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Hypertension occurs commonly and early in the natural history of autosomal dominant polycystic kidney disease (ADPKD), affecting both renal and patient outcome. Activation of the renin angiotensin aldosterone system due to cyst expansion and local renal ischaemia plays an important role in the development of ADPKD related hypertension and left ventricular hypertrophy (LVH), a known important risk factor for cardiovascular morbidity and mortality. The aim of this study was to investigate the effects of an angiotensin converting enzyme (ACE) inhibitor, enalapril, on renal function, blood pressure and LVH in hypertensive ADPKD patients.

Recent in vitro studies have suggested that the presence of extracellular acidosis is protective against the development of oxygen-deprivation injury in several tissues. Because cellular Ca accumulation after renal ischemia may represent a major pathogenic event leading to cellular damage, the purpose of the present study was to examine the effect of extracellular acidosis on 45Ca uptake and desaturation in normal and hypoxic isolated rat proximal tubules. At pH 7.4, an increase in 45Ca uptake was observed in proximal tubules after 30 min of hypoxia. In addition, 45Ca desaturation was increased significantly in hypoxic tubules at pH 7.4. The alterations in 45Ca uptake and desaturation in hypoxic tubules at pH 7.4 were accompanied by significant signs of cellular injury as assessed by the amount of lactate dehydrogenase (LDH) released by the tubules at the end of the hypoxic period (22.5% above control tubules, P less than 0.01) as well as in morphological changes consistent with hypoxic cell injury. In contrast, when maintained at pH 6.9 throughout the study, no difference in 45Ca uptake or desaturation was observed between the control and hypoxic tubules; the hypoxic proximal tubules exhibited a smaller increase in LDH release (12.7% above control tubules) and did not develop the morphological changes observed at pH 7.4. Thus, during hypoxia and reoxygenation at pH 7.4, the increased 45Ca uptake may contribute in part to cellular injury in rat proximal tubules.

A protective effect of verapamil against hypoxic renal proximal tubule injury has been demonstrated in vitro. In contrast to other cytoprotective agents such as glycine or alanine, the protective effect of verapamil is associated with better maintenance of cellular ATP and potassium levels. These findings suggested a possible direct effect of verapamil on the mitochondria in addition to known effects of verapamil on membrane Ca channels. In the present study, the direct effects of verapamil on Ca-induced swelling, respiratory dysfunction, Ca uptake rate and phospholipase activity of renal cortical mitochondria were determined. Verapamil (100 microM) significantly inhibited Ca-induced mitochondrial swelling and partially prevented the associated reduction in respiratory control ratio (State 3/State 4: Ca + verapamil: 2.8 +/- 0.1 vs. Ca alone, 2.0 +/- 0.2; P < .01). A phospholipase A2 inhibitor, dibucaine (100 microM), significantly inhibited Ca-induced mitochondrial swelling and attenuated the decrease in respiratory control ratio (Ca + dibucaine: 2.9 +/- 0.1 vs. Ca alone, 2.0 +/- 0.2; P < .001). Neither agent, either alone or combined, completely prevented the respiratory dysfunction. Either verapamil or dibucaine attenuated the mitochondrial Ca uptake rate and reduced the rate of Ca-stimulated polyunsaturated free fatty acid accumulation; verapamil treatment also was associated with diminished net release of saturated and monounsaturated free fatty acids. These findings demonstrate that verapamil exerts a protective effect against Ca-induced mitochondrial damage which may be mediated in part by its effect to suppress mitochondrial Ca uptake and mitochondrial phospholipase activity.

