in plasma renin activity in rats fed the low-NaCl K-free diet, 2) led to complete blockade of the pressor response to a 50-ng injection of angiotensin I but not ANG II, 3) did not affect daily water intake in rats consuming the control salt diet, 4) did not reduce basal water intake in rats fed the low-NaCl K-free diet below values measured in control animals, and 5) did not abolish water intake in response to osmotic stimulation. However, enalapril treatment abolished the increase in water intake that occurs in animals fed the low-NaCl K-free diet. In a double crossover study using two groups of rats fed the low-NaCl K-free diet, enalapril prevented increased water intake in rats initially fed the low-NaCl K-free diet and rapidly inhibited increased water intake in rats fed the low-NaCl K-free diet after the high water intake had been established.(ABSTRACT TRUNCATED AT 250 WORDS)

Summarization strategies of hearing-impaired and normally hearing college students.

The purpose of this study was to compare the summary writing skills of hearing-impaired and normally hearing college students. Summarization was defined in terms of the following measures: deletion of trivial text information, inclusion of most important ideas, selection of topic sentences, creation of topic statements, and integration of information within and among several paragraphs. Inclusion of opinionated, incorrect, and uninterpretable information was measured also. Thirty hearing-impaired and 30 normally hearing students read and summarized two expository science passages that were controlled for the number of topic (main idea) sentences and that had been rated previously for the importance of "idea units." Students' factual comprehension also was assessed. Hearing-impaired and normally hearing students exhibited a similar pattern of use among several measured summarization strategies, except for the inclusion of opinions or comments in their summaries. Hearing-impaired students were not as sensitive as normally hearing students to importance of ideas and used the following summarization strategies significantly less often: inclusion of important ideas, selection of topic sentences, creation of topic statements, and integration of ideas within and among paragraphs. The results indicated that hearing-impaired college students have basic summarization skills but do not apply summarization strategies as effectively as normally hearing students.

Plasma AVP and renal concentrating defect in chloride depletion metabolic alkalosis.

These studies were undertaken to determine the effect of chronic chloride depletion metabolic alkalosis (Cl-DEP-MALK) on water intake, plasma arginine vasopressin (AVP) levels, and renal concentrating ability. Cl-DEP-MALK was induced by feeding a chloride-free diet to rats subjected to gastric drainage and to dogs treated with furosemide. All of the animals developed a urine concentrating defect, polydipsia, and a persistent reduction in plasma osmolality. However, AVP release was not suppressed. The results of osmotic loading experiments in dogs analyzed using either linear or log-linear models have shown that chronic Cl-DEP-MALK significantly alters the relation between plasma osmolality and plasma AVP. In the classic linear analysis the results suggest that Cl-DEP-MALK reduces the plasma osmolality at which plasma AVP can be detected, i.e., reduced "threshold," and increases the slope of the plasma osmolality-to-plasma AVP relation nearly twofold, i.e., increased "sensitivity." Finally, we provide evidence that the concentrating defect is not related to high water turnover or deficient endogenous AVP and is therefore nephrogenic.

Thick ascending limb response to dDAVP and atrial natriuretic factor in vivo.

Because of the inaccessibility of the thick ascending limb (TAL) to micropuncture, it has been difficult to evaluate the effects of ADH and atrial natriuretic factor (ANF) on TAL salt transport in vivo. The purpose of the present study is to assess the effects of these hormones on TAL NaCl reabsorption in vivo. Although passive NaCl permeability and unidirectional Na fluxes were not measured, the microstop-flow conductivity technique that was used in these studies controls solute delivery, permits the measurement of the rate of net NaCl reabsorption, and tests the capacity of the TAL to establish a limiting gradient. Despite the presence of a dramatic natriuretic response, a natural extract of ANF did not significantly increase the minimum NaCl concentration reached by the TAL after 60 s of stop-flow. However, 1-[desamino]-8-D-arginine vasopressin (dDAVP) increased by 23%, the rate at which luminal Na concentration declined, and reduced by 28% the minimum NaCl concentration achieved by the TAL. Therefore, these results do not demonstrate a significant direct peritubular effect of ANF on TAL NaCl permeability in outer cortical nephrons, but support a role for antidiuretic hormone in enhancing TAL NaCl reabsorption in vivo.

