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Little is known about the effects of nitric oxide (NO) and the cyclic GMP (cGMP)/protein kinase G (PKG) system on Ca2+signaling in vascular smooth muscle cells (VSMC) of resistance vessels in general and afferent arterioles in particular. We tested the hypotheses that cGMP-, Ca2+-dependent big potassium channels (BKCa2+) buffer the Ca2+response to depolarization by high extracellular KCl and that NO inhibits adenosine diphosphoribose (ADPR) cyclase, thereby reducing the Ca2+-induced Ca2+release. We isolated rat afferent arterioles, utilizing the magnetized microsphere method, and measured cytosolic Ca2+concentration ([Ca2+]i) with fura-2, a preparation in which endothelial cells do not participate in [Ca2+]iresponses. KCl (50 mM)-induced depolarization causes an immediate increase in [Ca2+]iof 151 nM. The blockers Nω-nitro-l-arginine methyl ester (of nitric oxide synthase), 1,2,4-oxodiazolo-[4,3- a]quinoxalin-1-one (ODQ, of guanylyl cyclase), KT-5823 (of PKG activation), and iberiotoxin (IBX, of BKCa2+activity) do not alter the [Ca2+]iresponse to KCl, suggesting no discernible endogenous NO production under basal conditions. The NO donor sodium nitroprusside (SNP) reduces the [Ca2+]iresponse to 77 nM; IBX restores the response to control values. These data show that activation of BKCa2+in the presence of NO/cGMP provides a brake on KCl-induced [Ca2+]iresponses. Experiments with the inhibitor of cyclic ADPR 8-bromo-cyclic ADPR (8-Br-cADPR) and SNP + downstream inhibitors of PKG and BKCa2+suggest that NO inhibits ADPR cyclase in intact arterioles. When we pretreat afferent arterioles with 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP; 10 μM), the response to KCl is 143 nM. However, in the presence of both IBX and 8-Br-cGMP, we observe a surprising doubling of the [Ca2+]iresponse to KCl. In summary, we present evidence for effects of the NO/cGMP/PKG system to reduce [Ca2+]i, via activation of BKCa2+and possibly by inhibition of ADPR cyclase, and to increase [Ca2+]i, by a mechanism(s) yet to be defined.

It is unknown if endothelin-A and -B receptors (ETAR and ETBR) activate the production of superoxide via NAD(P)H oxidase and subsequently stimulate the formation of cyclic adenine diphosphate ribose (cADPR) in afferent arterioles. Vessels were isolated from rat kidney and loaded with fura 2. Endothelin-1 (ET-1) rapidly increased cytosolic Ca2+concentration ([Ca2+]i) by 303 nM. The superoxide dismutase mimetic tempol, the NAD(P)H oxidase inhibitor apocynin, and nicotinamide, an inhibitor of ADPR cyclase, diminished the response by ∼60%. The ETBR agonist sarafotoxin 6c (S6c) increased peak [Ca2+]iby 117 nM. Subsequent addition of ET-1 in the continued presence of S6c caused an additional [Ca2+]ipeak of 225 nM. Neither nicotinamide or 8-bromo- (8-Br) cADPR nor apocynin decreased the [Ca2+]iresponse to S6c, but inhibited the subsequent [Ca2+]iresponse to ET-1. The ETBR blockers BQ-788 and A-192621 prevented the S6c [Ca2+]ipeak and reduced the ET-1 response by more than one-half, suggesting an ETBR/ETAR interaction. In contrast, the ETAR blocker BQ-123 had no effect on the S6c [Ca2+]ipeak and obliterated the subsequent ET-1 response. ET-1 immediately stimulated superoxide formation (measured with TEMPO-9-AC, 68 arbitrary units) that was inhibited 95% by apocynin or diphenyl iodonium. S6c or IRL-1620 increased superoxide by 8% of that caused by subsequent ET-1 addition. We conclude that ETAR activation of afferent arterioles increases the formation of superoxide that accounts for ∼60% of subsequent Ca2+signaling. ETBR activation appears to result in only minor increases in superoxide production. Nicotinamide and 8-Br-cADPR results suggest that ET-1 (and primarily ETAR) causes the activation of vascular smooth muscle cell-ADPR cyclase.

In afferent arteriolar vascular smooth muscle cells, ANG II induces a rise in cytosolic Ca2+([Ca2+]i) via inositol trisphosphate receptor (IP3R) stimulation and by activation of the adenine diphosphate ribose (ADPR) cyclase to form cyclic ADPR, which sensitizes the ryanodine receptor (RyR) to Ca2+. We hypothesize that ANG II stimulation of NAD(P)H oxidases leads to the formation of superoxide anion (O2−·), which, in turn, activates ADPR cyclase. Afferent arterioles were isolated from rat kidney with the magnetized microsphere and sieving technique and loaded with fura-2 to measure [Ca2+]i. ANG II rapidly increased [Ca2+]iby 124 ± 12 nM. In the presence of apocynin, a specific inhibitor of NAD(P)H oxidase assembly, the [Ca2+]iresponse was reduced to 35 ± 5 nM ( P < 0.01). Tempol, a superoxide dismutase mimetic, did not alter the [Ca2+]iresponse to ANG II at a concentration of 10−4M (99 ± 12 nM), but 10−3M tempol reduced the response to 32 ± 3 nM ( P < 0.01). The addition of nicotinamide, an inhibitor of ADPR cyclase, to apocynin or tempol (10−3M) resulted in no further inhibition. Measurement of superoxide production with the fluorescent probe tempo 9-AC showed that ANG II caused an increase of 48 ± 20 arbitrary units; apocynin or diphenyl iodonium (an inhibitor of flavoprotein oxidases) inhibited the response by 94%. Hydrogen peroxide (H2O2) was studied at physiological (10−7M) and higher concentrations. In the presence of H2O2(10−7M), neither baseline [Ca2+]inor the response to ANG II was altered (125 ± 15 nM), whereas H2O2(10−6and 10−5M) inhibited the [Ca2+]iresponse to ANG II by 35 and 46%, respectively. We conclude that ANG II rapidly activates NAD(P)H oxidases of afferent arterioles, leading to the formation of O2−·, which then stimulates ADPR cyclase to form cADPR. cADPR, by sensitizing the RyR to Ca2+, augments the Ca2+response (calcium-induced calcium release) initiated by activation of the IP3R.