Summary The pathogenesis of the diabetic glomerular lesion is unknown. However, cumulative indirect evidence favors hemodynamic factors associated with the abnormal endocrine environment as the cause of diabetic angiopathy. Experimental evidence suggests that the increased hydrostatic pressures in capillary beds, a hallmark of the early stages of insulin-dependent diabetes, are associated with macromolecular leakage leading to the typical thickening of glomerular capillary basement membrane and increased glomerular mesangial matrix even prior to the occurrence of systemic hypertension. Patients with renal or carotid artery stenosis seem to be protected against diabetic nephropathy and retinopathy on the stenosed side. The first signal of diabetic nephropathy even before deterioration of the renal function is microalbuminuria detected by sensitive methods such as radioimmunoassay. Not only in hypertensive, but even in normotensive diabetic patients with microalbuminuria antihypertensive therapy has been shown to reduce albumin excretion rate and to slow the progression of diabetic nephropathy. Once overt diabetic nephropathy has been established, hypertension is a constant accompaniment of the disease. Thus, hypertension may be a cause as well as a result of diabetic nephropathy. Tight control of blood sugar in close association with antihypertensive treatment reducing blood pressure to a lower normal limit, possibly with agents that specifically decrease glomerular capillary hydraulic pressure are the corner stone in protection against progression of the diabetic angiopathy.

Summary This presentation briefly reviews the cellular mechanism of ischemic acute renal failure (ARF) with particular emphasis on the role of Ca2+ and calcium entry blockers (CEB). Vascular consequences of an ischemic renal insult including vasoconstriction, diminished glomerular permeability, loss of autoregulation, and hypersensitivity to renal nerve stimulation may relate to increased cellular Ca2+ concentration in the renal afferent arteriole and glomerular mesangial cells. Evidence is also presented that the ischemic injury to tubular plasma membranes is associated with increased Ca2+ uptake. With an ischemic insult of a short duration, the renal mitochondria are able to buffer the increased cellular Ca2+. However, after an ischemic insult of long duration, the Ca2+ overloaded mitochondria deteriorate, adenosine triphosphate (ATP) synthesis decreases, and cell death follows. If a sufficient number of renal tubular cells undergo this cell death, tubular obstruction, i.e. the maintenance phase of ARF, occurs.

Summary In all patients with hyponatremia, an etiologic diagnosis must be made. A diagnostic classification of hyponatremic states is proposed, which is based on extracellular fluid volume status. A spot urinary sodium concentration will be helpful in confirming the diagnosis. Treatment should concentrate on correction of etiologic factors and fluid restriction after any extracellular fluid volume depletion is treated. A regimen of rapid correction of hyponatremia is outlined for serious, symptomatic cases. It consists of diuretic induced diuresis, accompanied by hypertonic sodium replacement of urinary losses. Finally, the usefulness of demeclocycline hydrochloride is characterized.

Activation of the mitogen-activated protein (MAP) kinase pathway is believed to play a critical role in normal and pathophysiological proliferation of mesangial cells. Recent studies have shown that MAP kinase activation by growth factors in other cell types involves activation of the low-molecular-weight G protein Ras and the protooncogene serine kinase c-Raf-1. In this study, the role of this pathway in rat renal mesangial cells was assessed. Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), as well as phorbol esters (PMA) rapidly activated MAP kinase three- to fourfold in these cells. PDGF and EGF, but not PMA, were able to activate c-Raf-1 and Ras activity. Stimulation of mesangial cells with the inflammatory mediator prostaglandin E2 (PGE2) or elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) by treatment with forskolin markedly blunted activation of MAP kinase induced by PDGF and EGF, but not by PMA. Consistent with this observation, PGE2 abolished growth factor-induced activation of c-Raf-1. However, Ras activation induced by growth factors was not affected by PGE2 and forskolin. These results suggest that MAP kinase activation can occur by at least two separate pathways in mesangial cells. Tyrosine kinase receptors activate MAP kinase through activation of Ras and Raf. This pathway can be blocked by PGE2 and elevation of cAMP, presumably by interfering with the ability of Ras to activate Raf. In addition, activation of protein kinase C by phorbol esters can activate MAP kinase in a Ras/Raf-independent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The interrelationship between parasympathetic neural tone, renin secretion and vasopressin release was examined by observing the effect of bilateral cervical vagotomy on renin secretion in intact and acutely hypophysectomized dogs undergoing a water diuresis. 2. In intact dogs bilateral cervical vagotomy decreased the mean renin secretion from 1245 to 682 units/min (P < 0.01) as urinary osmolality increased from 95 to 414 mosmol/kg (P < 0.001). In contrast, in acutely hypophysectomized dogs cervical vagotomy failed to alter renin secretion significantly (834 to 893 units/min) and urinary osmolality was also unchanged (78 to 71 mosmol/kg). 3. The results suggest that a diminution in vagal tone may significantly alter renin secretion by stimulating vasopressin release. Exogenous vasopressin was associated with changes in urinary osmolality and renin secretion which were qualitatively similar to those seen after cervical vagotomy. 4. We suggest that there is a neurohumoral reflex mechanism by which a fall in parasympathetic tone increases the release of vasopressin, which, in turn, suppresses renin secretion. The results are also compatible with the hypothesis that vasopressin inhibits renin release by a direct effect on the juxtaglomerular cells.

