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Because of its cardio-protective effects, a low-Na, high-K diet (LNaHK) is often warranted in conjunction with diuretics to treat hypertensive patients. However, it is necessary to understand the renal handling of such diets in order to choose the best diuretic. Wild-type (WT) or Renal Outer Medullary K channel (ROMK) knockout mice (KO) were given a regular (CTRL), LNaHK, or high-K diet (HK) for 4-7 days. On LNaHK, mice treated with either IP furosemide for 12 hrs, or given furosemide in drinking water for 7 days, exhibited decreased K clearance. We used free-flow micropuncture to measure the [K + ] in the early distal tubule (EDT [K + ]) before and after furosemide treatment. Furosemide increased the EDT [K + ] in WT on CTRL but decreased that in WT on LNaHK. Furosemide did not affect the EDT [K + ] of KO on LNaHK or WT on HK. Furosemide-sensitive Na + excretion was significantly greater in mice on LNaHK than those on CTRL or HK. Patch clamp analysis of split-open TALs revealed that 70-pS ROMK exhibited a higher open probability (Po) but similar density in mice on LNaHK, compared with CTRL. No difference was found in the density or Po of the 30 pS K channels between the two groups. These results indicate mice on LNaHK exhibited furosemide-sensitive net K + secretion in the TAL that is dependent on increased NKCC2 activity and mediated by ROMK. We conclude that furosemide is a K-sparing diuretic by decreasing the TAL net K + secretion in subjects on LNaHK.

The relationships between renal blood flow (RBF), tissue oxygenation, and inflammatory injury in atherosclerotic renovascular disease (ARVD) are poorly understood. We sought to correlate RBF and tissue hypoxia with glomerular filtration rate (GFR) in 48 kidneys from patients with ARVD stratified by single kidney iothalamate GFR (sGFR). Oxygenation was assessed by blood oxygenation level dependent magnetic resonance imaging (BOLD MRI), which provides an index for the levels of deoxyhemoglobin within a defined volume of tissue (R2*). sGFR correlated with RBF and with the severity of vascular stenosis as estimated by duplex velocities. Higher cortical R2* and fractional hypoxia and higher levels of renal vein neutrophil-gelatinase-associated-lipocalin (NGAL) and monocyte-chemoattractant protein-1 (MCP-1) were observed at lower GFR, with an abrupt inflection below 20 ml/min. Renal vein MCP-1 levels correlated with cortical R2* and with fractional hypoxia. Correlations between cortical R2* and RBF in the highest sGFR stratum (mean sGFR 51 ± 12 ml/min; R = –0.8) were degraded in the lowest sGFR stratum (mean sGFR 8 ± 3 ml/min; R = –0.1). Changes in fractional hypoxia after furosemide were also absent in the lowest sGFR stratum. These data demonstrate relative stability of renal oxygenation with moderate reductions in RBF and GFR but identify a transition to overt hypoxia and inflammatory cytokine release with severely reduced GFR. Tissue oxygenation and RBF were less correlated in the setting of reduced sGFR, consistent with variable oxygen consumption or a shift to alternative mechanisms of tissue injury. Identifying transitions in tissue oxygenation may facilitate targeted therapy in ARVD.

Inhibitors of kidney urea transporter (UT) proteins have potential use as salt-sparing diuretics (‘urearetics’) with a different mechanism of action than diuretics that target salt transporters. To study UT inhibition in rats, we screened about 10,000 drugs, natural products and urea analogs for inhibition of rat UT-A1. Drug and natural product screening found nicotine, sanguinarine and an indolcarbonylchromenone with IC50 of 10–20 μM. Urea analog screening found methylacetamide and dimethylthiourea (DMTU). DMTU fully and reversibly inhibited rat UT-A1 and UT-B by a noncompetitive mechanism with IC50 of 2–3 mM. Homology modeling and docking computations suggested DMTU binding sites on rat UT-A1. Following a single intraperitoneal injection of 500 mg/kg DMTU, peak plasma concentration was 9 mM with t1/2 of about 10 hours, and a urine concentration of 20–40 mM. Rats chronically treated with DMTU had a sustained, reversible reduction in urine osmolality from 1800 to 600 mOsm, a 3-fold increase in urine output, and mild hypokalemia. DMTU did not impair urinary concentrating function in rats on a low protein diet. Compared to furosemide-treated rats, the DMTU-treated rats had greater diuresis and reduced urinary salt loss. In a model of Syndrome of Inappropriate Antidiuretic Hormone secretion, DMTU treatment prevented hyponatremia and water retention produced by water-loading in dDAVP-treated rats. Thus, our results establish a rat model of UT inhibition and demonstrate the diuretic efficacy of UT inhibition.

Inactivation of the tissue kallikrein gene in mice impairs renal handling of potassium due to enhanced H, K-ATPase activity, and induces hyperkalemia. We investigated whether the R53H loss-of-function polymorphism of the human tissue kallikrein gene affects renal potassium handling. In a crossover study, 30 R53R homozygous and 10 R53H heterozygous healthy males were randomly assigned to a low-sodium/high-potassium or a high-sodium/low-potassium diet to modulate tissue kallikrein synthesis. On the seventh day of each diet, participants were studied before and during a 2-h infusion of furosemide to stimulate distal potassium secretion. Urinary kallikrein activity was significantly lower in R53H than in R53R subjects on the low-sodium/high-potassium diet and was similarly reduced in both genotypes on high-sodium/low-potassium. Plasma potassium and renal potassium reabsorption were similar in both genotypes on an ad libitum sodium/potassium diet or after 7 days of a high-sodium/low-potassium diet. However, the median plasma potassium was significantly higher after 7 days of low-sodium/high-potassium diet in R53H than in R53R individuals. Urine potassium excretion and plasma aldosterone concentrations were similar. On the low-sodium/high-potassium diet, furosemide-induced decrease in plasma potassium was significantly larger in R53H than in R53R subjects. Thus, impaired tissue kallikrein stimulation by a low-sodium/high-potassium diet in R53H subjects with partial tissue kallikrein deficiency highlights an inappropriate renal adaptation to potassium load, consistent with experimental data in mice.

Cells in the hyperosmotic kidney medulla, express a transcriptional activator termed tonicity responsive enhancer binding protein (TonEBP). Genes targeted by TonEBP protect kidney cells from the deleterious effects of hyperosmolality by inducing the expression of organic osmolytes and molecular chaperones, and other genes that mediate urine concentration such as aquaporin-2 and urea transporters. We tested here the effect of hypertonicity and hyperosmotic salt in the renal medullary interstitium on the expression TonEBP. When massive water diuresis was induced in rats the medullary sodium concentrations did not change, neither did TonEBP expression. In these animals the medullary tonicity was unchanged despite the production of dilute urine. On the other hand, treatment with the loop diurectic furosemide resulted in a dose-dependent decrease in the medullary sodium concentration causing a reduction in interstitial tonicity. Here, TonEBP expression was blunted in the outer and inner medulla which was due, in part, to decreased mRNA abundance. As expected, the expression of TonEBP target genes in the renal medulla also decreased in response to furosemide. Hence TonEBP expression in the renal medulla is stimulated by interstitial hypertonicity.

Inhibition of renin with an active site inhibitor, aliskiren, lowers blood pressure (BP) in diabetic patients. Here, we studied the time course of the antihypertensive and antiproteinuric effect of renin inhibition in 15 patients with type 2 diabetes and elevated urinary albumin/ creatinine ratios (UACRs) to check whether aliskiren can decrease proteinuria. After a 4-week washout of previous medications, patients received aliskiren and furosemide daily for 28 days followed by a 4-week withdrawal period. Twenty-four-hour BPs were measured at baseline throughout treatment and withdrawal periods. The UACR was significantly reduced after 2 -4 days of treatment with another significant reduction after 28 days. Systolic blood pressure (SBP) was significantly lower after 7 days with no further reduction after 28 days. The BP returned toward baseline 3 days after withdrawal, whereas the UACR was still significantly reduced compared with baseline 12 days after withdrawal. Our study shows that aliskiren reduced 24 h SBP, and this was associated with a reduction in albuminuria in type 2 diabetic patients.

Distal renal tubular acidosis (RTA) can lead to rickets in children or osteomalacia in adults if undetected. This disorder is normally diagnosed by means of an oral ammonium chloride-loading test; however, the procedure often leads to vomiting and abandonment of the test. In this study, we assess an alternative, more palatable approach to test urinary acidification. This was achieved by the simultaneous oral administration of the diuretic furosemide and the mineralocorticoid fludrocortisone to increase distal tubular sodium delivery, principal cell sodium reabsorption, and α -intercalated cell proton secretion. We evaluated 11 control subjects and 10 patients with known distal RTA by giving oral ammonium chloride or furosemide/fludrocortisone in random order on separate days. One control and two patients were unable to complete the study owing to vomiting after NH 4 Cl; however, there were no adverse effects with the furosemide/fludrocortisone treatment. The urine pH decreased to less than 5.3 in the controls with both tests, whereas none of the patients was able to lower the urine pH below 5.3 with either test. We conclude that the simultaneous administration of furosemide and fludrocortisone provides an easy, effective, and well-tolerated alternative to the standard ammonium chloride urinary acidification test for the diagnosis of distal RTA.

Cyclosporine A attenuates the natriuretic action of loop diuret- ics by inhibition of renal COX-2 expression. Background. It is known that inhibition of cyclooxygenase (COX) impairs the renal actions of loop diuretics. Recently, we found that cyclosporine A (CsA) inhibits renal COX-2 expres- sion. Therefore, we examined the interferences of CsA with the renal actions of loop diuretics. Method. We investigated the renal effects of furosemide administration (12 mg/day subcutaneously) in male Sprague- Dawley rats receiving in addition vehicle, CsA (15 mg/kg × day), rofecoxib (10 mg/kg × day), or a combination of both. Results. CsA, rofecoxib, and their combination lowered the furosemide-induced increase of prostaglandin E2 (PGE2) and of 6-keto prostaglandin F1a (6-keto PGF1a) excretion by 55% and by 70%. They also lowered furosemide stimulated renal excretion of sodium and water by about 65% and 60%. Basal as well as furosemide-induced stimulation of plasma renin activity (PRA) and of renal renin mRNA was further enhanced by CsA. In contrast, rofecoxib attenuated the furosemide-induced rise of PRA and of renin mRNA, both in the absence and in the presence of CsA. In addition, the increase in plasma 6-keto PGF1a levels by furosemide was further enhanced by CsA and was attenuated by rofecoxib. Conclusion. Taken together, our data suggest that CsA acts as an antinatriuretic, likely by the inhibition of COX-2-mediated renal prostanoid formation. Since the furosemide-induced stim- ulation of the renin system is not attenuated by CsA but by COX-2 inhibition, we speculate that extrarenal COX-2-derived prostanoids may be involved in the stimulation of the renin sys- tem by CsA and by loop diuretics.