ANG II induces a rise in cytosolic Ca2+([Ca2+]i) in vascular smooth muscle (VSM) cells via inositol trisphosphate receptor (IP3R) activation and release of Ca2+from the sarcoplasmic reticulum (SR). The Ca2+signal is augmented by calcium-induced calcium release (CICR) and by cyclic adeninediphosphate ribose (cADPR), which sensitizes the ryanodine-sensitive receptor (RyR) to Ca2+to further amplify CICR. cADPR is synthesized from β-nicotinamide adenine dinucleotide (NAD+) by a membrane-bound bifunctional enzyme, ADPR cyclase. To investigate the possibility that ANG II activates the ADPR cyclase of afferent arterioles, we used inhibitors of the IP3R, RyR, and ADPR cyclase. Afferent arterioles were isolated from rat kidney with the magnetized microsphere and sieving technique and loaded with fura-2 to measure [Ca2+]i. In Ca2+-containing buffer, ANG II increased [Ca2+]iby 125 ± 10 nM. In the presence of the IP3R antagonists TMB-8 and 2-APB, the peak responses to ANG II were reduced by 74 and 81%, respectively. The specific antagonist of cADPR 8-Br ADPR and a high concentration of ryanodine (100 μM) inhibited the ANG II-induced increases in [Ca2+]iby 75 and 69%, respectively. Nicotinamide and Zn2+are known inhibitors of the VSM ADPR cyclase. Nicotinamide diminished the [Ca2+]iresponse to ANG II by 66%. In calcium-free buffer, Zn2+reduced the ANG II response by 68%. Simultaneous blockade of the IP3and cADPR pathways diminished the [Ca2+]iresponse to ANG II by 83%. We conclude that ANG II initiates Ca2+mobilization from the SR in afferent arterioles via the classic IP3R pathway and that ANG II may lead to activation of the ADPR cyclase to form cADPR, which, via its action on the RyR, substantially augments the Ca2+response.

SUMMARY In vascular smooth muscle (VSM) of Squalus acanthias , endothelin-1 (ET-1) signals via the ET B receptor. In both shark and mammalian VSM, ET-1 induces a rise in cytosolic Ca 2+ concentration ([Ca 2+ ] i ) via activation of the inositol trisphosphate (IP 3 ) receptor (IP 3 R) and subsequent release of Ca 2+ from the sarcoplasmic reticulum (SR). IP 3 R-mediated release of SR Ca 2+ causes calcium-induced calcium release (CICR) via the ryanodine receptor (RyR), which can be sensitized by cyclic adeninediphosphate ribose (cADPR). cADPR is synthesized from NAD + by a membrane-bound bifunctional enzyme, ADPR cyclase. We have previously shown that the antagonists of the RyR, Ruthenium Red, high concentrations of ryanodine and 8-Br cADPR, diminish the [Ca 2+ ] i response to ET-1 in shark VSM. To investigate how ET-1 might influence the activity of the ADPR cyclase, we employed inhibitors of the cyclase. To explore the possibility that ET-1-induced production of superoxide (O 2 .- ) might activate the cyclase, we used an inhibitor of NAD(P)H oxidase (NOX), DPI and a scavenger of O 2 .- , TEMPOL. Anterior mesenteric artery VSM was loaded with fura-2AM to measure [Ca 2+ ] i . In Ca 2+ -free shark Ringers, ET-1 increased [Ca 2+ ] i by 104±8 nmol l -1 . The VSM ADPR cyclase inhibitors, nicotinamide and Zn 2+ , diminished the response by 62% and 72%, respectively. Both DPI and TEMPOL reduced the response by 63%. The combination of the IP 3 R antagonists, 2-APB or TMB-8, with DPI or TEMPOL further reduced the response by 83%. We show for the first time that in shark VSM, inhibition of the ADPR cyclase reduces the [Ca 2+ ] i response to ET-1 and that superoxide may be involved in the activation of the cyclase.

In mammals, endothelin receptors are sub-classified into ET(A) receptors (ET(A)R), which are purely constrictive in vascular smooth muscle (VSM), and ET(B)R, which may produce constriction in VSM or dilatation by stimulating the production of nitric oxide (NO) from endothelial cells. In contrast, previous studies suggested that shark VSM is stimulated exclusively by ET(B)R. The Ca(2+) signaling pathways utilized by shark VSM in response to stimulation by endothelin-1 (ET-1) have not previously been investigated. We measured cytosolic Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded VSM of anterior mesenteric artery of Squalus acanthias and show that the ET(B)R agonists IRL 1620 and sarafotoxin S6c (SRX) increase [Ca(2+)](i) in VSM to the same extent as ET-1 and ET(B)R appears to be the only ETR subtype in sharks. To investigate the participation of the inositol trisphosphate (IP(3)) receptors (IP(3)R), we utilized two inhibitors of the mammalian IP(3)R, TMB-8 and 2-APB. In Ca(2+)-free Ringer, these agents inhibit the response to ET(B)R agonist stimulation by 71%. The ryanodine-sensitive receptor (RyR) may be activated by low concentrations of ryanodine, by abrupt local increases of [Ca(2+)](i), (calcium-induced calcium release) or by cyclic adeninediphosphate ribose (cADPR). We employed three inhibitors of activation of the RyR, Ruthenium Red, 8-Br cADPR and high concentrations of ryanodine; these agents blocked the [Ca(2+)](i) response to ET(B)R agonist stimulation by a mean of 39%. These data show for the first time that in VSM of the shark, ET(B)R activation stimulates both IP(3)R and RyR, and that cADPR is involved in RyR activation.