1. The effects of intravenous chlorthiazide and frusemide on urinary osmolality were compared in 19 hyponatraemic oedamatous patients. 2. Frusemide (1 mg/kg) caused production of a dilute urine (urine/plasma osmolality ratio, Uosm./Posm., 1.64–0.84, P < 0.01) whereas chlorthiazide (10 mg/kg) did not (Uosm./Posm. 1.54–1.34, not significant). 3. The osmolar clearance (Cosm.) was higher after frusemide than after chlorthiazide (11.45 vs 4.99 ml/min, P < 0.01). When the doses of frusemide (0.25–0.5 mg/kg) and chlorthiazide (20 mg/kg) were chosen to give a similar Cosm. (7.25 vs 7.48 ml/min, not significant), the Uosm./Posm. was still lower after frusemide (2.20–1.00, P < 0.001) than after chlorthiazide (1.75–1.26, P < 0.01). 4. Exogenous vasopressin did not increase the low Uosm./Posm. after frusemide (1.00–1.00, not significant) but increased the ratio after chlorthiazide (1.34–1.68, P < 0.001). 5. These results indicate that frusemide, but not chlorthiazide, leads to the excretion of a dilute urine in hyponatraemic oedematous patients. This dilution is not due to a greater solution excretion but is associated with a resistance to the action of vasopressin.

1. The effect of chronic bile-duct ligation on systemic and renal haemodynamics and on the capacity to dilute the urine was studied in conscious rats. Sham-operated rats served as controls. 2. In the rats with bile-duct ligation, the maximal urinary diluting capacity was impaired, despite an expanded plasma volume, a normal mean arterial pressure and cardiac output, and normal intrarenal determinants of water excretion including distal delivery of fluid and function of the diluting segment. 3. In contrast, maximal urinary dilution capacity was intact in rats with congenital central diabetes insipidus and chronic bile-duct ligation. 4. It is concluded that the defect in urinary dilution in rats with chronic bile-duct ligation is dependent on antidiuretic hormone.

The possible role of an increased calcium influx in mediating hypoxia-induced injury in isolated rat proximal tubules was examined. Reduction of extracellular calcium delayed the development of proximal tubule cell injury as reflected by the release of lactate dehydrogenase (LDH). This protection, as assessed by LDH release, was not associated with improvements of cell ATP or potassium concentrations. The calcium channel blocker verapamil also delayed the development of cell injury due to hypoxia. This protective effect was evident in the presence of normal or low extracellular calcium. Specifically, hypoxic tubules incubated in low calcium medium were further protected by the addition of verapamil. The protection with verapamil was evident not only by diminished LDH release, but also by improvements in cell ATP and potassium concentration. Further experiments demonstrated that verapamil also increased cell potassium levels in control oxygenated tubules incubated in normal calcium medium. Thus, the results demonstrate that low calcium medium delays the onset of cell membrane injury during hypoxia as assessed by LDH release. The protective effects of verapamil can be demonstrated in the presence of normal or low calcium media, and involve not only diminished LDH release but also increased cell ATP and potassium concentrations. Therefore, in addition to any effect on calcium influx, the effect of verapamil appears to involve a protective effect at cell membrane or mitochondria sites.