Effect of the calcimimetic NPS R-467 on furosemide-induced nephrocalcinosis in the young rat. Background Furosemide induces nephrocalcinosis in both humans and animals. We showed previously that parathyroidectomy protected against the development of furosemide-induced nephrocalcinosis in young rats, indicating a possible role for parathyroid hormone (PTH) in its pathogenesis. Calcimimetic agents such as NPS R-467 are potent and selective agonists at the calcium-sensing receptor in parathyroid glands and inhibit PTH secretion. Methods To determine whether NPS R-467 could, like parathyroidectomy, prevent furosemide-induced nephrocalcinosis, we studied 35 6-week-old male Sprague-Dawley rats, divided into five groups. Group A served as control, group B received intraperitoneally furosemide (40mg/kg), groups C, D, and E received furosemide and NPS R-467 intraperitoneally at doses of 10, 20, and 40 μmol/kg, respectively, daily for 8days. During the last 3days, animals were placed in metabolic cages for measurement of urine output, food, and water intake. Blood and kidneys were collected on day 8, 60 to 90 minutes after the last doses. Kidney calcium content was measured and nephrocalcinosis scoring (0 to 5) was assessed histologically. Results Furosemide increased urine output and fluid intake, and decreased body weight gain similarly in all groups. Serum PTH levels (mean ± SD) were significantly higher in furosemide-treated control animals (276 ± 226pg/mL vs. 64 ± 21pg/mL); NPS R-467 induced a dose-dependent decrease in PTH levels (52 ± 51pg/mL, 18 ± 7pg/mL, and 13 ± 3pg/mL in groups C, D, and E, respectively). Plasma Ca 2+ was slightly, but significantly lower in all three NPS R-467 treated groups (5.1 ± 0.4mg/dL, 4.8 ± 0.3mg/dL, and 4.5 ± 0.3mg/dL in groups C, D, and E, respectively) compared to 5.7 ± 0.1mg/dL and 5.5 ± 0.2mg/dL in groups A and B, respectively. Furosemide treatment induced a substantial increase in kidney calcium content (1819 ± 664 μg/g dry weight vs. 126 ± 26 μg/g dry weight) and nephrocalcinosis scoring (5.0 ± 0.0 vs. 0.0 ± 0.0). Treatment with NPS R-467 ameliorated the furosemide-induced increase in kidney calcium content (673 ± 312 μg/g, 361 ± 188 μg/g, and 563 ± 291 μg/g) and nephrocalcinosis scoring (2.2 ± 1.2, 0.7 ± 0.8, and 1.0 ± 1.2) in groups C, D, and E, respectively. Conclusion The calcimimetic agent NPS R-467 prevents the development of hyperparathyroidism and attenuates nephrocalcinosis in the furosemide-treated young rat.

Short-term modulation of distal tubule fluid nitric oxide in vivo by loop NaCl reabsorption. Background Intrarenal nitric oxide (NO) production and signaling effects are influenced by NaCl loading. To gain further insight into NO mechanisms we determined whether rat distal tubular fluid (DTF) [NO] and collected NO may acutely change when NaCl loop delivery is altered. Methods An NO microelectrode was used to measure real-time DTF [NO] and DT-collected NO. With proximal flow blocked (open system), 150mmol/L NaCl, with and without 10 −4 mol/L furosemide was perfused with measurement of loop [Cl] reabsorption. Using a closed system, DTF [NO] was also determined using several different loop perfusates. Results In the open system, perfusion with 40 nL/min of 150mmol/L NaCl to which 10 −4 mol/L furosemide was added, DT [NO] and DT-collected NO was approximately twice that measured with perfusion of 150mmol/L NaCl alone, while loop Cl reabsorption decreased by half. In the closed system, perfusion at 10 nL/min of 150mmol/L NaCl + furosemide 10 −4 mol/L also induced a significant rise in DTF [NO] and collected NO. Perfusion of 10 −3 mol/L S-methyl-L-thiocitrulline (SMTC) with 150mmol/L NaCl, induces a significant drop in DT [NO], but without a significant increase in collected NO. Furthermore, with addition of 10 −3 mol/L SMTC to the 150mmol/L NaCl + 10 −4 furosemide perfusate, the rise in DT [NO] was prevented. Analysis of covariance showed that flow changes within, or between all groups, had no significant additional effect. Conclusion In both open and closed loop perfusion systems, 10 −4 mol/L furosemide inhibition of NaCl transport stimulates net loop NO emission independent of flow; 10 −3 mol/L SMTC + 150mmol/L NaCl reduces DT [NO], but not DT-collected NO. Short-term net NO emission from the entire loop, as collected in distal tubule fluid, increases with inhibition of loop NaCl transport.

Role of excess volume in the pathophysiology of hypertension in chronic kidney disease.BackgroundThe pathophysiology of hypertension in patients with chronic kidney disease (CKD) is largely attributed to positive sodium balance. It is unclear how loop diuretics affect fluid volume compartments, especially with respect to their antihypertensive effect.MethodsSubjects with CKD were administered a single therapeutically equivalent dose of an oral loop diuretic (furosemide or torsemide in randomized crossover design). We measured acute volume changes over 12 hours using biophysical and hormonal biomarkers and then 24-hour ambulatory blood pressure after daily diuretic therapy for 3 weeks.ResultsSingle-dose administration of loop diuretic decreased extracellular water (ECW) by 1.7L [95% confidence interval (95% CI) 1.2, 2.2, P < 0.001], total body water (TBW) by 1.2L (95% CI 0.5, 1.9, P < 0.001), and increased natural log (ln) plasma renin activity (PRA) from -1.2 ± 1.3ng/mL/hour to -0.5 ± 1.5ng/mL/hour (P < 0.001). Daily loop diuretic administration resulted in reduced ECW from 24.2 ± 6.4L to 22.3 ± 5.2L (P = 0.02) and TBW from 54.3 ± 12.7L to 51.6 ± 11.9L (P < 0.001) in 1 week. After 3 weeks of diuretic therapy, whereas ECW reduction persisted at 22.8 ± 5.1L (P = 0.05), TBW trended toward baseline level at 52.7 ± 11.8L. A concomitant increase in ln PRA from –1.0 ± 1.3ng/mL/hour to 0.4 ± 1.9ng/mL/hour (P < 0.001) and ln plasma aldosterone (PA) from 2.0 ± 0.8ng/dL to 2.3 ± 0.8ng/dL (P < 0.005) and fall in ln brain natriuretic peptide (BNP) from 4.3 ± 0.9 pg/mL to 3.7 ± 1.0 pg/mL (P < 0.01) were seen at 1 week. Despite a trend toward restoration of TBW, changes in hormonal biomarkers were maintained at 3 weeks. Over these 3 weeks, furosemide reduced 24-hour ambulatory blood pressure from 147 ± 17/78 ± 11mm Hg to 138 ± 21/74 ± 12mm Hg (P = 0.021) and torsemide reduced it from 143 ± 18/75 ± 10mm Hg to 133 ± 19/71 ± 10mm Hg (P = 0.007).ConclusionPatients with CKD have elevated extracellular fluid volume that can be corrected acutely with loop diuretics. Persistent diuretic use results in dynamic changes in ECW and other body fluid compartments that translate into chronic blood pressure reduction.

Coadministration of albumin and furosemide in patients with the nephrotic syndrome.BackgroundIn patients with nephrotic syndrome, the natriuretic effect of furosemide (FU) is diminished. The effect of coadministration of FU and human albumin (HA) has remained controversial.MethodsIn a double-blind, placebo-controlled study, nine nephrotic patients (six males, 48±4years) on standardized sodium chloride intake, in random order on three separate days, received by intravenous administration for 60minutes either (a) 60mg FU plus a sham infusion, (b) 60mg FU plus 200ml of a 20% solution of HA, or (c) sham infusion plus 200ml of a 20% solution of HA. Urinary volume, sodium, albumin and FU excretion, renal hemodynamics, and plasma atrial natriuretic factor concentration were assessed.ResultsAdministration of FU alone significantly (P < 0.01) increased mean cumulative urinary sodium (259±30mmol) and volume excretion (2684±167ml) in the first eight hours as compared with the HA infusion alone (118±12mmol, 1827±141ml). The coadministration of FU and HA caused an even more marked increase (P < 0.01 vs. HA alone) of urinary sodium (312±28mmol) and volume excretion (3230±201ml); the difference to FU administration alone was significant (P < 0.05). Plasma atrial natriuretic factor, serum albumin concentration, and urinary albumin excretion increased significantly on both HA infusion days, whereas urinary excretion of FU remained unchanged with HA coadministration. Glomerular filtration rate (CIn) was not significantly affected by any of the infusion protocols, but effective renal plasma flow (CPAH) increased significantly on both HA infusion days.ConclusionsCoadministration of HA potentiates the action of FU in patients with the nephrotic syndrome, but only modestly. This effect is mediated by changes in renal hemodynamics.

Fluorescent determination of chloride (Cl - ) in nanoliter samples. Background Measurements of Cl - in nanoliter samples, such as those collected during isolated, perfused tubule experiments, have been difficult, somewhat insensitive, and/or require custom-made equipment. We developed a technique using a fluorescent Cl - indicator, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), to make these measurements simple and reliable. Methods This is a simple procedure that relies on the selectivity of the dye and the fact that Cl - quenches its fluorescence. To measure millimolar quantities of Cl - in nanoliter samples, we prepared a solution of 0.25 mM SPQ and loaded it into the reservoir of a continuous-flow ultramicrofluorometer, which can be constructed from commercially available components. Samples were injected with a calibrated pipette via an injection port, and the resultant peak fluorescent deflections were recorded. The deflections represent a decrease in fluorescence caused by the quenching effect of the Cl - injected. Results The method yielded a linear response with Cl - concentrations from 5 to 200 mM NaCl. The minimum detectable Cl - concentration was approximately 5 mM. The coefficient of variation between 5 and 200 mM was 1.7%. Resolution, defined as two times the standard error divided by the slope, between 10 and 50 mM and between 50 and 200 mM was 1 mM and 2.6 mM, respectively. Furosemide, diisothiocyanostilbene-2,2′-disulfonic acid and other nonchloride anions (HEPES, HCO 3 , SO 4 , and PO 4 ) did not interfere with the assay, whereas 150 mM NaBr resulted in a peak height greater than 150 NaCl. In addition, the ability to measure Cl - did not vary with pH within the physiological range. Conclusion We developed an easy, accurate, and sensitive method to measure Cl - concentration in small aqueous solution samples.

