Calcium Oxalate Stone Agglomeration Inhibition [tm] Reflects Renal Stone-Forming Activity.

Louisiana and other Gulf South states comprise a "Stone Belt" where calcium oxalate stone formers (CaOx SFs) are found at a high rate of approximately 5%. In these patients, the agglomeration of small stone crystals, which are visible in nearly all morning urine collections, forms stones that can become trapped in the renal parenchyma and the renal pelvis. Without therapy, about half of CaOx SFs repeatedly form kidney stones, which can cause excruciating pain that can be relieved by passage, fragmentation (lithotripsy), or surgical removal. The absence of stones in "normal" patients suggests that there are stone inhibitors in "normal" urines.At the Ochsner Renal Stone Clinic, 24-hour urine samples are collected by the patient and sent to the Ochsner Renal Stone Research Program where calcium oxalate stone agglomeration inhibition [tm] measurements are performed. Urine from healthy subjects and inactive stone formers has demonstrated strongly inhibited stone growth [tm] in contrast to urine from recurrent CaOx SFs. [tm] data from 1500 visits of 700 kidney stone patients have been used to evaluate the risk of recurrence in Ochsner's CaOx SF patients. These data have also been used to demonstrate the interactive roles of certain identified urinary stone-growth inhibitors, citrate and Tamm-Horsfall protein (THP), which can be manipulated with medication to diminish recurrent stone formation. Our goal is to offer patients both financial and pain relief by reducing their stones with optimized medication, using medical management to avoid costly treatments.

Agglomeration inhibition reflected stone-forming activity during long-term potassium citrate therapy in calcium stone formers.

OBJECTIVES: The agglomeration of preformed crystals of calcium oxalate has been hypothesized to be the rate-limiting step in renal stone-forming activity (SFA). The effect of urine on the in vitro inhibition of agglomeration of seed crystals of calcium oxalate monohydrate, designated [tm], has been used to monitor SFA in calcium oxalate stone formers (CaOxSF). The objective of the present study was to determine whether [tm] could be used to help monitor the long-term effectiveness of oral potassium citrate therapy (K-Cit-Rx) in CaOxSF. METHODS: Clinic and radiographic (or ultrasound) reports were evaluated for 80 patients, aged 20 to 72 years, 55 men and 25 women, who were treated with oral K-Cit for recurrent calcium oxalate urolithiasis at the Ochsner Stone Clinic between January 1992 and July 1996. Seventy-five of these patients had at least one 24-hour citrate excretion rate of less than 3.0 mm/day before or after K-Cit-Rx. SFA graded on a scale of -2 to +2 by radiographic criteria was combined with information on stone passage to evaluate clinical stone status, and 24-hour urine collections were evaluated for volume, pH, calcium, citrate, uric acid, oxalate, creatinine, and [tm] on free diet before and after 6 to 53 months of K-Cit-Rx. Historical information on procedures performed for urolithiasis before and on K-Cit-Rx was also reviewed. RESULTS: K-Cit-Rx resulted in increased urine pH (P <0.0001) and decreased calcium (P=0.0475), [tm] (P=0.0045), number of stones passed per year (P=0.0016), and remedial procedures per year (P <0.0001). Patients taking allopurinol in addition to K-Cit required higher doses (P <0.0001) of K-Cit to control their disease, had lower pretreatment urine pH (P=0.0493), and showed greater increase in urine citrate (P=0.0092) than those on K-Cit alone. Those taking high-dose K-Cit were younger (P=0.0363) and showed greater decrease in SFA (P=0.0005) than those taking lower doses. A small group of 10 medication refractory patients, who retained (n=9) or increased (n=1) their stone burden during K-Cit-Rx, was identified. Compared with the medication-responsive group, the refractory patients were older (P=0.0124), and had greatly increased SFA (P <0.0001) and higher (P=0.0347) urine pH before and during (P=0.0173) treatment (data not shown). CONCLUSIONS: The data confirm that [tm] can be used not only to verify previously documented stone formation rate but also to help evaluate the long-term effectiveness of therapy. In this report, changes in [tm] after K-Cit-Rx reflected decreased stone formation rate and decreased remedial procedures.

Urinary Tamm-Horsfall protein increased after potassium citrate therapy in calcium stone formers.