OBJECTIVE: Exercise capacity has been used as a noninvasive parameter for predicting cardiovascular events. It has been demonstrated previously in NIDDM patients that several risk factors (i.e., obesity, smoking, hypertension, and African-American race) are associated with an impaired exercise capacity. We studied 265 male and 154 female NIDDM patients who underwent graded exercise testing with expired gas analyses to determine the possible influences of diabetic neuropathy, nephropathy, and retinopathy on exercise capacity. RESEARCH DESIGN AND METHODS: Univariate and multiple linear regression analyses were performed to determine the relationship between diabetic neuropathy, urinary albumin excretion (UAE), and retinopathy with respect to peak oxygen consumption (VO2). Neuropathy was assessed by neurological symptom and disability scores, autonomic function testing, and quantitative sensory exams involving thermal and vibratory sensation. Three categories of UAE were used: normal albuminuria (< 20 micrograms/min), microalbuminuria (20-200 micrograms/min), and overt albuminuria (> 200 micrograms/min). Retinopathy was assessed by stereoscopic fundus photographs. Multiple linear regression analyses were then performed controlling for age, sex, length of diagnosed diabetes, duration of hypertension, race and ethnicity, GHb, BMI, and smoking to determine whether there was an independent effect of these diabetic complications on exercise capacity. RESULTS: Univariate analyses revealed that the presence of diabetic retinopathy (P = 0.03), neuropathy (P = 0.002), microalbuminuria (P = 0.04), and overt albuminuria (P = 0.06) were associated with a lower peak VO2. Multiple linear regression analyses were performed to determine independent relationships with peak VO2. The results revealed that increasing retinopathy stage (Parameter estimate [PE] = -0.59 +/- 0.3 ml.kg-1.min-1; P = 0.026) and increasing UAE stage (PE = -0.62 +/- 0.3 ml.kg-1.min-1; P = 0.04) were associated with a decrease in peak VO2. CONCLUSIONS: In the present study of NIDDM subjects, a significant independent association was demonstrated between diabetic nephropathy and retinopathy with exercise capacity. These results were obtained controlling for age, sex, length of diagnosed diabetes, hypertension, race, and BMI. Thus the findings in this large NIDDM population without a history of coronary artery disease indicate a potential pathogenic relationship between microvascular disease and exercise capacity.

Mitochondrial respiration, Ca2+ content, and Ca2+ kinetics have been found to be profoundly altered in ischemic acute renal failure (ARF). The effect of clamping the bilateral renal artery for 50 and 90 min on microsomal Ca2+ uptake was therefore examined in the rat. The 50-min clamping produced a reversible model of nonoliguric ARF, and the 90-min clamping produced a model of nonreversible oliguric ARF. In the 50-min nonoliguric model, ATP-dependent Ca2+ uptake by microsomes from renal cortex (nmol X mg protein-1 X 30 min-1) was significantly impaired immediately before release of the clamp and before return of renal blood flow (reflow) (191 +/- 11 vs. 83 +/- 11, P less than 0.005). However, in this nonoliguric model of ischemic ARF, microsomal uptake returned completely to normal after 1 h of reflow (sham 189 +/- 11 vs. 167 +/- 14 at 1 h, NS) and persisted at this normal level at 24 h (sham 166 +/- 14 vs. 150 +/- 13 at 24 h, NS). In the oliguric model of ARF the microsomal Ca2+ uptake also was impaired immediately after the clamp release (sham 191 +/- 11 vs. 93 +/- 11, P less than 0.001) as well as after 1 h of reflow (sham 189 +/- 11 vs. 129 +/- 12, P less than 0.005) but not at 24 h (sham 166 +/- 14 vs. 173 +/- 13, NS). The results indicate that impaired microsomal Ca2+ uptake occurs early in both oliguric and nonoliguric ARF and persists after 1 h of reflow in the oliguric model.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was undertaken in anesthetized rats to examine the effect of aortic constriction and volume expansion on proximal tubular sodium reabsorption during constancy of flow and fluid composition in the peritubular circulation of the kidney. Efferent arterioles and branch capillaries were perfused at 625 nl/min with an artificial perfusate containing 9 g per liter of albumin both before and during either aortic constriction or saline infusion. Results of recollection micropuncture studies in those tubules surrounded by artificially perfused capillaries were compared with results in control tubules in which the peritubular capillary flow and fluid composition were allowed to change during aortic constriction or volume expansion following saline infusion. Changes in single nephron filtration rate, fractional and absolute reabsorption induced by both aortic constriction and saline infusion were found to be qualitatively and quantitatively comparable in tubules with constant peritubular capillary microperfusion and in the control tubules with changing peritubular capillary environment due to the experimental maneuvers. Taken together, therefore, the present results indicate that with the use of the peritubular microperfusion technique no evidence is found to support a role of alteration in the peritubular environment in modulating the effect of aortic constriction or saline infusion on tubular sodium reabsorption in the rat nephron. Rather, these results provide indirect evidence in support of intraluminal factors as mediating these responses in tubular reabsorption to volume expansion and aortic constriction.