Low Na + diet inhibits Na + and water transport response to vasopressin in rat cortical collecting duct. Background We previously demonstrated that vasopressin (AVP) produces a sustained increase in Na + reabsorption by the isolated perfused cortical collecting duct (CCD) from rats on a normal diet, and that this effect is synergistic with that of pharmacological doses of deoxycorticosterone (DOC) or physiological levels of aldosterone. The present experiments examined the effect of AVP under the more physiological circumstances when plasma aldosterone was elevated by prior volume depletion. Methods Rats were volume depleted by a single dose of furosemide followed by a low-salt diet (0.3% NaCl) for four to nine days. Some of these rats were also implanted with a pellet containing 2.5mg DOC. Rats in a third group were not injected with furosemide but were implanted with the DOC pellet and maintained on a standard (∼1% NaCl) diet. CCD were perfused and the lumen-to-bath Na + flux ( J Na ), transepithelial voltage ( V T ), and osmotic water permeability ( P f ) were measured in the presence and absence of 200 pm AVP. Results Although Na + depletion by a single injection of furosemide and the low salt diet elevated plasma aldosterone and V T , J Na remained low and there was a decreased response to AVP in comparison with DOC-treated rats on a standard diet. In CCD from rats on the low salt-diet with DOC, J Na was less than observed in CCD from DOC-treated rats on a standard diet. AVP-dependent P f in CCD from rats on the low-salt diet, with or without DOC treatment, was also markedly lower. Conclusions We interpret the results to demonstrate that maximal rates of Na + reabsorption in the CCD depend not only on the synergistic stimulatory effects of aldosterone and AVP, but also require normal to high rates of salt delivery in vivo for the effects of the hormones on Na + transport to be maximized in vitro .

α-Adducin polymorphisms and renal sodium handling in essential hypertensive patients. The relationship between blood pressure and sodium (Na) excretion is less steep in hypertension caused by increased renal tubular reabsorption. We recently demonstrated that one mutation in rat α-adducin gene: (1) is responsible for approximately 50% of the hypertension of MHS rats, and (2) stimulates tubular Na-K pump activity when transfected in renal epithelial cell, suggesting that its pressor effect may occur because an increased tubular reabsorption. Linkage and association studies demonstrated that the α-adducin locus is relevant for human hypertension. A point mutation (G460W) was found in human α-adducin gene, the 460W variant (G/W) is more frequent in hypertensives than in normotensives. The aim of this study was to test whether acute changes in body Na may differently affect blood pressure in humans as a function of α-adducin genotype. The pressure-natriuresis relationship was analyzed in 108 hypertensives using two different acute maneuvers: Na removal (furosemide 25 mg p.o.) and, two days later, Na load (310 mmoles i.v. in 2 hr). We found that 80 patients were wild-type homozygous (G/G), 26 were G/W heterozygous, and 2 were W/W homozygous with similar blood pressure, age, body mass index, gender, plasma and urinary sodium and potassium. In basal condition G/W-W/W patients showed a lower plasma renin activity and fractional excretion of Na. In either case the pressure-natriuresis relationship was less steep in G/W-W/W than in G/G patients, obviously negative for Na depletion with furosemide (-0.011 ± 0.004 vs. -0.002 ± 0.002 mm Hg/μmol/min, P < 0.03), and positive for Na load (0.086 ± 0.02 vs. 0.027 ± 0.007 mm Hg/μmol/min, P < 0.001). The finding of reduced slope after Na depletion or Na load supports the hypothesis that, as MHS rats, humans bearing one W α-adducin variant display an increased of renal tubular sodium reabsorption.

Expression of neuronal type nitric oxide synthase and renin in the juxtaglomerular apparatus of angiotensin type-1a receptor gene-knockout mice. Angiotensin type-1a (AT1a) receptor gene-knockout ( AT1a -/- ) mice exhibit chronic hypotension and renin overproduction. In the kidneys of AT1a -/- mice, the activity of neuronal type nitric oxide synthase (N-NOS) was histochemically detected by nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase (NADPHd) reaction combined with N-NOS immunohistochemistry. The localization of renin was detected by immunohistochemistry and the results were analyzed morphometrically. The levels of N-NOS and renin mRNA in the renal cortical tissue were determined by reverse transcription-PCR and Northern blot analysis, respectively. In the renal sections from wild-type mice, NADPHd activity and N-NOS immunoreactivity were localized to the discrete region of the macula densa in contact with the parent glomerulus. In contrast, N-NOS-positive macula densa cells were distributed beyond the original location of the macula densa, occasionally extending to the opposite side of the distal tubules. The mean number of N-NOS positive macula densa cells was significantly increased in AT1a -/- mice (186 per 100 glomeruli) compared with wild-type mice (65 per 100 glomeruli). AT1a -/- mice showed 1.4-times higher N-NOS mRNA levels in the renal cortical tissues than wild-type mice. The plasma renin activity was significantly higher in AT1a -/- mice (205.5 ± 26.1 ng/ml/hr) than in wild-type mice (8.0 ± 0.2 ng/ml/hr). The renin-positive areas per glomerulus and renal renin gene expression were 12-times and 2.6-times higher in AT1a -/- mice than in wild-type mice, respectively. These abnormalities, however, were less remarkable in AT1a -/- mice compared with angiotensinogen-knockout mice. When AT1a -/- mice were fed a high-salt diet, the signal intensity of the NADPHd reaction and the number of positively-stained macula densa cells were significantly decreased. The levels of renal cortical N-NOS mRNA were also suppressed by the treatment. Dietary salt loading produced a parallel decrease in plasma renin activity, renal renin-immunoreactive areas, and the levels of renin mRNA without affecting systemic blood pressure. These results provide evidence for the possible involvement of N-NOS at the macula densa in the increased renin production in AT1a -/- mice.

Na+/H+ exchange and H-K ATPase increase distal tubule acidification in chronic alkalosis. We examined whether H+-ATPase, H+-K+-ATPase, and or Na+/H+ exchange mediates increased distal tubule acidification in animals with chronic metabolic alkalosis using pharmacological inhibitors of these H+ transporters in in vivo-perfused tubules of anesthetized rats. Chronic metabolic alkalosis was induced with furosemide followed by minimum electrolyte diet and HCO3 drinking water. The reduction in net HCO3 reabsorption was greater in distal tubules of alkalotic compared to control animals perfused with Schering 28080 to inhibit H+-K+-ATPase (-6.4 ± 0.9 vs. -1.4 ± 0.5 pmol/mm · min-1, P < 0.02) and with EIPA to inhibit Na+/H+ exchange (-11.1 ± 1.7 vs. -6.6 ± 0.9 pmol/mm · min-1, P < 0.01) but was similar in distal tubules of alkalotic and control animals perfused with bafilomycin to inhibit H+-ATPase. The greater reduction of distal tubule net HCO3 reabsorption in alkalotic compared to control animals induced by EIPA was eliminated by systemic infusion of the endothelin receptor antagonist bosentan (-4.6 ± 0.7 vs. -4.4 ± 0.7 pmol/mm · min-1, P = NS) but the greater reduction induced by Schering 28080 persisted. Urine endothelin-1 (ET-1) excretion was higher in animals with maintained alkalosis (164.5 ± 23.7 vs. 76.6 ± 10.8 fmol/day, P < 0.03), but decreased following KCl repletion to a value (86.7 ± 10.0 fmol/day, P < 0.02 vs. respective before-KCl value) that was not different from that for KCl-repleted control animals (79.9 ± 8.7 fmol/day, P = NS vs. KCl-repleted alkalotic animals). The data support that augmented distal tubule acidification in alkalotic animals is due to increased H+-K+-ATPase and Na+/H+ exchange activity, the latter stimulated by endogenous endothelins.

Effects of partial nephrectomy on the expression of osmolyte transporters. Na + / myo -inositol cotransporter (SMIT) and Na + /Cl − /betaine-γ-amino-n-butyric acid transporter (BGT-1) are the major osmolyte transporters that are regulated by extracellular osmolarity. We have recently shown localization and rapid regulation of the mRNAs for these transporters in rat kidney. In the present study, we examined the expression of SMIT and BGT-1 in partial nephrectomized rats in order to assess the change in local osmolarity following reduction of renal mass. Four weeks after 5/6 nephrectomy (NX), the rats were compared to sham-operated control animals (CONT). Northern analysis using RNA of whole kidney indicated that there were little differences in the levels of SMIT and BGT-1 mRNAs between the two groups. In situ hybridization revealed that signals for both transporter mRNAs were markedly reduced in the inner medulla of the remnant kidney. In contrast, these signals in the outer medulla increased following nephrectomy. SMIT signals in the cortex increased as well. Grain density, determined by counting grain number per cell, revealed that the signals in the inner medullary collecting ducts were markedly reduced whereas those in the thick ascending limbs of Henle (TAL) as well as macula densa cells were significantly increased. The signals in the TAL and macula densa were reduced by furosemide administration. The increased expression in NX rats may reflect the increased NaCl transport and high local osmolarity in this segment.