The dogfish shark Squalus acanthias regulates plasma osmolality and extracellular volume by secreting a fluid from its rectal gland which has a higher NaCl and lower urea concentration than plasma. We have previously identified the presence of a calcium-sensing receptor or polyvalent cation sensing receptor (CaSR) on vascular smooth muscle of the rectal gland artery (RGA) and rectal gland tubules (RGT). Activity of the CaSR is influenced by changes in ionic strength. This led us to speculate that the ingestion of invertebrate sea animals increased plasma ionic strength, resulting in inhibition of the receptor, relaxation of RGA, and reversal of inhibition of chloride secretion by the RGT. In contrast, ingestion of fish could diminish ionic strength and have the opposite effect. To study the effect of changes in extracellular ionic strength, shark Ringers solutions were adjusted to three different ionic strengths with NaCl, but the osmolarities were kept constant by varying the concentration of urea. High ionic strength inhibited and low ionic strength enhanced the response to increasing external Ca2+ from 2.5 to 4.7 mM in RGT. The increase in cytosolic Ca2+ ([Ca2+]i) of cells in low, normal, and high ionic strength Ringers solution was 344 ± 60, 201 ± 26, and 114 ± 15 nmol/L, respectively. The [Ca2+]i responses of RGA to external Ca2+ in Ringers of three different ionic strengths were 323 ± 43, 231 ± 14, and 56 ± 11 nmol/L, respectively. Activation of the CaSR by spermine was reduced by more than 50% by high ionic strength in both RGT and RGA. Whether the small changes in shark plasma ionic strength that occur after a shark ingests marine animals with lower and higher ionic strength modulates salt secretion by the rectal gland is not yet known. J. Exp. Zool. 301A:235–239, 2004. © 2004 Wiley-Liss, Inc.

Gordon Murray (1894–1976), a brilliant and innovative surgeon who spent the majority of his professional career at the University of Toronto, Ont., Canada, is properly credited with having performed the first successful hemodialyses in humans in North America. Neither he nor Kolff, working in the Netherlands, were aware of each other’s work during the middle 1940s when wartime hampered communication. Murray’s extensive investigations and experience in the use of heparin in vascular surgery laid the groundwork for the use of this anticoagulant with the artificial kidney. He first designed a coil dialyzer in which cellophane tubing was wound about a steel frame. His second-generation apparatus was a plate dialyzer. In all, he performed dialysis on 11 patients with presumed acute renal failure, 50% of whom survived. Those who died succumbed to sepsis or irreversible chronic renal failure. Not much has changed in 50 years.

Ryanodine receptor and capacitative Ca 2+ entry in fresh preglomerular vascular smooth muscle cells. Background A multiplicity of hormonal, neural, and paracrine factors regulates preglomerular arterial tone by stimulating calcium entry or mobilization. We have previously provided evidence for capacitative (store-operated) Ca 2+ entry in fresh renal vascular smooth muscle cells (VSMCs). Ryanodine-sensitive receptors (RyRs) have recently been identified in a variety of nonrenal vascular beds. Methods We isolated fresh rat preglomerular VSMCs with a magnetized microsphere/sieving technique; cytosolic Ca 2+ ([Ca 2+ ] i ) was measured with fura-2 ratiometric fluorescence. Results Ryanodine (3 μmol/L) increased [Ca 2+ ] i from 79 to 138 nmol/L ( P = 0.01). Nifedipine (Nif), given before or after ryanodine, was without effect. The addition of calcium (1 mmol/L) to VSMCs in calcium-free buffer did not alter resting [Ca 2+ ] i . In Ca-free buffer containing Nif, [Ca 2+ ] i rose from 61 to 88 nmol/L after the addition of the Ca 2+ -ATPase inhibitor cyclopiazonic acid and to 159 nmol/L after the addition of Ca 2+ (1 mmol/L). Mn 2+ quenched the Ca/fura signal, confirming divalent cation entry. In Ca-free buffer with Nif, [Ca 2+ ] i increased from 80 to 94 nmol/L with the addition of ryanodine and further to 166 nmol/L after the addition of Ca 2+ (1 mmol/L). Mn 2+ quenching was again shown. Thus, emptying of the sarcoplasmic reticulum (SR) with ryanodine stimulated capacitative Ca 2+ entry. Conclusion Preglomerular VSMCs have functional RyR, and a capacitative (store-operated) entry mechanism is activated by the depletion of SR Ca 2+ with ryanodine, as is the case with inhibitors of SR Ca 2+ -ATPase.