The present study was undertaken in anesthetized rats to evaluate the technique of efferent arteriolar and branch capillary microperfusion in the kidney. In contrary to previous suggestions, subsurface efferent arteriolar branches were demonstrated to occur prior to the surface site of insertion of the microperfusion pipette into an efferent arteriole. This finding, in addition to the presence of continued blood flow around the microperfusion pipette, provided an explanation for continued filtration and tubular reabsorption in nephrons under study. Moreover, additional microdissection studies demonstrated that a mean of 3.8 nephrons are surrounded by artificially perfused capillaries from a micropipette inserted into a single efferent arteriole. Thus, any obstructive effect of the microperfusion technique on post-glomerular blood flow might be expected to alter nephron function in only a minority of nephrons which are surrounded by artificial perfusate. The results also demonstrated the importance of avoiding osmotic gradients between the animal's plasma and the artificial perfusate. A method is also described in which the correlation betweenin vivo diagrams andin vitro microdissections allows assessment of the completeness of the artificial perfusion of capillaries surrounding the surface loops of nephrons under study.

The ABCD (Appropriate Blood Pressure Control in Diabetes) Trial is a large, prospective, randomized clinical trial of 950 patients with non-insulin-dependent diabetes mellitus (NIDDM) designed to compare the effects of intensive blood pressure control with moderate control on the prevention and progression of diabetic nephropathy, retinopathy, cardiovascular disease, and neuropathy in NIDDM. The secondary objective is to determine equivalency of the effects of a calcium channel blocker (nisoldipine) and an angiotensin-converting-enzyme inhibitor (enalapril) as a first-line antihypertensive agent in the prevention and/or progression of these diabetic vascular complications. The study consists of two study populations aged 40–74 years, 470 hypertensive patients (diastolic blood pressure of ≥ 90.0 mmHg at time of randomization) and 480 normotensive patients (diastolic blood pressure of 80.0 mmHg at time of randomization). The study duration is 5 years and is scheduled to end in May of 1998. Patients are randomized to receive either intensive antihypertensive drug therapy or moderate antihypertensive drug therapy. Patients are also randomized to nisoldipine or enalapril, with open-label medications added if further blood pressure control is necessary. The primary outcome measure is glomerular filtration rate as assessed by 24-h creatinine clearance. Secondary outcome measures are urinary albumin excretion, left ventricular hypertrophy, retinopathy, and neuropathy. Cardiovascular morbidity and mortality will also be evaluated. Given the data showing the impact of hypertension on complications in NIDDM, the ABCD Trial is designed to determine if intensive antihypertensive therapy will be more efficacious than moderate antihypertensive therapy on the outcome of diabetic complications in NIDDM. [Diabetologia (1996) 39: 1646–1654]