Nitric oxide (NO) exerts profound effects on renal physiology [1—41. Renal NO generation occurs in endothelial cells by the constitutive isoform of nitric oxide synthase (cNOS) and in macrophages, smooth muscle and mesangial cells by the inducible isoform (iNOS). The former system plays an important role in basal renal vascular tone and hemodynamic regulation, in mesangial contraction, renin release and tubuloglomerular feedback. The inducible NOS, widely distributed in the rat kidney, predominantly in the outer medulla and in cortical glomeruli [5], counterbalances vasoconstrictive, thrombogenic and proliferative stimuli associated with cytokine stimulation and inflammatory responses [6]. Intact nitrovasodilation is particularly important at the outer medulla, where tissue oxygen tension as low as 30 mm Hg normally exists [7]. Inhibition of NOS results in profound renal hypoperfusion, both at the cortex and outer medulla [8, 9], associated with substantial aggravation of medullary hypoxia [10]. Blockade of NO synthesis potentiates medullary hypoxic injury from radiocontrast and non-steroidal anti-inflammatory agents [9—111 and augments medullary hypoperfusion and damage during acute ureteral obstruction [121. Recently developed Clark-type selective NO microelectrodes enable real-time monitoring of NO concentrations in vitro in cell lines and incubated tissues [13—17], in organs maintained ex vivo [18] or in the living organism [19, 20]. With concomitant determination of intrarenal blood flow it could be a potent experimental tool for the evaluation of the role of NO in renal hemodynamics. In preliminary studies with the NO electrode, a paradoxical increase in medullary NO reading was noted following NOS inhibition. This led us to evaluate potential artifacts of NO monitoring, such as fluctuations in tissue oxygenation (since oxygen and reactive oxygen species scavenges NO with the production of NO2/NO3 , not sensed by the electrode) and temperature (which markedly affect the electrode current) [21]. Methods

Defective renal calcium reabsorption in genetic hypercalciuric rats. Idiopathic hypercalciuria is a frequent cause of calcium (Ca) containing kidney stones. We have previously shown that there is increased intestinal Ca absorption in selectively inbred genetic hypercalciuric stone forming (GHS) rats; however, excess Ca excretion persists when the rats are fed a low Ca diet indicating a defect in renal Ca reabsorption and/or increased bone resorption. To determine if GHS rats have a defect in renal Ca reabsorption we performed 14 C-inulin clearance studies on parathyroid-ectomized female GHS and control (Ctl) rats. After three baseline collections, chlorothiazide (CTZ) or furosemide (FUR) was infused and three more collections were obtained. Both GFR and filtered load of Ca did not differ among the groups; however, fractional and absolute excretion (U Ca V) of Ca was three times higher in GHS rats. The increased Ca excretion was not diminished by a low Ca diet. Urine flow rate nearly tripled in all rats after either FUR or CTZ. After CTZ, U Ca V was decreased to a greater extent in GHS compared to Ctl rats. After FUR, U Ca V was increased to a greater extent in Ctl rats compared to GHS rats. These data indicate that GHS rats have a defect in renal Ca reabsorption, in addition to increased intestinal Ca absorption. The effect of CTZ was greater, and that of FUR was smaller, in GHS compared with Ctl rats, suggesting that the defect in renal Ca handling might be at the level of the thick ascending limb.

Effects of cyclosporine A on Na,K-ATPase expression in the renal epithelial cell line NBL-1. The bovine renal epithelial cell line NBL-1 has been used to monitor the effects of cyclosporine A (CsA) on Na + ,K + -ATPase activity and expression. CsA at two single doses (0.6 mg/liter and 2.5 mg/liter) inhibits the ouabain-sensitive component of Rb + uptake, assumed to be Na + ,K + -ATPase, but increases the low activity of a furosemide-sensitive component corresponding to a Na + /K + /Cl - cotransporter. CsA addition also induces a slight decrease of α 1 subunit mRNA levels, without altering the already low β 1 subunit mRNA amounts. Hypertonic treatment of NBL-1 cells leads to a significant increase in both Na + ,K + -ATPase activity and α 1 subunit mRNA amounts, but does not modify β 1 subunit mRNA levels. The differential response of the α 1 and β 1 subunit genes may explain why hypertonic treatment does not result in higher α 1 protein expression, and supports the view that increased activity relies upon post-translational events, despite the likely transcriptional activation of the α 1 subunit gene. The addition of CsA does not alter the hypertonicity-mediated increase of Na + ,K + -ATPase activity but blocks the accumulation of α 1 subunit mRNA. In conclusion, CsA may compromise the ion handling by renal cells as a result of the inhibition of basal Na + ,K + -ATPase activity and the stimulation of Na + /K + /Cl - cotransport activity. Morevoer, this is the first report showing that CsA may affect the long-term adaptation of the pump by altering its subunit gene expression.

Chloride and fluid secretion by cultured human polycystic kidney cells. Epithelial cells cultured from the renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD) secrete fluid via a process stimulated by adenosine 3′,5′-cyclic monophosphate (cAMP). We have investigated the hypothesis that fluid secretion by these cells is dependent on cAMP-mediated chloride secretion. Individual cultured ADPKD cells were suspended within a polymerized collagen matrix and stimulated to form cysts. Individual cultured cysts were placed in a chamber on the stage of an inverted microscope equipped with epifluorescent and video analysis attachments. The rate of fluid secretion, cell volume and changes in intracellular Cl- were measured. In the absence of secretagogues, fluid was absorbed from the cyst cavity (-2.36 ± 0.64 nl/min/cm2 inner surface area). 8-Bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) plus 3-isobutyl-l-methlyxanthine (IBMX) induced a rapid reversal in the net movement of fluid to secretion (6.79 ± 1.28 nl/min/cm2). Bumetanide reversibly reduced fluid secretion to 0.95 ± 0.60 nl/min/cm2. Cell volume rapidly decreased by 7.5 ± 0.9% with the initiation of secretion and bumetanide caused an additional loss (4.2 ± 1.0%). Furosemide had a similar effect on forskolin-induced fluid secretion. Cellular chloride concentration was monitored with the use of the indicator, 6-methoxy-N-ethylquinolinium chloride (MEQ). Removal of Cl- from the bath reduced intracellular [Cl-] (MEQ fluorescence increased by 11.4 ± 2.3%). In cysts pretreated with furosemide to prevent Cl- entry, the application of forskolin caused a decrease in Cl- concentration (MEQ fluorescence increased by 9.3 ± 2.6%). Using monolayers of cultured ADPKD cells, grown on permeant supports, we compared the changes in short circuit current (ISC) induced by forskolin in the presence and absence of external Cl-. Forskolin increased ISC (from 8.9 ± 2.7 to 10.6 ± 2.7 µA/cm2) in the presence of Cl-, but did not significantly affect ISC in its absence. These data indicate that cultured ADPKD cells can direct fluid transport in either the absorptive or the secretory direction, and that cAMP stimulates secretion and this secretion is accompanied by a net loss of cell solute. Inhibition of secretion by bumetanide or furosemide caused an additional loss of cell solute, including Cl-. The ionic transepithelial current induced by forskolin is dependent on the presence of Cl-. These data support the thesis that chloride secretion drives fluid secretion by cultured ADPKD cells.

Expression of betaine transporter mRNA: Its unique localization and rapid regulation in rat kidney. Betaine is a major compatible osmolyte in the renal medulla. It is taken up into cells via the betaine γ -amino- n -butyric acid transporter (BGT-1). We investigated the localization of BGT-1 mRNA and its acute regulation by NaCl and furosemide administration. In situ hybridization revealed that BGT-1 mRNA is predominantly present in the outer medulla and papilla. Less intense signals were seen in the inner medulla and no signals were found in the cortex. Microscopic examination suggested that intense signals were present in the medullary thick ascending limbs of Henle's loop (MTAL) and the inner medullary collecting ducts (IMCD). A reverse transcription and polymerase chain reaction assay of individual microdissected segments along the nephron confirmed its localization. Intraperitoneal administration of NaCl rapidly increased the signal in the MTAL, and furosemide prevented the increase in BGT-1 mRNA by NaCl loading. In contrast, BGT-1 mRNA in the IMCD is less sensitive to these kinds of acute regulation. These results suggest that BGT-1 expression in the MTAL is rapidly regulated in response to the magnitude of NaCl absorption, as suggested for the expression of Na + / myo -inositol cotransporter.

Regulation of renin release is impaired after nitric oxide inhibition. The aim of the present study was dual: first to establish that a preparation of afferent arterioles freshly isolated from the rat kidney is a suitable model to study renin release and synthesis, and second to investigate the effect(s) of nitric oxide (NO) inhibition on renin release in this model. Purification of renal microvessels was based on iron oxide infusion into the kidneys and separation of the afferent arterioles from glomeruli and connective tissue with a magnet. These microvessels express preprorenin mRNA, contain renin granules and release renin as evidenced by RT-PCR, immunocytochemistry and measurement of renin activity, respectively. Renin secretion was increased in isolated afferent arterioles after in vivo treatment with the diuretic furosemide (+300%) or in vitro treatment with the adenylyl cyclase activator forskolin (+50%), indicating that this vascular preparation responds appropriately to regulators of the renin-angiotensin system. Furthermore, in afferent arterioles isolated from control rats, renin release was positively correlated with total renin content (r = 0.85). In afferent arterioles isolated from rats chronically treated with the NO-synthase inhibitor N G -nitro-L-arginine methyl ester (L-NAME), forskolin was ineffective in modifying renin release despite stimulation of cAMP levels. In addition, the correlation between renin release and tissue renin content was disrupted. Similar results were obtained when cortical slices were used instead of afferent arterioles, suggesting that this defect in the regulation of renin release is independent of the presence of macula densa cells. To verify that the lack of regulation of renin release after L-NAME treatment was due to NO inhibition, the NO donor 3-morpholino-syndonimin-hydrochloride (SIN-1) was administered in afferent arterioles or cortical slices from kidneys of L-NAME-treated rats. In both preparations, SIN-1 reversed the L-NAME effect and re-established the responsiveness of renin release to forskolin and the relationship between renin release and renin content. These data indicate that the adenylyl cyclase-mediated mechanism regulating renin release is impaired when NO synthesis is inhibited.

Contribution of tubuloglomerular feedback to renal arteriolar angiotensin II responsiveness. The purpose of the present study was to test the hypothesis that a component of the afferent arteriolar vasoconstrictor response to angiotensin II (Ang II) requires an intact tubuloglomerular feedback (TGF) mechanism. Enalaprilat-treated male Sprague-Dawley rats served as tissue donors for study of renal microvascular function using the in vitro blood-perfused juxtamedullary nephron technique. Arteriolar lumen diameter responses to exogenous Ang II were determined before and after TGF blockade (papillectomy or 50 µ m furosemide). Before TGF blockade, 10nm Ang II significantly reduced diameters of both midafferent (53 ± 5%) and efferent (43 ± 9%) arterioles. TGF blockade did not alter baseline diameter of either arteriole, but significantly blunted the mid-afferent vasoconstriction evoked by 10nm Ang II (44 ± 7% inhibition by papillectomy; 43 ± 10% inhibition by furosemide). Similar behavior was observed at afferent arteriolar sites near the glomerulus; however, efferent arteriolar Ang II responsiveness was not altered by papillectomy. The impact of TGF blockade on afferent arteriolar Ang II responsiveness was most prominent at high peptide concentrations (10nm), while not significantly influencing the response to 1nm Ang II. In contrast, the afferent vasoconstrictor effect of norepinephrine was unaffected by papillectomy. These data indicate that the vasoconstrictor influence of exogenous Ang II on afferent, but not efferent, arterioles of intact juxtamedullary nephrons includes both TGF-dependent and TGF-independent components.