Calcium entry via voltage-gated L-type channels is responsible for at least half of the increase in cytosolic calcium ([Ca2+]i) in afferent arterioles following agonist stimulation. We sought the presence of capacitative calcium entry in fresh vascular smooth muscle cells (VSMC) derived from rat preglomerular vessels. [Ca2+]iwas measured using fura-2 ratiometric fluorescence. Vasopressin V1 receptor agonist (V1R) (10−7M) increased [Ca2+]iby ∼100 nM. A calcium channel blocker (CCB), nifedipine or verapamil (10−7M), inhibited the response by ∼50%. V1R in the presence of CCB increased [Ca2+]ifrom 106 to 176 nM, confirming that calcium mobilization and/or entry may occur independent of voltage-gated channels. In nominally Ca2+-free buffer, V1R increased [Ca2+]ifrom 94 to 129 nM, denoting mobilization; addition of CaCl2(1 mM) further elevated [Ca2+]ito 176 nM, indicating a secondary phase of Ca2+entry. Similar responses were obtained when CCB was present in calcium-free buffer or when EGTA was present. In nominally Ca2+-free medium, the sarcoplasmic reticulum Ca2+-ATPase inhibitors (SRCAI), thapsigargin and cyclopiazonic acid (CPA), increased [Ca2+]ifrom 97 to 128 and 143 nM, respectively, and to 214 and 220 nM, respectively, when 1 mM extracellular Ca2+was added. In the presence of verapamil, the results with CPA acid were nearly identical. In Ca2+-free buffer, the stimulatory effect of V1R or SRCAI on the Ca2+/fura signal was quenched by the addition of Mn2+(1 mM), demonstrating divalent cation entry. These studies provide evidence for capacitative (store- operated) calcium entry in VSMC freshly isolated from rat preglomerular arterioles.

Thomas Graham (1805-1869), who is credited with seminal work on the nature of the diffusion of gases and of osmotic forces in fluids, can properly be called the father of modern dialysis. His apparatus to study the behavior of biological fluids through a semipermeable membrane clearly presaged the artificial kidney in clinical use today. In 1913, John Abel and coworkers reported the first application of the principles of diffusion to remove substances from the blood of living animals. Unaware of Abel's work, Georg Haas (1886-1971) performed the first human dialysis in the German town of Giessen in 1924. But it was not until 1945 that Willem Johan Kolff, working under extremely difficult wartime conditions in The Netherlands, achieved the first clinically successful hemodialysis in a human patient.

Cardiovascular consequences of correction of the anemia of renal failure with erythropoietin. The purpose of this study was to define the physiologic responses of the heart and peripheral circulation to chronic anemia using noninvasive measurements while eliminating confounding biochemical, pharmacologic and physiologic variables. Stable chronic hemodialysis patients were studied at the University Hospital based chronic dialysis unit and echocardiography laboratory before and after therapy with human recombinant erythropoietin (rHuEPO). Subjects included maintenance hemodialysis patients free of left ventricular regional wall motion abnormalities discernible by echocardiography, rhythm disturbance, significant valvular or ischemic heart disease. Two-dimensional echocardiograms and simultaneous targeted M-mode echocardiograms, phonocardiograms and externally acquired subclavian artery pulse tracings were used to measure whole blood viscosity, arterial blood gases and ionized calcium, complete blood count, electrolytes, creatinine, blood urea nitrogen (BUN), and inorganic phosphate. All measurements were made immediately post-dialysis before and after therapy with rHuEPO. The interval between pre- and post-rHuEPO studies was 8.3 ± 2.3 months. We found that post-dialysis hematocrit rose from 24.7 ± 0.9 to 36.4 ± 0.9%, hemoglobin from 83 ± 3 to 121 ± 3 g/liter and whole blood viscosity from 2.87 ± 0.11 to 3.71 ± 0.18 centipoise (all, P < 0.001 after therapy with rHuEPO). The remaining biochemical measurements did not change. Heart rate fell from 83 ± 3 to 77 ± 3 beats/min (P = 0.013). Left ventricular preload and afterload were not statistically different before and after rHuEPO. Total vascular resistance rose from 1313 ± 84 to 1568 ± 129 dynes · sec · cm-5, P = 0.029. Cardiac output and cardiac index fell by 12 and 15% (P = 0.024 and 0.030), respectively. Left ventricular contractility assessed using load and heart rate independent indices fell after therapy with rHuEPO (P = 0.003) in the nine patients in whom it was measured. In conclusion, correction of the anemia of chronic renal failure in maintenance hemodialysis patients with rHuEPO reduces measurements of global left ventricular systolic function by decreasing the heart rate and contractile state without significantly altering chamber loading conditions. The net effect is a decrease in the hyperdynamic circulatory state that typically characterizes chronic anemia.

Whether parathyroid hormone (PTH) has a clinically important effect on myocardial performance is unclear. Previous investigations of cardiac function before and after parathyroidectomy have failed to control for ionized calcium, other biochemical parameters, or heart rate and cardiovascular loading conditions. We performed load- and rate-independent measurements of myocardial contractility in seven stable hemodialysis patients before and after surgical parathyroidectomy under identical conditions of blood ionized calcium (Ca2+), electrolytes, pH, PO2, and hematocrit. Mid-molecule PTH decreased from 44 +/- 8 to 2 +/- 1 ng/mL. Aortic systolic and diastolic pressures, left ventricular chamber dimensions, end systolic wall stress, left ventricular contractility at a common level of afterload, and contractile reserve evaluated with dobutamine were similar before and after parathyroidectomy. Thus, PTH appears not to have a direct effect on myocardial contractile state in dialysis patients.