OBJECTIVES: To evaluate the effect of oral potassium citrate therapy on urinary excretion rates of citrate. Tamm-Horsfall protein (THP), and on calcium oxalate monohydrate crystal agglomeration inhibition [tm], in patients with recurrent calcium stone formation. METHODS: To evaluate the effect of oral therapy with potassium citrate on urinary citrate, THP, and [tm], 24-hour urine samples were collected before and at least 2 months after initiation of oral potassium citrate therapy in 33 calcium stone-forming patients who had no dietary restrictions. The citrate concentration was measured by an adaptation of a citrate lyase method. Urinary disaggregated THP concentration was determined with a quantitative enzyme-linked immunosorbent assay. The [tm] was determined by observing the effects of patients' urine, before and after oral potassium citrate therapy, on the uptake of 45Ca2+ onto the surfaces of added preformed calcium oxalate crystals in a supersaturated solution of calcium oxalate, using the in vitro kinetic method described by other investigators. RESULTS: We observed an increased urinary excretion rate of citrate from a mean of 1.9 mmol/24 h prealkali to 2.6 mmol/24 h postalkali (P < 0.0004) and of THP from a mean of 94.0 mg/24 h prealkali to 199.3 mg/24 h postalkali (P < 0.0016). A corresponding increase in [tm] from a mean of 177.1 minutes prealkali to 221.0 minutes postalkali (P < 0.024) was also observed. CONCLUSIONS: To our knowledge this is the first report correlating increased urinary citrate with THP excretion rate following oral alkalinization with potassium citrate in calcium stone formers. Of clinical importance is the corresponding increase in [tm], which was previously shown to be inversely related to stone-forming activity. Moreover, urinary citrate and THP are known to have a synergistic effect on [tm]. Our data suggest that the effectiveness of potassium citrate therapy in calcium stone-forming patients may, at least in part, be due to increased levels of THP.

Calcium oxalate stone agglomeration reflects stone-forming activity: citrate inhibition depends on macromolecules larger than 30 kilodalton.

To evaluate the clinical utility of in vitro calcium oxalate monohydrate (COM) crystallization kinetics measurements and to determine the effect of quantitative removal of urinary Tamm-Horsfall glycoprotein on such measurements, we examined 24-hour, room temperature urine collections of patients from our Stone Clinic and of normal subjects from our research laboratories at Ochsner Medical Institutions in New Orleans, LA, and compared their COM kinetic parameters in vitro before and after urine ultrafiltration (30 kd). Data from 53 calcium oxalate stone-forming patients (26% women; mean age, 47 years) who demonstrated radiographic or other evidence of forming at least one stone were compared with data from 22 healthy volunteers (25% women; mean age, 40 years). Hypercalciuria (> 7.5 mm/24 hr), hyperoxaluria (> 0.5 mm/24 hr), and hypocitraturia (< 2.0 mm/24 hr) were present in 38%, 26%, and 26% of the patient population, respectively. Urinary creatinine, urate, calcium, citrate, phosphate, oxalate, pH, volume, total immunoreactive-disaggregated Tamm-Horsfall glycoprotein, and the urine's effects on COM solubility, percent crystal growth inhibition, and crystal agglomeration inhibition [tm] were determined. Calcium oxalate monohydrate agglomeration inhibition, [tm], was reduced in stone-forming patients. It decreased with increasing stone frequency, making [tm] a useful tool for measuring the risk of stone recurrence. Urinary Tamm-Horsfall glycoprotein and citrate concentrations were linearly related to COM agglomeration inhibition. Their effects were synergistic. Tamm-Horsfall glycoprotein removal from urine reduced COM agglomeration inhibition dramatically. Alkali therapy increased urinary citrate concentration and increased [tm].(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine 5'-triphosphate, phorbol ester, and pertussis toxin effects on atrial natriuretic peptide stimulation of guanylate cyclase in a human renal cell line.