Glomerular mesangial cells are located between and within glomerular capillary loops, phenotypically resemble vascular smooth muscle cells, and play an essential role in regulating glomerular filtration and growth [1–6]. In response to locally-produced and circulating growth factors [that is, insulin-like growth factors (IGF) including insulin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF)], mesangial cells demonstrate both contractile and mitogenic responses. Stimulation of growth factor receptor-linked tyrosine kinase activity is required for tyrosine autophosphorylation of growth factor receptors, receptor internalization, inositol phospholipid turnover, changes in intracellular pH and [Ca 2+ ] i , and DNA synthesis and mitosis [1–3, 5]. Mesangial cells also show contractile and mitogenic responses to vasoactive peptides which are not traditionally considered growth factors [that is, angiotensin II (Ang II) and endothelin-1 (ET)][1, 2, 4, 6]. ET and Ang II increase tyrosine-specific, protein phosphorylation without these ligands having direct receptor-linked, tyrosine kinase activity. In addition, Ang II stimulates mesangial cell expression of both PDGF and ET [1, 2, 4]. Our recent patch clamp studies have characterized ion channels in the membranes of cultured rat glomerular mesangial cells which are stimulated by classic renal growth factors, including PDGF and insulin [7–10], and vasoactive peptides which possess mitogenic effects, including Ang II and ET [9, 11–13] (Fig. 1). These ion channels promote extracellular Ca 2+ entry via classic, voltage-activated Ca 2+ channels and/or receptor-operated, Ca 2+ -permeable channels.

Acute changes in urinary excretion of nitrite + nitrate do not necessarily predict renal vascular NO production. NO 2 + NO 3 (NO x ), the stable oxidation products of NO, and cGMP are widely accepted as indices of in vivo NO production. Whether acute changes in urinary excretion of nitrite + nitrate (U NOX V) can be taken to reflect acute changes in renal and/or systemic NO production is not known. The present studies were conducted in the conscious rat to investigate the effect on acute changes in U NOx V, of maneuvers that (a) enhance NO production and (b) act as diuretics. L-arginine (L-arg) and acetylcholine (Ach) produce equivalent NO dependent falls in renal vascular resistance (RVR), but a much greater increase in U NOX V is seen with L-arg. D-arg does not stimulate NO and has no renal vasodilatory effect, but produces a large rise in U NOX V, and SNP lowers BP but not RVR and results in a reduced U NOX V. None of the diuretics employed should stimulate the NO system or lower RVR; however, the proximally acting agents, acetazolamide and D-arg increased U NOx V, while the loop diuretic furosemide had little effect. H 2 O diuresis (a distal event) led to a fall in U NOx V. These data suggest that NO x is reabsorbed extensively in the proximal tubule and that inhibition of proximal reabsorption leads to an increase in U NOx V. Also, our results show that the relationship between U NOx V and U cGMP V is unpredictable. Therefore, we conclude that measurements of acute changes in U NOx V and/or U cGMP V should be interpreted cautiously, since they may reflect altered tubular handling of NO x rather than the acute activity of the systemic and/or renal NO systems.

Characterization of Na+ transport across the cell membranes of the ascending thin limb of Henle's loop. In the ascending thin limb of Henle's loop (ATL), intracellular Na+ is extruded by Na+/K+ ATPase in the basolateral membrane. To further characterize Na+ transport across the cell membranes of the ATL, the intracellular sodium concentration ([Na+]i) was monitored using a sodium-sensitive fluorescent probe, SBFI, in the in vitro microperfused hamster ATL. Basal [Na+]i was 19.0 ± 1.2 mM (N = 24). Removal and replacement of luminal Na+ did not change [Na+]i in the presence of Na+ in the bathing fluid. In contrast, luminal Na+ removal reduced [Na+]i from 11.6 ± 0.9 to 6.3 ± 0.8 mM in the absence of peritubular Na+ (P < 0.0005, N = 21). Replacement of luminal Na+ increased [Na+]i to 12.6 ± 0.9 mM. In the absence of Na+ in the bath, the addition of 1 µM benzamil, 0.1 mM 5-(N,N-dimethyl)-amiloride (DMA), 0.1 mM furosemide, or 0.1 mM trichlormethiazide to the lumen did not change [Na+]i or the rate of change in [Na+]i (d[Na+i/dt) after removal and replacement of luminal Na+. Decreases in luminal pH in a Hepes-buffered solution and luminal HCO3- did not affect [Na+]i. In the absence of peritubular Na+, DMA in the bathing fluid decreased [Na+]i from 11.4 ± 1.3 to 6.4 ± 1.2 mM (P < 0.01, N = 5) and completely inhibited the changes in [Na+]i after removal and replacement of luminal Na+. Removal of peritubular Na+ reduced [Na+]i from 18.8 ± 1.2 to 11.3 ± 0.7 mM (P < 0.0001, N = 23). Addition of DMA in the bathing fluid reduced [Na+]i and inhibited the changes in [Na+]i after removal and replacement of peritubular Na+. Addition of benzamil, furosemide or trichlormethiazide to the bathing fluid did not alter [Na+]i or the changes in [Na+]i after removal and replacement of peritubular Na+. Decreases in peritubular pH in a Hepes-buffered solution and peritubular HCO3- did not affect [Na+]i. These results indicate that Na+ transport across the cell membranes of the ATL is mediated by the Na+/H+ antiporter in the basolateral membrane together with Na+/K+ATPase in the basolateral membrane and demonstrate the absence of Na+ permeability in the luminal membrane of the ATL.

Increased expression of Na + /H + exchanger in the injured renal tissues of focal glomerulosclerosis in rats. The renal mRNA expression of Na + /H + exchanger (NHE) and the effects of NHE inhibitor, amiloride, on renal injury were investigated in adriamycin (ADR)-induced glomerulosclerosis model in rats, which progressively developed extensive glomerulosclerosis and interstitial fibrosis. NHE-1 mRNA from the cortex of the ADR rats progressively increased at weeks 4 and 8 and then peaked at week 16, which paralleled with the degree of glomerular sclerosis and interstitial fibrosis. The interstitial fibrosis in the ADR-rats was prevented by a daily administration of amiloride. A simultaneous analysis of the effects of a high salt diet on NHE-1 mRNA expression or renal injury was performed in the ADR rats at weeks 2 and 8. Renal or glomerular hypertrophy was observed in the control or ADR rats fed an 8% NaCl diet at week 2 and 8 compared to a 1% NaCl diet, while the NHE-1 mRNA expression was not up-regulated by an 8% NaCl diet at week 2. At week 8, the NHE-1 mRNA expression or glomerulosclerosis and interstitial fibrosis were enhanced in the ADR rats fed an 8% NaCl diet compared to a 1% NaCl diet. This histological aggravation by an 8% NaCl diet was prevented by a daily administration of amiloride but not by furosemide. In conclusion, the increased NHE-1 mRNA expression and the preventive effects of amiloride on the renal lesions suggest a potential importance of NHE in the progressive process of ADR-nephropathy. The high salt diet had a hypertrophic and destructive effect on kidney or glomeruli in the ADR rats. The hypertrophic effect of a high salt diet did not seem to be mediated through NHE activation.

Effects of atrial natriuretic peptide in clinical acute renal failure. Fifty-three consenting patients meeting clinical and urine composition criteria for established intrinsic ARF were assigned to two treatment groups. Group I patients were treated with human atrial natriuretic peptide (ANP) with or without diuretics. Groups II patients were treated with or without diuretics and with no ANP. Age, sex, etiology of ARF, entry serum creatinines (SCr) (Group I, 5.3 ± 1.8; Group II, 5.1 ± 2.1 mg/dl) and creatinine clearances (CCr) (Group I, 9.9 ± 2.1; Group II, 9.2 ± 2.1 ml/min) were similar. Thirty patients received ANP [0.20 µg/kg/min i.v. × 24hr (N = 20) or 0.08 µg/kg/min i.a. × 8hr (N = 10)] and furosemide, 0.5 mg/kg/hr × 24hr or mannitol, 12.5g every six hours × 4, or no diuretic; 23 Group II patients received diuretics as above or no diuretic in a similar distribution to Group I. CCr (verified with simultaneous inulin clearances × 12, r = 0.93, P < 0.001) increased significantly by eight hours of ANP treatment to 17.1 ± 3.2ml/min and by 24 hours after discontinuing ANP to 21.0 ± 4.4ml/min (both P < 0.05). There was no corresponding increase in CCr in Group II. Dialysis was required in 23% of Group I and in 52% of Group II patients (different at P < 0.05). Mortality rates of 17% for Group I and 35% for Group II were not significantly different (P = 0.11). It is concluded that parenteral ANP increases CCr and reduces need for dialysis in patients with established intrinsic ARF.

Loop diuretics reduce hypoxic damage to proximal tubules of the isolated perfused rat kidney. The straight portion (S3) of the proximal tubule lies in close proximity to the thick ascending limbs (TALs) at the cortico-medullary junction. Since a delicate balance exists between oxygen demand and the limited oxygen supply in this region, we hypothesized that reduction of thick limb metabolic activity might augment oxygen availability to S3 segments, which depend heavily upon aerobic metabolism, and prevent hypoxic damage. The degree of functional deterioration and morphological damage to S3 was assessed in isolated rat kidneys perfused with an erythrocyte-free medium. Bumetanide (10−5M) and furosemide (10−4M) reduced S3 fragmentation from 9.8 ± 3.9% of tubules in controls to 0 and 1.4 ± 0.9%, respectively (P < 0.0005). Tubular glucose reabsorption was better preserved in kidneys exposed to loop diureties than in control kidneys (P < 0.01), and urinary alkaline phosphatase (P < 0.05) and the total amount of LDH released into the perfusate and urine (P < 0.01) were lower in the treatment groups. Morphological damage to S3 was closely correlated with medullary TAL necrosis (r = 0.66, P < 0.001), urinary alkaline phosphatase excretion (r = 0.89, P < 0.001) and glycosuria (r = 0.83, P < 0.001). We conclude that under hypoxic conditions TALs and S3 segments may compete with each other for a limited oxygen supply. Reduction of active transport in the mTAL might augment oxygen availability to S3 segments and improve their survival.