In afferent arterioles, the signaling events that lead to an increase in cytosolic Ca2+ concentration ([Ca2+]i) and initiation of vascular contraction are increasingly being delineated. We have recently studied angiotensin II (ANG II)-mediated effects on sarcoplasmic reticulum (SR) mobilization of Ca2+ and the role of superoxide and cyclic adenosine diphosphoribose in these processes. In the current study we investigated the participation of transient receptor potential canonical channels (TRPC) and a Na+/Ca2+ exchanger (NCX) in Ca2+ entry mechanisms. Afferent arterioles, isolated with the magnetized polystyrene bead method, were loaded with fura-2 to measure [Ca2+]i ratiometrically. We observed that the Ca2+-dependent chloride channel blocker niflumic acid (10 and 50 μ M) affects neither the peak nor plateau [Ca2+]i response to ANG II. Arterioles were pretreated with ryanodine (100 μM) and TMB-8 to block SR mobilization via the ryanodine receptor and inositol trisphosphate receptor, respectively. The peak [Ca2+]i response to ANG II was reduced by 40%. Addition of 2-aminoethoxydiphenyl borane to block TRPC-mediated Ca2+ entry inhibited the peak [Ca2+]i ANG II response by 80% and the plateau by 74%. Flufenamic acid (FFA; 50 μM), which stimulates TRPC6, caused a sustained increase of [Ca2+]i of 146 nM. This response was unaffected by diltiazem or nifedipine. KB-R7943 (at the low concentration of 10 μM) inhibits reverse (but not forward) mode NCX. KB-R7943 decreased the peak [Ca2+]i response to ANG II by 48% and to FFA by 38%. We conclude that TRPC6 and reverse-mode NCX may be important Ca2+ entry pathways in afferent arterioles.

We have previously shown that in afferent arterioles, angiotensin II (ANG II) involves activation of the inositol trisphosphate receptor (IP3R), activation of adenine diphosphoribose (ADPR) cyclase, and amplification of the initial IP3R-stimulated release of cytosolic Ca2+ ([Ca2+]i) from the sarcoplasmic reticulum (SR) (Fellner SK, Arendshorst WJ. Am J Physiol Renal Physiol 288: F785–F791, 2004). The response of the ryanodine receptor (RyR) to local increases in [Ca2+]i is defined as calcium-induced calcium release (CICR). To investigate whether Ca2+ entry via voltage-gated channels (VGCC) can stimulate CICR, we treated fura 2-loaded, freshly isolated afferent arterioles with KCl (40 mM; high KCl). In control arterioles, peak [Ca2+]i increased by 165 ± 10 nM. Locking the RyR in the closed position with ryanodine (100 μM) inhibited the [Ca2+]i response by 59% ( P < 0.01). 8-Br cADPR, a specific blocker of the ability of cyclic ADPR (cADPR) to sensitize the RyR to Ca2+, caused a 43% inhibition. We suggest that the lower inhibition by 8-Br cADPR ( P = 0.02, ryanodine vs. 8-Br cADPR) represents endogenously active ADPR cyclase. Depletion of SR Ca2+ stores by inhibiting the SR Ca2+-ATPase with cyclopiazonic acid or thapsigargin blocked the [Ca2+]i responses to KCl by 51% ( P not significant vs. ryanodine or 8-Br cADPR). These data suggest that about half of the increase in [Ca2+]i induced by high KCl is accomplished by activation of CICR through the ability of entered Ca2+ to expose the RyR to high local concentrations of Ca2+ and that endogenous cADPR contributes to the process.

Prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis in lepidopteran prothoracic glands (PGs), thus indirectly controlling molting and metamorphosis. PTTH triggers a signal transduction cascade in PGs that involves an early influx of Ca2+. Although the importance of Ca2+ has been long known, the mechanism(s) of PTTH-stimulated changes in cytoplasmic Ca2+ [Ca2+]i are not yet well understood. PGs from the fifth instar of Manduca sexta were exposed to PTTH in vitro. The resultant changes in [Ca2+]i were measured using ratiometric analysis of a fura-2 fluorescence signal in the presence and absence of inhibitors of specific cellular signaling mechanisms. The phospholipase C (PLC) inhibitor U-73122 nearly abolished the PTTH-stimulated increase in [Ca2+]i, as well as PTTH-stimulated ecdysteroidogenesis and extracellular-signal regulated kinase phosphorylation, thus establishing a role for PLC and implicating inositol trisphosphate (IP3) in PTTH signal transduction. Two antagonists of the IP3 receptor, 2-APB and TMB-8, likewise blocked the [Ca2+]i response by a mean of 92%. We describe for the first time the presence of Ca2+ oscillations in PTTH-stimulated cells in Ca2+-free medium. External Ca2+ entered PG cells via at least two routes: store-operated (capacitative) Ca2+ entry channels and L-type voltage-gated Ca2+ channels. We propose that PTTH initiates a transductory cascade typical of many G-protein coupled receptors, involving both Ca2+ mobilization and entry pathways.

We report a patient with presumed recurrence of IgA nephropathy in three successive cadaver transplants. Failure to recognize the cause of progressive renal failure in the first two transplants may have been associated with less than optimal treatment for his hypertension and nephrotic syndrome. His course illustrates the importance of biopsy-documented diagnosis of progressive dysfunction in kidney transplants.