The inhibitory effect of phorbol-12-myristate-13-acetate (PMA) on the Ca2+-mobilization mechanisms by arginine vasopressin (AVP) and angiotensin II (AII) was analysed in rat vascular smooth muscle cells (VSMC) in culture. PMA inhibited the Ca2+-mobilizing effect of both AVP and AII in a dose-dependent manner, including the rise in cytosolic free Ca2+ ([Ca2+]i) and Ca2+ efflux. In addition, inositol trisphosphate (IP3) production induced by AVP or AII was more than 50% reduced by PMA. The involvement of protein kinase C was implicated by the diminution of the PMA effect by the specific protein kinase C inhibitor isoquinoline-sulphonyl-O-2-methylpiperazine (H7) and the lack of effect of an inactive phorbol. Thus, these results suggest that there is a blocking site that is common or similar for both AVP and AII signal transduction, and that it is a substrate for protein kinase C. This blocking action of protein kinase C occurred at least in part by inhibition of IP3 production and, subsequently, a reduction in cytosolic Ca2+ release. In the presence of ionomycin, which produces an increase in [Ca2+]i that is not altered by PMA, 45Ca2+ efflux was increased instead of inhibited by PMA, thus suggesting that protein kinase C activation also stimulates a Ca2+-extrusion mechanism in VSMC.

Proximal tubules (PSTs) of the S1, S2 and S3 segments and cortical collecting tubules (CCTs) were microdissected individually from rabbit kidneys and cultured for 7 days in hormonally defined media. Anoxia was induced by incubation of cultures in normal medium for 45 min at 25 degrees C in an atmosphere of nitrogen and cell death was measured by nigrosine dye uptake. After 45 min of anoxia and a 4- to 6-hr incubation in normal Ca++-containing media, cells from all segments were dead. Addition of calcium channel blockers verapamil and nifedipine (5 X 10(-7) and 10(-6) M, respectively) for the first 2 hr after anoxia to the incubation media was associated with a 60 +/- 8 and 33 +/- 7% survival of PST cells (5 hr after anoxia), p less than .05. Verapamil at 5 X 10(-8) M caused a 42 +/- 4% survival whereas nifedipine at 10(-7) M was not effective on the survival rate of PST cells (5 hr after anoxia). These calcium channel blockers also afforded protection from anoxic cell death for CCT cells. The role of calmodulin in anoxic cell injury was studied by means of calmodulin binding drugs, trifluoperazine and W7 [N-(6-aminohexyl)-5-chloronapthalene-sulfonamide]. Addition of trifluoperazine (5 X 10(-7) M) and W7 (5 X 10(-7) M to both PST and CCT cells during the 2-hr reflow period after 45 min of anoxia increased viability by 58 +/- 3 and 62 +/- 3%, respectively (P less than .05) at 5 hr postanoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide (NO), among several other functions, may play a role in hypoxia and reoxygenation injury due to its free radical nature and high reactivity with the superoxide radical to yield peroxynitrite, an oxidant molecule. The present study was undertaken to evaluate a potential role for NO, either endogenous or exogenous, in a model of hypoxia/reoxygenation (H/R) in freshly isolated rat proximal tubules. NO synthase activity, as assessed by conversion of L-[3H]arginine to L-[3H]citrulline, was detected in normoxic tubules. This activity could be inhibited by N-nitro-L-arginine methyl ester (L-NAME), a NO synthase inhibitor, and was stimulated by 15 min of hypoxia. The injury in proximal tubules caused by 15 min of hypoxia followed by 35 min of reoxygenation was completely prevented by L-NAME as assessed by release of lactate dehydrogenase, whereas D-NAME, which does not inhibit NO synthase, had no effect. In contrast, L-arginine (NO substrate) enhanced the H/R injury. These effects were paralleled by nitrite/nitrate production. In separate experiments, the addition of sodium nitroprusside, a NO donor, to proximal tubules enhanced the H/R injury; this effect could be blocked by hemoglobin, a NO scavenger. Also, addition of nitroprusside reversed L-NAME protection against H/R injury. These results demonstrate that NO is synthesized in rat proximal tubules and participates as one of the mediators in rat tubular H/R injury.