We examined adenosine 5'-triphosphate (ATP), pertussis toxin (PT) and phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, modulation of atrial natriuretic peptide (ANP)-stimulated cell-membrane guanylate cyclase (ANP-s-GC) activity and ANP stimulation of whole-cell cGMP accumulation (ANP-s-cGMP) in an ANP-receptor-transduction cell model, the human renal cell line (SK-NEP-1). Acute and long-term effects of PMA on PKC isotype activity are different: Acute (20-min) PMA activation of PKC inhibits ANP-s-cGMP and ANP-s-GC; whereas, long-term (36-h) PMA treatment inhibits slightly less by only partially down-regulating PKC activity, the type-III PKC isotype being 36-h resistant. Long-term 10(-7)M PMA treatment of cells neither affected membrane basal GC activity nor ANP-s-GC activity but partially inhibited ATP enhancement of ANP-s-GC. This partial inhibition was completely reversed by the PKC inhibitor H7 and a PKC inhibitory antibody but only partially reversed by the antibody to the catalytic domain of PKC type III. The EC50 for ATP and its non-phosphorylating analog ATP gamma S in the presence of acute PMA inhibition of ANP-s-cGMP was similar (approximately 10(-9)). This enhancement of PMA inhibition was two orders of magnitude more sensitive (EC50 10(-7)M) than inhibition of ANP-s-cGMP that we previously reported for acute PMA treatment of whole SK-NEP-1 cells. The three- to four-fold ATP enhancement of cell membrane ANP-s-GC was not blocked by 12-hour preincubation of cells with 150 ng/mL PT but was completely blocked if 2-x-10(-7)M PMA was then added for 20 minutes, indicating that acute activation of PKC by PMA does not require a functional "G-type" protein. Acute PMA inhibition of ANP-s-cGMP was reversed by permeabilizing SK-NEP-1 cells to a specific PKC inhibitory peptide, further confirming that PMA inhibition was mediated through PKC activation. These data demonstrated that ANP-s-GC and ANP-s-cGMP were modified through non-phosphorylating interactions with ATP, by multiple PMA activatable PKC isoforms, and that neither were affected by PT-sensitive guanine-nucleotide-binding (G)-protein(s).

Different ATP effects on natriuretic peptide receptor subtypes in LLC-PK1 and NIH-3T3 cells.

We have observed different ATP interactions in two guanylate cyclase (GC)-coupled natriuretic peptide (NP) receptor subtypes, designated NPR-A and NPR-B. The NPR-A is selectively expressed by LLC-PK1 epithelial cells and the NPR-B by NIH-3T3 fibroblast cells. In LLC-PK1 membranes, ATP-Mg2+ potentiated ANP-stimulated GC activity (ANP-s-GC). In contrast, in NIH-3T3 membranes, ATP-Mg2+ inhibited ANP-s-GC but enhanced CNP-stimulated GC activity (CNP-s GC). ATP in the presence of Mn2+ inhibited LLC-PK1 and NIH-3T3 membrane ANP-s-GC and CNP-s-GC. These are the first data suggesting that the ATP-Mg2+ produces different effects between membrane NPR-A and -B subtypes. We have also demonstrated that GC of NPR-B is sensitive to methylene blue.

Calcium and calmodulin regulate atrial natriuretic factor stimulation of cyclic GMP in a human renal cell line.

We examined calcium and calmodulin regulation of atrial natriuretic factor stimulation of particulate-membrane guanylate cyclase (ANF-s-GC) in SK-NEP-1 cells. W7 and trifluoropiperazine, but not W5, inhibited whole cellular ANF-stimulated cyclic GMP accumulation (ANF-s-cGMP). EGTA and LaCl3 decreased ANF-s-GC and calmodulin reversed this inhibition. A23187-induced inhibition of ANF-s-cGMP was only partly reversible by IBMX. H7 or staurosporine counteracted the inhibitory effect of A23187. Calcium inhibited basal and ANF-s-GC. These data suggest that at low concentrations of calcium, ANF-s-GC was calcium-calmodulin dependent but high concentrations of calcium inhibited ANF-s-GC through phosphodiesterase, through inhibition of GC, and probably through protein kinase C.

Phorbol and calcium decreased atriopeptin response in a human renal cell line.