SUMMARY The elasmobranch Squalus acanthias controls plasma osmolality and extracellular fluid volume by secreting a hypertonic fluid from its rectal gland. Because we found a correlation between extracellular Ca2+concentration and changes in cytosolic Ca2+([Ca2+]i), we sought the possible presence of a calcium-sensing receptor in rectal gland artery and tubules. Cytosolic Ca2+ of both tissues responded to the addition of external Ca2+ (0.8-5.3 mmol l-1) in a linear fashion. Spermine,Gd3+ and Ni2+, known agonists of the calcium-sensing receptor, increased [Ca2+]i. To assess the participation of inositol triphosphate (IP3) generation, sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ depletion, and activation of store-operated Ca2+ entry, we utilized thapsigargin and ryanodine to deplete Ca2+ SR/ER stores and the inhibitory reagents TMB-8 and 2-APB to block IP3 receptors. In each case, these agents inhibited the[Ca2+]i response to agonist stimulation by approximately 50 %. Blockade of L-channels with nifedipine had no significant effect. Increases in ionic strength are known to inhibit the calcium-sensing receptor. We postulate that the CaSR stimulates Ca2+-mediated constriction of the rectal gland artery and diminishes cyclic AMP-mediated salt secretion in rectal gland tubules during non-feeding conditions. When the shark ingests sea water and fish, an increase in blood and interstitial fluid ionic strength inhibits the activity of the calcium-sensing receptor, relaxing the rectal gland artery and permitting salt secretion by the rectal gland tubules.

Effects of acidosis on muscle contractile function have been studied extensively. However, the relative effects of different types of extracellular acidosis on left ventricular (LV) contractile function, especially the temporal features of contraction, have not been investigated in a single model. We constituted perfusion buffers of identical ionic composition, including Ca2+concentration ([Ca2+]), to mimic physiological control condition (pH 7.40) and three types of acidosis with pH of 7.03: inorganic (IA), respiratory (RA), and lactic (LA). Isolated rabbit hearts ( n = 9) were perfused with acidotic buffers chosen at random, each preceded by the control buffer. Under steady-state conditions, instantaneous LV pressure (Pv) and volume (Vv) were recorded for a range of Vv. The results were as follows. 1) LV passive (end-diastolic) elastance increased with IA and RA. However, this increase may not be a direct effect of acidosis; it can be explained on the basis of myocardial turgor. 2) Although LV inotropic state (peak active Pvand elastance) was depressed by all three acidotic buffers, the magnitude of inotropic depression was significantly less for LA. 3) Temporal features of Pvwere altered differently. Whereas IA and RA reduced time to peak Pv( tmax) and hastened isovolumic relaxation at a common level of LV wall stress, LA significantly increased tmaxand retarded relaxation. These results and a model-based interpretation suggest that cooperative feedback (i.e., force-activation interaction) plays an important role in acidosis-induced changes in LV contractile function. Furthermore, it is proposed that LA-induced responses comprise two components, one due to intracellular acidosis and the other due to pH-independent effects of lactate ions.

Prevention and treatment of cardiovascular disease and, in particular, ischemic heart disease (IHD) in an increasingly elderly and diabetic population of patients with end-stage renal disease (ESRD) pose a challenge to all members of the renal failure treatment program. The patient described herein presents such a challenge, illustrating risk factors for IHD, the dilemma of performing surgery given his age and comorbid conditions, and the impact of IHD on his quality of life and capacity to function independently. The coordinated efforts of the nephrologist, cardiologist, cardiac surgeon, rehabilitation specialist, nutritionist, primary nurse, and social worker all contributed to a successful intervention.

Hypertension arising from retained native kidneys complicates the management of recipients of renal transplants. Reluctance to administer angiontensin-converting enzyme inhibitor (ACEI) drugs to patients taking cyclosporine has reopened the question of performing native nephrectomies for poorly controlled, renin-dependent hypertension. We report the first published cases of simultaneous bilateral laparoscopic nephrectomies in 2 patients: 1 in preparation for living-related donor transplantation and the other ten months following cadaver transplantation in a patient whose end-stage renal disease was from malignant nephrosclerosis. Both had very severe hypertension resistant to multiple drugs and both became normotensive with little or no antihypertensive medication following nephrectomies. A bilateral nephrectomy is currently feasible using a laparoscopic approach.

Serum levels of oxalate are elevated in uremic patients on dialysis. The effect of living related donor kidney transplants on serum and urine oxalate levels was studied in 8 patients. Serum and urine oxalate levels were measured prior to transplant, on the day of transplant and daily for 5 days postoperatively, and the results compared to those in 11 normal subjects. All transplanted kidneys functioned immediately. Serum oxalate fell from 55 +/- 9 mumol/l (484 +/- 79 micrograms/dl) before transplant to 21 +/- 3 mumol/l (185 +/- 26 micrograms/dl) the day after transplant, and to 9 +/- 2 mumol/l (79 +/- 18 micrograms/dl) 72 h after transplant. Serum oxalate in normal subjects was 9 +/- 2 mumol/l (79 +/- 18 micrograms/dl). During the initial 24 h after transplant urine oxalate averaged 1,244 +/- 150 mumol/l (109.5 +/- 13.2 mg), but fell to levels not statistically different from normal by 72 h after transplant. Rapid clearance of oxalate after transplant leads to transient hyperoxaluria until normal levels of serum oxalate are reached.

Hyperlipidemia is one of many atherogenic risk factors encountered by patients undergoing chronic hemodialysis (HD). We have studied lipid profiles in these patients and have found less hypertriglyceridemia in those undergoing high-flux HD than those receiving traditional HD. Mean +/- SEM triglyceride level was 1.62 +/- 0.15 mmol/L (143.3 +/- 13.6 mg/dL) in high-flux dialysis patients, 2.39 +/- 0.27 mmol/L (211.6 +/- 24.1 mg/dL) in conventional dialysis patients, and 1.55 +/- 0.13 mmol/L (137.1 +/- 11.5 mg/dL) in normal age- and sex-matched controls. In addition, we found that in patients undergoing high-flux HD, females had higher high-density lipoprotein2 (HDL2) levels (0.62 +/- 0.03 mmol/L [23.8 +/- 1.3 mg/dL]) than males (0.33 +/- 0.04 mmol/L [12.9 +/- 1.7 mg/dL]) (P0.01). The mechanism(s) responsible for divergent lipid profiles in subsets of HD patients deserves further investigation. Whether reductions of hypertriglyceridemia and/or increases of HDL2 will diminish the incidence of cardiovascular disease in dialysis patients is unknown.