The role of the lysosomal proteases cathepsins B and L and the calcium-dependent cytosolic protease calpain in hypoxia-induced renal proximal tubular injury was investigated. As compared to normoxic tubules, cathepsin B and L activity, evaluated by the specific fluorescent substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, was not increased in hypoxic tubules or the medium used for incubation of hypoxic tubules in spite of high lactate dehydrogenase (LDH) release into the medium during hypoxia. These data in rat proximal tubules suggest that cathepsins are not released from lysosomes and do not gain access to the medium during hypoxia. An assay for calpain activity in isolated proximal tubules using the fluorescent substrate N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was developed. The calcium ionophore ionomycin induced a dose-dependent increase in calpain activity. This increase in calpain activity occurred prior to cell membrane damage as assessed by LDH release. Tubular calpain activity increased significantly by 7.5 min of hypoxia, before there was significant LDH release, and further increased during 20 min of hypoxia. The cysteine protease inhibitor N-benzyloxycarbonyl-Val-Phe methyl ester (CBZ) markedly decreased LDH release after 20 min of hypoxia and completely prevented the increase in calpain activity during hypoxia. The increase in calpain activity during hypoxia and the inhibitor studies with CBZ therefore supported a role for calpain as a mediator of hypoxia-induced proximal tubular injury.

The present study examines the binding and postbinding cellular processing and recycling of the V1 arginine vasopressin (AVP) receptor in cultured vascular smooth muscle cells (VSMCs). The surface binding of AVP to VSMCs was temperature dependent and reached equilibrium within 60 min at 4 degrees C. Displacement studies with unlabeled AVP or a specific V1 AVP antagonist revealed a single class of V1 receptors (Bmax, 1.99 pmol [corrected] per mg of protein; Kd, 2.15 nM). Incubation of VSMCs with unlabeled 10 nM AVP to promote receptor internalization resulted in a time- and temperature-dependent loss of AVP surface binding. At 37 degrees C, maximum loss of binding sites (65%) occurred within 20 min. Recovery of AVP binding occurred rapidly (t1/2, 15-20 min at room temperature) and was uninfluenced by inhibiting protein synthesis with cycloheximide. Pretreating VSMCs with chloroquine prevented AVP receptor recycling, indicating that the AVP-receptor complex requires endosomal processing. The biological competence of the recycled AVP receptor was shown by AVP-induced Ca2+ uptake. The results of these studies therefore indicate that, after surface binding, the AVP-receptor complex internalizes and dissociates in an endosomal compartment. It is demonstrated that in VSMCs biologically active V1 AVP receptors recycle back to the cell surface, thus attenuating the loss of AVP surface binding sites.

Arginine vasopressin (AVP) has been shown to stimulate tyrosine phosphorylation and activation of p42 mitogen-activated protein (MAP) kinase (p42MAPK) in vascular smooth muscle cells (VSMC). In VSMC, AVP increases free intracellular Ca2+ concentration ([Ca2+]i) and activates protein kinase C (PKC) through activation of phospholipase C. The contribution of PKC and [Ca2+]i in p42MAPK regulation was therefore determined. Activation of PKC by phorbol 12-myristate 13-acetate (PMA) stimulated tyrosine phosphorylation and activation of p42MAPK to the same extent as AVP. Inhibition of PKC by staurosporine or downregulation of PKC by PMA pretreatment abolished AVP-induced stimulation of p42MAPK. When [Ca2+]i was elevated to the same level as with AVP, using either ionomycin (0.1 microM) or thapsigargin (0.1 microM), MAP kinase was only partially activated. Elevation of [Ca2+]i to supraphysiological levels by 1 microM ionomycin stimulated MAP kinase activity to the same extent as AVP. This effect was blocked by downregulation of PKC. The intracellular Ca2+ chelator BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] blocked AVP-induced [Ca2+]i increase but did not affect AVP stimulation of p42MAPK. Thus AVP-induced activation of p42MAPK requires only the activation of PKC but not an increase in [Ca2+]i.