A 58-year-old woman underwent an evaluation for renal impairment. She had no family history of nephrocalcinosis, end-stage renal failure, or polycystic kidney diseases. She had a history of chronic edema of the lower limbs, which had been treated with 120 mg/day furosemide for more than 10 years. She had no history of back pain or hematuria. On physical examination, the patient was 152 cm tall and weighed 41 kg, and her blood pressure was 110/60 mm Hg and heart rate was 96 b.p.m. She had pitting edema in both legs. A urinalysis was negative for both hematuria and proteinuria. The blood analyses showed a potassium level of 2.9 mEq/l, blood urea nitrogen level of 27 mg/dl, creatinine level of 1.26 mg/dl, and uric acid level of 8.3 mg/dl. An abdominal X-ray showed bilateral renal calcification (Figure 1). Axial computed tomography of the abdomen showed bilateral nephrocalcinosis and cysts in the cortical and corticomedullary regions (Figure 2). Additional metabolic evaluations were also performed with the patient still taking furosemide. Her serum levels of calcium (9.3 mg/dl) and phosphate (4.1 mg/dl) were normal, although her serum intact parathyroid hormone (PTH) level was elevated (110 pg/l). Her urine volume was 2500 ml/24 h and calcium excretion was 262 mg/24 h. After stopping furosemide treatment, her serum potassium level increased to 3.6 mEq/l and urinary calcium excretion and serum intact PTH level were normalized (152 mg/24 h and 60 pg/l, respectively). The differential diagnosis should include nephronophthisis and medullary sponge kidney, but the facts that the cysts were cortical and urine calcium and serum intact PTH levels normalized after discontinuation of the loop diuretic are inconsistent with these entities. Loop diuretics cause chronic hypokalemia and hypercalciuria, which can induce renal cyst formation and nephrocalcinosis, respectively, and consequently chronic kidney injury.

Bradykinin causes selective efferent arteriolar dilation during angiotensin I converting enzyme inhibition. We studied the effects of interruption of the renin-angiotensin system (RAS) in rats that were volume depleted by water deprivation for 48 hours (AWD) with/without furosemide (AWD + F), a condition known to activate RAS. Following baseline micropuncture, AWD rats (N = 6) were treated with a specific angiotensin II type 1 receptor antagonist (AURA; 4 mg/kg body wt bolus i.v. and then continuous infusion) and glomerular hemodynamics compared to those obtained during angiotensin I converting enzyme inhibitor treatment (ACEI; 24 mg/kg bolus i.v. and then continuous infusion). Systemic blood pressure decreased equally following AIIRA and ACEI. Single nephron glomerular filtration rate (SNGFR) increased from baseline following AIIRA (24 nl/min vs. 30, P < 0.025). While a decrease in efferent arteriolar resistance (RE) reduced glomerular capillary pressure (PGc 67 mm Hg vs. 60, P < 0.05), this change in RE together with decrease in afferent arteriolar resistance (RA), enhanced glomerular plasma flow rate (QA; 80 nl/min vs. 111). Antagonizing angiotensin II receptor increased QA which, together with the tendency to increase glomerular capillary ultrafiltration coefficient, Kf, served to improve glomerular filtration. By contrast, although inhibition of the angiotensin I converting enzyme caused greater vasodilatation, no increase in SNGFR occurred. The lack of response in filtration after ACEI was due to a further fall in PGC to 52 mm Hg (P < 0.01 vs. AIIRA), reflecting profound reduction in RE. Since ACEI but not AIIRA potentiates bradykinin activity we examined effects of a specific bradykinin antagonist (Hoe). In a separate group of AWD+F rats (N = 6), ACEI again caused significant renal vasodilatation without affecting filtration rate. The ACEI-induced decrease in resistances was substantially antagonized by inhibition of bradykinin activity. Thus, Hoe significantly increased RE, effectively raising PGC by 9 mm Hg (P < 0.05 vs. ACEI) and thus secondarily effecting increased filtration (SNGFR 22 nl/min vs. 28, P < 0.05, ACEI vs. Hoe). In contrast to these results with ACEI, treatment with AIIRA significantly increased GFR, and addition of bradykinin antagonist (N = 4) did not cause further increase in filtration. Thus, in conditions where high PGC is crucial in maintaining GFR, activation of bradykinin by ACEI can compromise GFR secondary to bradykinin's selective vasodilatory action on the efferent arteriole.

An 83-year-old male was evaluated by the Renal Service because of an increase in serum creatinine from 1.7 mg/dl to 3.5 mg/dl over a 48-hour period after cardiac catheterization. He had hypertension for more than 30 years and non-insulin-dependent diabetes mellitus for approximately 25 years. One year ago, he sustained an inferior wall myocardial infarction and subsequently underwent two percutaneous transluminal coronary angioplasty procedures. Since that time he had been free of chest pain and had had minimal evidence of congestive cardiac failure. The current admission was prompted by progressive shortness of breath over several days; on presentation to the emergency room he was found to have atrial fibrillation and severe pulmonary edema. He was intubated and admitted to the coronary care unit. A non-Q-wave myocardial infarction was diagnosed and, after a prolonged course marked by recurrent episodes of supraventricular tachyarrythmias, he became hemodynamically stable. Blood urea nitrogen and creatinine were 24 mg/dl and 1.9 mg/dl, respectively, at the time of admission and 23 mg/dl and 1.7 mg/dl, respectively, when he was discharged from the coronary care unit. Cardiac catheterization was performed to evaluate his coronary circulation; normal saline was administered intravenously prior to the procedure (50 mllhr) and mannitol, 25 g, was administered intravenously immediately after the procedure. Intravenous normal saline was continued at a rate of 100 mI/hr for 8 hours post catheterization but, despite positive fluid balance, the serum creatinine increased to 2.3 mg/dl. Over the subsequent 24 hours, his urine output decreased to 900 ml124 hr and the serum creatinine increased further to 3.5 mg/dl. His blood pressure remained normal, and he had no signs of hemodynamic instability over the 48-hr post-catheterization period. Medications included digoxin, a beta-blocker, subcutaneous heparin, and enteric-coated aspirin; no known nephrotoxic agents had been administered, other than the contrast agent (50 ml Omnipaque, a low osmolar agent) used during the cardiac catheterization procedure. On physical examination, the patient was alert, cooperative, and in no obvious distress. His blood pressure was 140/80mm Hg in the supine position; the pulse rate was 90 beats/mm and regular; and he was afebrile. Cardiac examination disclosed an S4 gallop with no murmurs, and his lungs were clear to auscultation. Abdominal examination was unremarkable and peripheral edema was absent. Laboratory studies revealed: serum sodium, 133 mEq/liter; potassium, 4.2 mEq/liter; chloride, 101 mEq/Iiter; total carbon dioxide, 26 mM/liter; blood urea nitrogen, 37 mg/dl; serum creatinine, 3.5 mg/dl; calcium, 8.0 mg/dl; phosphorus, 4.4 mg/dl; and albumin, 2.6 g/dl. The hemoglobin was 10.9 g/dl; hematocrit, 32.7%; white blood cell count, 8300 mm3; and platelet count, 315,000 mm3. Urinalysis revealed: pH, 5.0; protein, 30 mg/dl; occasional red blood cells/high-power field, and occasional granular casts. Over the subsequent 72 hours, his fluid intake was adjusted to match his urine output. The serum creatinine increased to 4.2 mg/dl, and his urine output gradually increased to 1400 ml/24 hr without additional therapy. The following day he experienced another acute myocardial infarction, became markedly hypotensive, and required intubation. Intravenous furosemide, 40 mg/hr over 10 hrs, was given on each of 5 successive days; the urine output increased to 2000 ml/24 hr. The serum creatinine peaked at 5.6 mg/dl but fell during the following week to the admission value of 1.7 mg/dl. He subsequently underwent successful triple-vessel coronary bypass surgery without additional renal insult. He was discharged home 12 days postoperatively with stable renal function.

Effect of furosemide-induced hypokalemic metabolic alkalosis on renal transport enzymes. Hypokalemic metabolic alkalosis is one of the most common complications of chronic furosemide administration. In this study we examined acid-base composition and ATPase enzyme activities in medullary thick ascending limb of Henle's loop (MTAL) and collecting tubule (CCT and MCT) after seven days of chronic furosemide therapy. All of the studies were conducted in adrenal intact (AI) rats or in adrenalectomized (ADX) glucocorticoid replete rats replaced with a physiological dose of aldosterone (Aldo). Furosemide (F) was administered to each rat by mini-osmotic pump. In the AI + F group, plasma Aldo was high and obvious metabolic alkalosis occurred (HCO3- = 37 ± 2 mEq/liter vs. 22 ± 2 mEq/liter in controls, P < 0.005); activities of H-K-ATPase, H-ATPase, and Na-K-ATPase were increased approximately twofold in both CCT and MCT. In the ADX + F group (HCO3- = 28 ± 2 mEq/liter, P < 0.05 from control), H-ATPase activity was normal in CCT and it was slightly increased in MCT. CCT and MCT H-K-ATPase activities were markedly increased (∼ twofold). Na-K-ATPase actiyity was the same as control in CCT but it was increased in MCT. In ADX + F + Vanadate (V) group which also had normal Aldo levels, acid-base changes were modest (20 ± 2 mEq/liter, NS from control); in CCT and MCT H-K-ATPase and Na-K-ATPase activities were markedly reduced, but H-ATPase activity in MCT was increased. In all three experimental groups Na-K-ATPase activity in MTAL was reduced fivefold. Hypokalemia developed in both intact and ADX animals receiving furosemide. Thus, metabolic alkalosis in chronic furosemide therapy is associated with stimulation of all three collecting tubule ATPases. The high aldosterone level likely stimulates the H-ATPase in both CCT and MCT; and in the former it also stimulates Na-K-ATPase activity. The hypokalemia probably activates H-K-ATPase in both CCT and MCT. Furosemide also appears to directly stimulate H-ATPase activity in the MCT.