Hyperlipidemia poses a risk for cardiovascular disease in both hemodialysis and renal transplantation patients. Although lipid profiles differ between the 2 populations, we evaluated the possibility that both groups have similar abnormalities of lipoprotein(a) [Lp(a)]. Mean serum Lp(a) and standard error of the mean (SEM) in hemodialysis and transplant recipients was 16.6 +/- 4.7 and 18.3 +/- 3.6 mg/dl, respectively, compared with 10.7 +/- 4.1 mg/dl in healthy controls, p less than 0.05. That serum Lp(a) levels are significantly elevated in dialysis and renal transplantation patients suggests at least 1 common pathogenic mechanism for the high incidence of atherosclerosis in both groups.

To determine the cause of an outbreak of acute illness and death in a long-term hemodialysis unit.A retrospective cohort and case-control study of patients receiving hemodialysis and a laboratory study of a model deionization system to purify water for hemodialysis.An outpatient hemodialysis unit of a university hospital.12 patients who became severely ill after hemodialysis treatment and 20 patients who did not become ill after receiving hemodialysis treatment in the same unit.Medical and dialysis unit records were reviewed to identify and characterize cases. Fluids for dialysis were tested for toxic substances, and fluoride was measured in patients' serum. Resistivity and fluoride were measured in effluent from a model deionization system operated in the same way as the system associated with illness.During five consecutive hemodialysis shifts, 12 of 15 patients receiving dialysis treatment in one room became acutely ill, with severe pruritus, multiple nonspecific symptoms, and/or fatal ventricular fibrillation (3 patients). None of 17 patients treated in the adjacent room became ill (P0.0001). Death was associated with longer hemodialysis time and increased age compared with other patients who became ill. Serum concentrations of fluoride in the sick patients were markedly increased to as high as 716 mumol/L, and the source of fluoride was the temporary deionization system used to purify water for hemodialysis only in the affected room. Operation of a model deionization system showed how fluoride was adsorbed and then displaced in a massive efflux.Because deionization systems are used widely in hemodialysis and can cause fatal fluoride intoxication, careful design and monitoring are essential.

In 24 patients, sonograms were obtained of 25 vascular accesses, 16 of which were bovine grafts, 7 arteriovenous fistulae, 1 a polytetrafluoroethylene graft, and 1 an external Silastic shunt. On the sonograms, the lumen, course, and insertions of the vascular grafts and fistulae were outlined, anterior and posterior aneurysms demonstrated, hematomas differentiated from aneurysms, and intrinsic distinguished from extrinsic lesions. The authors conclude ultrasonography is a valuable adjunct to clinical evaluation in assessing certain complications of vascular access.

Store-operated Ca2+ entry is exaggerated in fresh preglomerular vascular smooth muscle cells of SHR.BackgroundRegulation of preglomerular vasomotor tone vessels ultimately control glomerular filtration rate, sodium reabsorption and systemic blood pressure. To gain insight into the complex renal hemodynamic factors that may result in hypertension, we studied calcium signaling pathways.MethodsFresh, single, preglomerular vascular smooth muscle cells (VSMC) were isolated from 5- to 6-week-old SHR and WKY utilizing a magnetized microsphere/sieving technique. Cytosolic Ca2+ ([Ca2+]i) was measured with fura-2 ratiometric fluorescence. To examine store-operated calcium entry (SOC), VSMC were activated in calcium-free buffer containing nifedipine. To deplete the sarcoplasmic reticulum (SR) of Ca2+, vasopressin-1 receptor agonist [V1R; inositol trisphosphate (IP3)-mediated mobilization], ryanodine (non-IP3 induced mobilization), and cyclopiazonic acid (CPA; Ca2+-ATPase inhibition) were utilized. Addition of external calcium followed by quenching of the fura/Ca2+ signal with Mn2+ permitted assessment of divalent cation entry via SOC.ResultsV1R caused greater mobilization in SHR than WKY (P < 0.01) as well as greater calcium entry (P < 0.001). Ryanodine and CPA both caused SR calcium depletion that was not statistically different between strains, but absolute calcium entry through SOC was more than double in SHR following either maneuver (P < 0.001). 2-Amino-ethoxybiphenyl borane (2-APB), an inhibitor not only of IP3 receptors, but also of SOC, blocked calcium entry in the ryanodine and CPA experiments independent of IP3. As well, Gd3+, a selective inhibitor of SOC, inhibited the Ca2+ response. We also studied L-channel calcium entry stimulated by V1R. The total calcium response was greater in SHR as was the absolute inhibition by nifedipine. As a percent of the total response, participation of L-type channels sensitive to nifedipine was about 45% in both strains of rat.ConclusionUtilizing three separate mechanisms to deplete the SR of Ca2+ in order to activate SOC, we show for the first time, that SOC is exaggerated in preglomerular VSMC of young SHR.

The nephrotic syndrome developed in a 47-year-old woman in association with severe hypercalcemia (23.5 mg/dl) from primary hyperparathyroidism. Other causes for hypercalcemia were sought and were not found. The nephrotic syndrome remitted spontaneously within two weeks of normalization of the serum calcium level. Kidney biopsy specimens showed deposition of electron-dense material, thought to be calcium, in the glomerular basement membranes initially and in the mesangium as well six months later.