The effects of anoxia or hypoxia on Ca++ uptake and lactic dehydrogenase (LDH) release were examined in freshly isolated rat proximal tubules (rPT). Both Ca++ uptake and LDH release were increased above control after only 10 min of either anoxia or hypoxia in rPT. The increase in Ca++ uptake was through voltage-sensitive, slow Ca++ channels, because pretreatment with chemically dissimilar calcium channel blockers (CCB), either verapamil or flunarizine, prevented the increased Ca++ uptake and reduced the LDH release from the anoxic and hypoxic rPT. After 20 min of hypoxia, however, verapamil pretreatment did not significantly reduce the high Ca++ uptake rate, thus, suggesting that this increase in Ca++ permeability was occurring through pathways other than the slow Ca++ channels. The increase in LDH release was only slightly decreased by verapamil after 20 min of hypoxia. After 20 min of anoxia in rPT, Ca++ uptake was no longer increased, but the increased LDH release persisted. These effects of anoxia were unaltered by verapamil. These results, thus, suggest that early membrane injury to isolated rPT in suspension, which is associated with 10 min of either anoxia or hypoxia, involves increased cellular Ca++ uptake through voltage-sensitive Ca++ channels and protection is afforded by CCB. In contrast, after 20 min of anoxia or hypoxia, rPT membrane damage persisted and was only partially reversed by CCB. The rPT injury induced by 20 min of O2 deprivation, thus, involves factors independent of voltage-sensitive Ca++ channels.

To assess more completely the long-term effect of a long-acting calcium channel blocker, anipamil was given p.o. to rats with subtotal (five-sixths) nephrectomy. The mortality rates in anipamil-treated and control groups were 5% and 20%, respectively, at 6 weeks after separation (P less than .01) and 10% and 55%, respectively, at 10 weeks after separation (P less than .01). Mean arterial blood pressure was also more well controlled in the anipamil-treated group (144 +/- 36 vs. 192 +/- 35 mm Hg; n = 20; P less than .05 at week 5). In paired experiments, the degree of renal impairment in the placebo- and anipamil-treated groups, just before the onset of preterminal azotemia, was determined. Rats that were treated with anipamil had lower serum creatinine concentrations, compared with the placebo controls, at 4 to 6 weeks after separation (0.55 +/- 0.02 vs. 0.87 +/- 0.02 mg/100 ml; P less than .05). To dissociate this beneficial effect of anipamil from mean arterial blood pressure control, experiments were also performed to assess the effects of hydralazine on the remnant kidney model of chronic renal failure. Rats with remnant kidneys were divided into three groups and treated with anipamil (2 mg/kg/day), hydralazine (80 mg/liter in drinking water) or control. The anipamil-treated group exhibited significantly greater protection of renal function than did the hydralazine-treated group for the same level of blood pressure control. Thus, a long-acting calcium channel entry blocker such as anipamil may afford an additional cytoprotective effect in the prevention of progression of chronic renal failure beyond the antihypertensive effects of the agent.

1. A frog skin was incorporated into the blood circulation of a dog. Each side of the skin was supplied with blood at a constant temperature, flow and pressure. 2. In the experiments in which the blood volume of the dog was expanded with equilibrated blood, there was a fall in the short-circuit current across all eight frog skins. The fall in current began 10 min after the start of the transfusion and reached its lowest value 15–30 min after the end of the transfusion. The dog showed a simultaneous rise in urinary sodium excretion. 3. In the experiments in which the blood volume was not expanded, there was no change in the trend in the short-circuit current in five of the eight skins. There was a fall in current across the other three skins; the pattern of this fall differed from that which occurred when the blood volume was expanded. There was no rise in the rate of urinary sodium excretion in any of the dogs. 4. It is concluded that when a dog's blood volume is expanded the dog alters the concentration of some circulating substance, and that this change causes a fall in the rate of active sodium transport across the frog skin.