NaCl transport by Madin Darby canine kidney (MDCK) cyst epithelial cells. The mechanism of NaCl transport across the epithelium of intact MDCK cysts grown in a collagen gel matrix was investigated. Double-barreled microelectrodes were used to measure basolateral membrane PD (V b1 ), transepithelial PD (V t ), and intracellular (Cl i ) and intralumenal (Cl cy ) Cl - activities in cysts under different conditions. In a control Ringer's solution (RS), Cl i (60 ± 1mM) and Cl cy (107 ± 2mM) exceeded the values corresponding to electrochemical equilibrium across the basolateral membrane and epithelium, respectively. Cl i was reduced by superfusing the cysts with a low Cl - RS (Cl i , 20 ± 3mM), a low Na + RS (Cl i , 40 ± 4mM), or by adding amiloride to the control RS (Cl i , 46 ± 1mM). Cl i was unaffected by removal of either K + or HCO 3 - from the RS or by adding furosemide or SITS to the control RS. V b1 in the control RS was -50 ± 2mV and was affected only by removal from the RS of K + (V b1 , -31 ± 3mV) or HCO 3 - (V b1 , -29 ± 4mV) or by the addition of SITS to the control RS (V b1 , -59 ± 5mV). V t in control RS was -2 ± 0.2mV (lumen negative), and was increased by reducing bath Na + (V t , -37 ± 2mV) but not by reducing bath Cl - . These data indicate that Cl - is secreted in a basolateral to apical direction by the cyst epithelium. Basolateral Cl - transport probably occurs mainly by an electroneutral Cl - /HCO 3 - exchanger. Transepithelial Na + transport seems to occur via a paracellular route which appears to be cation selective. These experiments also support the existence, in the basolateral membrane, of a Na + /K + ATPase, a Na + /H + exchanger, and possibly a Na + /HCO 3 - / CO 3 2- transporter.

Effects of acute and chronic uremia on active cation transport in rat myocardium. As abnormalities of active cation transport could contribute to the genesis of uremic cardiomyopathy, we investigated myocardial sodium pump function in rats with acute renal failure (ARF) and with a model of experimental chronic renal failure (CRF) that has metabolic similarities to advanced chronic uremia in humans. CRF rats were hypertensive and had left ventricular hypertrophy (33% higher heart:body weight ratio; P < 0.01) at four weeks compared to pair-fed sham-operated rats. Importantly, both ouabain- and furosemide-sensitive 86Rb uptake rates were unchanged in left ventricular myocardial slices from CRF, and the intracellular sodium concentration was not different from that of control rats even though skeletal muscle sodium was increased, as we found previously (J Clin Invest 81:1197, 1988). Insulin-stimulated, ouabain-sensitive 86Rb influx was also preserved. There also were no abnormalities in myocardium cation transport in rats with ARF. However, [3H]ouabain binding was decreased 45% in CRF rats (P < 0.01); it was unchanged in acute uremia. Decreased ouabain binding in chronic uremia was due entirely to fewer low affinity [3H]ouabain binding sites (the binding affinity for ouabain was unaffected). We conclude that in chronic, (but not acute) renal failure, sodium pump number is reduced in myocardium but intracellular sodium is unchanged and active cation flux rates are maintained. These results emphasize that in rats with chronic uremia, intracellular sodium homeostasis is preserved in myocardium, despite the presence of marked abnormalities of active cation transport in skeletal muscle that are characteristic of chronic uremia.

Glomerular angiotensinase A in the rat: Increase of enzyme activity following renal ablation. Angiotensinase A (aminopeptidase A; ATA) is an angiotensin II splitting exopeptidase, which is localized in endothelial and epithelial cells of the glomerular tuft. In order to investigate the influence of a reduction in renal mass on enzyme activity, ATA activity was measured in isolated rat glomeruli five and 14 weeks after 1-1/3 nephrectomy. Glomerular ATA activity in remnant kidneys increased significantly after five weeks following ablation compared with glomeruli of two kidney control rats (5.34 ± 4.02 vs. 1.71 ± 1.96mU /mg protein, P < 0.05). After 14 weeks, however, this difference was no longer present. Treatment of rats with enalapril or saralasin inhibited the increase of ATA seen five weeks after renal ablation, whereas indomethacin had no effect on enzyme activity. Furthermore, normal two kidney rats, treated with furosemide, revealed a higher glomerular ATA than two kidney controls (5.5 ± 2.64 vs. 2.1 ± 1.7 mU/mg protein, P < 0.05). In vitro superfusion of isolated glomeruli with enalaprilate or furosemide from rats after renal ablation did not influence enzyme activity, however, superfusion with 0.05mM angiotensin II or 0.05mM saralasin significantly reduced ATA. Our results suggest that glomerular ATA might be involved in the early regulation of the intrarenal renin-angiotensin system and could modify glomerular adaptation to reduce renal mass by affecting angiotensin II degradation.

The progression of chronic renal failure is facilitated by the presence of arterial hypertension. Antihypertensive therapy has been shown to effectively protect the kidney from continuous functional derangement [1, 2]. The degree of proteinuria has also been shown to be a prognostic factor for the progression of chronic renal insufficiency [3, 4]. Recently, experimental [5, 6] and preliminary clinical data [7] have shown that converting enzyme inhibitors are effective in slowing the progression of chronic renal failure. These drugs also have the capacity to reduce glomerular proteinuria [8, 9], and through this mechanism they also have the ability to change the rate of progression of chronic renal failure. On the other hand, the measurement of the progression of chronic renal failure remains as a matter of debate [10, 11]. The usefulness of the slopes of the reciprocal of serum creatinine has been questioned [10–12] and there seems to be general agreement in the sense that sequential measurements of isotope glomerular filtration rate or inulin clearance are the most reliable methods to determine the rate of progression of chronic renal failure. This paper contains our experience on long-term effects of the therapy with the angiotensin converting enzyme, captopril, on the glomerular filtration rate and on the rate of progression of renal insufficiency of patients with chronic renal failure and arterial hypertension. Two previous publications of our group [7, 9] contain the initial results of this study obtained during the first year of follow-up in Group 1 and the first six months in Group 2. Before switching their therapy to captopril, patients were divided in two groups: Group 1, patients with primary non-glomerular disease who exhibited a progressive decline in renal function; their blood pressure was adequately controlled by means of standard triple therapy. They were initially classified by the finding of a negative and significant slope of the reciprocal of serum creatinine. Group 2 were patients diagnosed by a renal biopsy as having a primary glomerular disease with chronic renal failure, who presented with an adequate control of their arterial hypertension with one or two standard drugs. The long-term comparison of captopril versus standard therapy on the progression of renal failure and on non-diabetic glomerular proteinuria, as well as an evaluation of the different methods employed to evaluate glomerular filtration rate are presented.

Tubuloglomerular feedback dependence of autoregulation in rat juxta-medullary afferent arterioles. Experiments were performed in blood-perfused juxtamedullary nephrons in vitro to evaluate the tubuloglomerular feedback (TGF) dependence of autoregulatory vasoconstriction in mid-to-late (mAA) and juxtaglomerular (jAA) afferent arterioles. Video-metric measurements were made of perfusion pressure (PP) dependent changes in lumen diameter of superficial vessels before and after acute inhibition of the TGF mechanism by direct microinfusion of 0.1mM furosemide solution into the macula densa (MD) segment. When PP was raised from 60 to 123 ± 7mm Hg in seven vessels, jAA diameter decreased by 29 ± 3% (SEM, N = 7). During furosemide infusion with the same change in PP, jAA diameter decreased only 7 ± 2%. After calcium channel blockade with 1 micromolar nimodipine, jAA lumen diameter increased by 21 ± 7%. A similar pattern of responses was observed in eight jAA where TGF was inhibited with an oil block at the MD. mAA autoregulatory responses were also blunted by TGF inhibition. Raising PP from 60 to 120mm Hg resulted in 15 ± 2% and 7 ± 2% decreases in mAA luminal diameter before and after TGF inhibition. These results demonstrate that the autoregulatory responses in mid- and juxtaglomerular afferent arteriolar segments are mediated by both TGF and a TGF-independent myogenic mechanism.

Effects of specific COX-2-inhibition on renin release and renal and systemic prostanoid synthesis in healthy volunteers. Background The renin-angiotensin system plays a critical role in cardiovascular function, but little is known about the effects of specific cyclooxygenase 2 (COX-2) inhibition on this system in healthy humans under physiologic conditions. Methods Twenty-one healthy female volunteers received, in a randomized, double-blind, crossover study, celecoxib 200mg twice a day, indomethacin 50mg three times a day, or placebo for 4 days and a single dose, each, on day 5. On day 5 of each treatment, the following parameters were assessed with subjects in an upright position before and after administration of 20mg furosemide intravenously: plasma renin activity (PRA), plasma aldosterone, serum and urine electrolytes, and creatinine. Index metabolites of prostanoids were analyzed by gas chromatography-tandem mass spectrometry in 24-hour urine on day 4 and in 2-hour urines before and after furosemide administration. Results Baseline and furosemide-stimulated PRA were reduced to a similar degree by celecoxib and indomethacin. Plasma aldosterone and urinary excretion of potassium showed changes consistent with the alteration of PRA. Urinary excretion rates of prostaglandin E 2 , (PGE 2 ), 7α-hydroxy-5, 11-diketotetranor-prosta-1,16-dioic acid (PGE-M), and 2,3-dinor-thromboxane B 2 (TxB 2 ) were not reduced by celecoxib, whereas indomethacin led to a decrease of 40%, 45%, and 80%, respectively. Both active treatments inhibited urinary excretion of 2,3-dinor-6-keto-PGF 1α and 6-keto-PGF 1α by 60% and 40%, respectively. Conclusion Renin-release in healthy humans with normal salt intake is COX-2 dependent. While COX-1 is critical for renal and systemic PGE 2 production, renal prostacyclin synthesis is apparently COX-2 dependent. Finally, the previously demonstrated shift of the thromboxane-prostacyclin balance toward prothrombotic thromboxane by specific COX-2 inhibition is confirmed.

Validation of quantitative BOLD MRI measurements in kidney: Application to unilateral ureteral obstruction. Background Blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) provides a measure of deoxyhemoglobin content and therefore an indirect measure of the partial oxygen pressure (pO 2 ). The main purpose of this study was to examine the relationship between the apparent relaxation rate (R2*) in the pig kidney by BOLD imaging and renal tissue pO 2 levels measured directly by oxygen-sensitive microelectrodes. Second, BOLD imaging was applied to kidneys in pigs subjected to acute unilateral ureteral obstruction (UUO) to examine whether this condition is associated with changes in intrarenal oxygenation. Methods Oxygen-sensitive microelectrodes were inserted in the cortex and medulla of pig kidneys ( N = 6). Different arterial and intrarenal levels of pO 2 were obtained by stepwise changing the oxygen-to-nitrogen ratio supplied by a respirator. Simultaneous BOLD MRI measurements using an R2*-sensitive Echo Planar Imaging (EPI) sequence were performed on the contralateral kidney. In another group of pigs ( N = 3) BOLD imaging was performed following 24 hours of UUO. Results When the inhaled oxygen fraction was 5% to 70%, R2* was linearly related to pO 2 levels (cortex ΔR2*/ΔpO 2 =-1.2 ms −1 kPa −1 , and medulla ΔR2*/ΔpO2 =–1.7 ms −1 kPa −1 ). Twenty-four hours of UUO was associated with an increased R2* in the cortex and a decreased R2* in medulla as compared with baseline, which remained augmented after the release of UUO, indicating that pO 2 levels were reduced in the cortex and increased in the medulla during and after release of obstruction. Conclusion BOLD MRI provides noninvasive estimates of regional renal oxygen content and our study demonstrates that this technique may provide a useful tool in UUO which is associated with altered renal oxygen consumption.