Eighty-three chronic hemodialysis patients were tested for human immunodeficiency virus (HIV) infection. Testing included screening enzyme immunoassay (EIA) for HIV antibodies, competitive EIA for envelope and core antibodies, EIA for HIV antigen, and lymphocyte culture. Five (6%) of the patients had positive screening EIA at low reactivity. Four of these five had antibodies to H-9 cellular antigens. Comparison of the five seropositive patients to matched controls showed no significant differences in number of lymphocytes or helper/suppressor ratio. Six months later, the five patients had negative screening EIA results using a kit with a manufacturing change approved by the Food and Drug Administration that provided improved specificity. In addition, their Western blot analysis was negative. We conclude that (1) false-positive screening EIA results are more common in chronic hemodialysis patients than other populations; (2) evaluation of chronic hemodialysis patients for HIV infection requires confirmatory tests; and (3) newer EIA screening kits appear to have improved specificity.

We examined the urine for lymphocytes in 60 renal allograft recipients in the immediate posttransplant period using a simple staining technique with methylene blue. 37 acute rejection episodes associated with deterioration in renal function were observed. Other causes of decreased renal function, such as acute tubular necrosis, vascular occlusion or urologic obstruction were carefully excluded. 34 (92%) of the 37 acute rejection episodes were accompanied by significant lymphocyturia. Lymphocyturia was recognized concomitantly with the rise in serum creatinine in 20 of the 34, whereas in 14 it preceded the rise in serum creatinine by a period of 3.5 +/- 2.5 days. 24 (71%) of the 34 acute rejection episodes were reversed by high dose steroid administration and only 2 of them showed persistent lymphocyturia following treatment. On the other hand, 9 of the 10 nonresponders to steroid therapy showed persistent lymphocyturia. All the nonresponders eventually required maintenance dialysis. Detection of lymphocyturia is not only of value in the diagnosis of acute allograft rejection, but is also useful in determining allograft survival in the immediate posttransplant period.

• A patient with cryptogenic cirrhosis and disseminated sporotrichosis developed acute renal failure immediately following the administration of amphotericin B on four separate occasions. The abruptness of the renal failure and its reversibility within days suggests that there was a functional component to the renal dysfunction. We propose that amphotericin, in the setting of reduced effective arterial volume, may activate tubuloglomerular feedback, thereby contributing to acute renal failure. ( Arch Intern Med 1987;147:593-595)

To determine the effect of variations in blood ionized calcium (Ca2+) on myocardial contractility independent of changes in loading conditions and other biochemical variables.Hemodialysis done in a randomized, double-blind manner with dialysates differing in calcium concentration only. Left ventricular contractility was assessed using the load- and heart rate-independent relationship between end-systolic wall stress (sigma es) and rate-corrected velocity of fiber shortening (Vcfc).In-hospital dialysis unit and echocardiography laboratory of a university medical center.Seven patients with stable, chronic renal failure maintained on regular hemodialysis.Each patient was hemodialyzed three times within 1 week with dialysates differing in calcium concentration only. Ultrafiltration was adjusted to achieve the same postdialysis weight. Immediately after dialysis, two-dimensionally targeted M-mode echocardiographic and calibrated carotid pulse tracings were recorded over a wide range of left ventricular end-systolic wall stress values (a measure of left ventricular afterload) generated by either methoxamine or nitroprusside.After dialysis, three statistically distinct levels of Ca2+ were achieved. When Ca2+ was 1.34 +/- 0.03 mmol/L, Vcfc, calculated at a common level of afterload (sigma es = 50 g/cm2), was 1.01 +/- 0.05 cir/sec; at low Ca2+ (1.02 +/- 0.02 mmol/L), Vcfc fell to 0.89 +/- 0.04 cir/sec (P less than 0.001 compared with medium); at high Ca2+ (1.68 +/- 0.07 mmol/L) Vcfc rose to 1.10 +/- 0.03 circ/sec (P less than 0.001 compared with medium and low).Variations in Ca2+ are directly correlated with clinically significant changes in myocardial contractility.

• Of 90 patients undergoing regular dialysis, 42% had elevated levels of creatine phosphokinase (CPK). The BB isoenzyme was not detected, and only one patient had CPK MB. The elevation of CPK MM level did not correlate with values for calcium, phosphorus, calcium times phosphorus product, dry weight, or parathyroid hormone. Elevated levels of the enzyme correlated directly with muscle weakness in male patients and with hypothyroidism (depressed free thyroxin index) and inversely with treatment with vitamin D3supplements. We conclude that high CPK levels in uremia are secondary to skeletal muscle abnormalities and that hypothyroidism and vitamin D3deficiency may contribute to so-called uremic myopathy. (Arch Intern Med141:181-183, 1981)

The precise role of phenacetin, its metabolites, associated impurities, and fellow travelers, aspirin and caffeine, in producing interstitial nephritis remains to be clearly defined. 1,2 Despite continuing disagreement regarding the pathogenesis of the renal disease, it is clear that abuse of analgesic compounds is associated with a high incidence of chronic interstitial nephritis or pyelonephritis or both and in many instances with papillary necrosis as well. 3-6 Gsell and co-workers 2 have described the clinical picture of analgesic nephropathy, stressing the physiological consequences of insidiously progressive renal failure. Other writers have focused upon the situations in which analgesic abuse is found to occur 5,7,8 or upon some of the associated clinical findings of the syndrome. Noteworthy is the common occurrence of migraine headache, 5 gastritis and peptic ulcer, 5,7,9 anemia, 4 and psychiatric disease. 4,10 These and other features of the clinical syndrome of analgesic abuse have recently been reviewed