Potassium depletion enhances renal compensatory hypertrophy in the nephrectomized rat.

The purpose of the present investigation was to determine whether dietary K depletion would stimulate further renal growth in rats subject to 5/6 nephrectomy. In 36 rats renal mass was reduced by left 2/3 nephrectomy and right complete nephrectomy. Rats were fed either a normal or K-free diet. Two weeks later, total renal mass, water, protein and RNA content were measured. Twenty-nine rats served as the controls for the two surgical procedures, for treatment on the K-free diet or control diet, and for repeated cardiac blood sampling. Two weeks of dietary K depletion in the sham-operated rats caused a 13% increase in total renal mass compared to sham-operated controls. There was no difference in the weight of the removed right kidney or body weight before initiation of the diet in nephrectomized rats. However, after 2 weeks of consuming the K-free diet, there was a 48% increase in the weight of the remnant left kidney in the K-depleted rats compared to the nephrectomized control rats (K-depleted 2.31 +/- 0.147 g vs. control 1.56 +/- 0.059 g, p less than 0.001). Renal tissue water content was only slightly elevated (3%) in the K-depleted nephrectomized rats and could not account for the 48% increase in renal mass in these animals. In addition, RNA content mg/g dry weight was 75% higher in K-depleted nephrectomized rats compared to their nephrectomized controls. We conclude that K depletion causes a greater increase in renal mass and RNA content than would otherwise occur in response to nephrectomy in the rat.

Distal tubule bicarbonate accumulation in vivo. Effect of flow and transtubular bicarbonate gradients.

We have performed microperfusion studies on distal tubules of normal and alkalotic rats in an attempt to demonstrate in vivo bicarbonate secretion. All perfusion solutions were free of phosphate and other nonbicarbonate buffers. In both normal and alkalotic rats, distal perfusions elicited significant tCO2 entry only at high flow (24 nl/min). Even when perfusate tCO2 concentration closely matched plasma tCO2 concentration (30 mM tCO2), significant tCO2 entry again occurred at high flow. This was associated with a rise of the perfusate tCO2 concentration, which indicated net entry of tCO2 against a concentration gradient. In this "symmetrical" perfusion situation, acetazolamide blockade prevented tCO2 entry. Accordingly: distal tubule tCO2 entry is demonstrable in both alkalotic and normal rats at high flow rates; increasing perfusate tCO2 concentration can suppress tCO2 entry; and entry can occur in the absence of a gradient and this effect can be blocked by acetazolamide.

Neuroendocrine factors mediating polydipsia induced by dietary Na, Cl, and K depletion.

We investigated whether the increased intake of water during dietary electrolyte depletion is related to activation of the renin-angiotensin system. Young adult male rats were fed a low Na-, Cl-, K-free (low-salt) diet for 2 wk during which measurements were made of daily water intake and urine volume, plasma osmolality (Posm) and electrolytes, and plasma renin activity (PRA) and angiotensin I (ANG I) concentration. Water intake and urine output increased on day 3 of the low-salt diet, reached a maximum on day 4, and remained elevated, paralleling the time course of increases in PRA and ANG I plasma concentrations. Posm was normal after 2 days on the low-salt, although it was significantly lower by day 11. Renal concentrating ability was not different from controls after 6 days, but was significantly reduced after 11 days of treatment. Electrolytic lesions of the subfornical organ (SFO) abolished the low-salt diet-induced polydipsia, but had no effect on the diet-induced increases in PRA and plasma ANG I concentration. These data demonstrate that polydipsia induced by feeding a low-salt diet can develop in the presence of a normal or reduced Posm and precedes the development of a renal concentrating defect. The primary polydipsia is associated with elevated PRA and ANG I and appears to be mediated by angiotensin receptors in the SFO.