BSC1 inhibition complements effects of vasopressin V 2 receptor antagonist on hyponatremia in SIADH rats. Background Severe hyponatremia is most frequently caused by the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Although the expressional alteration of the kidney-specific apical water channel, aquaporin 2 (AQP2), in the collecting duct has been demonstrated to be involved in the development of hyponatremia and the subsequent physiologic reaction that is resistant to arginine vasopressin (AVP; vasopressin escape) in SIADH, the complete pathogenesis of and the appropriate medical treatment for hyponatremia have yet to be elucidated. Methods Hyponatremia was induced in male Sprague-Dawley rats by water loading and subcutaneous infusion of 1-deamino-8-D-arginine vasopressin (dDAVP). For the treatment, a selective AVP V 2 receptor antagonist (OPC-31260) and/or a loop diuretic (furosemide) were administered orally. Protein expression of AQP2 and rat bumetanide-sensitive cotransporter (rBSC1) was examined by Western blotting during the hyponatremia and the subsequent treatment. Results We noted a markedly high expression of rBSC1 during the development of hyponatremia, and a relatively low expression during vasopressin escape. OPC-31260 administration elevated serum sodium level in a dose-dependent manner. The therapeutic effect, however, declined with increasing number of treatment days, and doses higher than 15 mg/kg/day induced severe toxicity. The physiologic parameters and the alterations of AQP2 and rBSC1 expression during the treatment demonstrated reactions that were completely opposite to those of vasopressin escape. Combination of a furosemide (100 mg/kg/day) and a low dose of OPC-31260 (5 mg/kg/day) additively elevated serum sodium level and sustained the elevated serum sodium level by significantly reducing sodium accumulation in the renal medulla. Conclusion AVP-induced alterations of rBSC1 expression, as well as those of AQP2, are involved in the pathogenesis of SIADH. The pharmacologic blockade of AVP stimulus in SIADH limits its therapeutic efficacy by discontinuing the vasopressin escape, and the selective inhibition of rBSC1 complements this limitation.

Control of renin gene expression in 2 kidney-1 clip rats. This study was done to investigate the mechanisms that underly the changes of renal renin gene expression upon hypoperfusion of one kidney. To this end the left renal arteries of male Sprague-Dawley rats were clipped with 0.2mm silver clips and renal renin mRNA levels were assayed by RNase protection during the first ten days after clipping. Unilateral reduction of renal blood flow led to transient maximal fivefold increases of renin mRNA levels in the clipped kidneys and to sustained suppression of renin gene expression to 20% of the control value in the contralateral intact kidneys. Inhibition of prostaglandin (PG) formation by meclofenamate or EDRF synthesis by L-NAME markedly attenuated the increase of renin mRNA levels in response to clipping, and a combination of PG/EDRF inhibition almost abolished the increase of renin mRNA levels. Inhibition of PG/EDRF formation did not change the suppression of renin mRNA levels in the contralateral intact kidneys. Neither did renal denervation nor inhibition of macula densa function by furosemide prevent the suppression of renin gene expression in response to unilateral renal artery clipping. Only converting enzyme inhibition by ramipril and blockade of Ang II-AT1 receptors by losartan attenuated the decrease of renin mRNA levels in the contralaterals to clipped kidneys. These findings suggest that intact PG and EDRF synthesis represent stimulatory signals for renin gene expression that are required for the elevation of renin mRNA levels upon unilateral renal hypoperfusion. The suppression of the renin gene in the intact contralateral kidney appears to require the presence of Ang II but seems not to be regulated by this parameter, suggesting the existence of an as yet unidentified factor that acts in concert with Ang II to suppress renin gene expression.

Effects of enhanced oxygen release from hemoglobin by RSR13 in an acute renal failure model.BackgroundAcute renal failure is believed to be caused, in some circumstances, by impaired oxygen delivery to the outer medulla. This study examined the effect of RSR13, a synthetic allosteric modifier of hemoglobin oxygen-binding affinity, on renal function in a setting of acute renal failure in rats.MethodsAn in vivo model of acute renal failure in the rat produced by reduced renal mass, salt restriction, volume depletion, prostaglandin inhibition, and radiocontrast administration was used. A computer-based simulation of oxygen tensions along the nephron was utilized to interpret the findings. Mechanistic studies were subsequently performed using oxygen-sensitive electrodes and 31P nuclear magnetic resonance (NMR) spectroscopy to define the effect of RSR13 on renal function in the setting of compromised acute renal failure.ResultsRSR13 did not attenuate acute renal failure in this model; rather, serum creatinine increased to a greater degree in the RSR13-treated rats than in rats receiving saline vehicle as the control (P < 0.05). Simulations explained this finding under conditions of severe medullary hypoxia. Mechanistic studies demonstrated marked worsening of medullary hypoxia following RSR13 under conditions similar to our experimental model. Furosemide pretreatment to reduce the imbalance between oxygen supply and demand markedly attenuated the basal-medullary hypoxia produced in the presence of indomethacin and RSR13 (P < 0.01). Additionally, 31P NMR studies demonstrated renal adenosine 5′-triphosphate (ATP) depletion in rats with acute renal failure treated with RSR13 (45% decrease, P < 0.01); again, this effect of RSR13 was completely prevented by pretreatment with furosemide.ConclusionsUnder conditions of severe renal medullary hypoxia, induced in part by indomethacin-mediated reductions in outer medullary blood flow, the administration of RSR13 can exacerbate acute renal dysfunction. However, reducing the rate of oxygen consumption by inhibiting sodium transport with furosemide pretreatment or post-treatment appears to be functionally protective.

Effects of furosemide on medullary oxygenation in younger and older subjects. Renal medullary hypoxia is characteristic of mammalian kidneys and can be assessed noninvasively in animals and humans by blood oxygen level-dependent magnetic resonance imaging (BOLD MRI). Water diuresis has been shown to improve medullary oxygenation in young human subjects but not in elderly subjects, a difference attributed to a decline in renal prostaglandin production with age. Loop diuretics such as furosemide also increase medullary oxygenation in experimental animals, by inhibiting active transport and oxygen consumption in the medullary thick ascending limb. We examined, using BOLD MRI, this response to furosemide in eight younger (23 to 34 years) and eight elderly (64 to 81 years) healthy women. We also attempted to assess the role of prostaglandins in age-related differences, using ibuprofen to inhibit prostaglandin E 2 synthesis. Renal medullary oxygenation, initially low, increased during furosemide diuresis in younger subjects. In the older population, however, furosemide usually elicited little or no change in oxygenation of the renal medulla, despite profuse diuresis. Ibuprofen did not inhibit the action of furosemide to improve medullary pO 2 in younger subjects. Conclusions The action of loop diuretics to improve medullary oxygenation, apparent in younger subjects, is blunted by normal aging. Inhibition of prostaglandin synthesis did not counteract the effect of furosemide in younger subjects, suggesting that a decline in prostaglandin E 2 production with age is not the central cause of this age-related defect.

Factors maintaining a pH gradient within the kidney: Role of the vasculature architecture.BackgroundThe architecture of the vasa rectae produces significant oxygen (O2) “shunting” and marked decreases in renal medullary pO2 values. We hypothesized that carbon dioxide (CO2) trapping and increases in medullary pCO2 along with decreases in medullary pH values should also accompany this O2 shunting.MethodsWe developed computer simulations employing a model of gas exchange through the countercurrent vasculature that predicted trapping of CO2 along with O2 shunting. To test the validity of this model directly, medullary pH was measured by using needle electrodes in the in situ kidney before and after the administration of mannitol or furosemide, or by decreasing blood flow with a transient decrease of renal perfusion pressure with a suprarenal clamp. Data are expressed as mean ± SD.ResultsMedullary pH was lower than cortical pH (7.20 ± 0.09 vs. 7.39 ± 0.08, P < 0.01). Mannitol caused a decrease in medullary pH to 7.02 ± 0.07 (P < 0.01), whereas furosemide increased medullary pH to 7.31 ± 0.09 (P < 0.01). Brief periods of severe hypotension decreased medullary pH to 6.90 ± 0.09 (P < 0.01).ConclusionsThese data demonstrate that a significant pH gradient exists within the kidney parenchyma. This gradient is related to the metabolic activity of the thick ascending limb of Henle and the countercurrent vascular architecture, and may be relevant to a variety of physiological phenomena involved in volume, electrolyte, and acid-based homeostasis.

24-hour urinary protein excretion was 4.6 g. The patient initially was treated for edema with furosemide, 40 mg/day, and hydrochlorothiazide, 12.5 mg/day. Because the edema did not respond to treatment, the furosemide dose was progressively increased over the following two weeks to 200 mg/day; the thiazide dose remained the same. Because of the finding of hyponatremia, diuretics were withdrawn after his admission to the emergency room. The patient was treated with water restriction and oral sodium repletion; his confusion and somnolence gradually resolved over the first five days in hospital. He left the hospital 10 days after admission with normal serum electrolytes and arterial blood gases.

Dietary salt regulates expression of Tamm-Horsfall glycoprotein in rats. Background Tamm-Horsfall glycoprotein (THP) is a unique protein that is produced exclusively by cells of the thick ascending limb of Henle's loop (TALH). This study examined whether dietary salt altered renal THP production. Methods Male Sprague-Dawley rats were examined on days 1, 4, and 15 following placement in metabolic cages on diet that contained 0.3%, 1.0% or 8.0% NaCl. THP expression was quantified using Northern hybridization and Western blotting analysis. Results An increase in dietary salt produced sustained increases in relative steady-state mRNA and protein levels of THP in the kidney. Addition of furosemide, but not chlorothiazide, to animals on the 8.0% NaCl diet further augmented steady-state mRNA levels of THP. Conclusions An increase in dietary salt and the loop diuretic, furosemide, increased expression of THP in the rat. The data support the involvement of this unique protein in the function of the TALH during changes in dietary salt. These findings also suggest that restriction of dietary salt may be beneficial in cast nephropathy in multiple myeloma and recurrent nephrolithiasis, two diseases in which THP can play an important pathogenetic role.