We investigated regulation of atrial natriuretic factor (ANF)-stimulated cellular cGMP accumulation (ANF-s-cGMP) in an ANF-responsive human renal cell line, SK-NEP-1. Dose-response data indicated that the EC50 for ANF(99-126) was 1.1 x 10(-9) M. Brain natriuretic peptide (10(-6) M) increased cGMP to a level indistinguishable from that of ANF (10(-6) M). [Met-(O)]ANF was only half as potent as ANF, and atriopeptin I (10(-6) M) did not increase cGMP over basal levels. Preincubation of SK-NEP-1 cells with ANF, but not atriopeptin I (API), for two hours or longer, caused a concentration-dependent down-regulation of ANF-s-cGMP. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, and A23187 and its 4-bromo derivative, calcium ionophores, inhibited ANF-s-cGMP in a dose-dependent manner. A23187 inhibition was calcium dependent and promoted net cGMP degradation. Thirty-six hour preincubation with PMA, a procedure used to down-regulate PKC, abolished acute PMA inhibition of ANF-s-cGMP without having an effect on ANF-s-cGMP or on 4-bromo-A23187 inhibition thereof. These data indicate that PKC activation specifically inhibited ANF-s-cGMP but that PKC was not required for ANF-s-cGMP in SK-NEP-1 cells. Thus structurally related ANF peptides, protein kinase C (PKC) activators, calcium ionophores are potential modulators of ANF-s-cGMP in cells from this human renal cell line.

The opposing effects of calmodulin, adenosine 5'-triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells.

In the present study, we investigated the effects of calmodulin, adenosine 5'-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) (a protein kinase C activator) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation. In conclusion, these results demonstrated that PMA (activated protein kinase C) inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.

Increased secretion of erythropoietin in human renal carcinoma cells in response to atrial natriuretic factor.

The present studies were undertaken to assess the effects of atrial natriuretic factor (ANF) on erythropoietin (Ep) secretion in Ep-producing renal carcinoma (RC) cells using a sensitive radioimmunoassay for Ep. Human ANF produced a significant dose-related increase in Ep secretion at concentrations of 10(-7) and 10(-6) M when compared with vehicle controls. ANF (greater than or equal to 10(-9) M) also significantly increased the intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration after 5-min incubation with the RC cells. Scatchard analysis of the human 125I-labeled ANF binding data indicated that the RC cells contain a single class of binding sites with a dissociation constant (Kd) of 93 +/- 1 pM and a binding capacity of 2,190 +/- 750 sites/cell. Incubation of the RC cells with 8-bromo-cGMP in concentrations of 10(-7)-10(-5) M also produced a significant dose-related enhancement of Ep secretion. These findings suggest that the increase in Ep secretion in response to ANF can be attributed, at least in part, to activation of guanylate cyclase, which is coupled to specific ANF receptors on the RC cell.

Atrial natriuretic factor effects on cyclic nucleotides in a human renal cell line.

The natriuretic effects of atrial peptide hormones have been attributed, at least in part, to their stimulation of guanylate cyclase activity in renal cell membranes. The effects of atrial natriuretic factor (ANF) on stimulation of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) accumulation were investigated in cloned human kidney tumor (hKT) cells and parent cells from a human renal tumor epithelial cell line (SK-NEP-1). Human ANF-(99-126) (10(-6)M) stimulated (p less than 0.001) cellular cGMP accumulation in a dose-dependent manner from a basal level of 0.26 +/- 0.04 to 3.73 +/- 0.81 pmol/mg protein/5 mi (mean +/- SEM, n = 13). ANF stimulation of cGMP accumulation was specific, in that high concentrations (10(-6)M) of atriopeptin I [rat ANF-(103-123)], angiotensin II, arginine vasopressin, and amiloride (10(-4)M) did not increase basal cGMP. Amiloride (10(-4)M) enhanced (p less than 0.01, n = 6) the ANF stimulation of cGMP accumulation (1.24 +/- 0.39 pmol/mg protein/5 min), particularly at low doses of ANF (10(-10)M) where stimulation by ANF without amiloride (0.34 +/- 0.08 pmol/mg protein/5 min) was barely distinguishable from a basal level (0.19 +/- 0.02 pmol/mg protein/5 min) of cGMP accumulation. The stimulatory effect of ANF (1.59 +/- 0.07 pmol/mg protein/5 min) was attenuated (0.75 +/- 0.06 pmol/mg protein/5 min, p less than 0.01, n = 6) by preincubation of the cells with pertussis toxin but not by cholera toxin. ANF (4.56 +/- 0.93 pmol/mg protein/5 min, n = 8) did not affect cAMP accumulation (4.32 +/- 0.98 pmol/mg protein/5 min) in hKT cells. This is the first report of an ANF responsive human renal cell line, and its use should facilitate investigation of ANF-receptor interactions.

Amiloride enhances atrial natriuretic factor stimulation of cGMP accumulation in rat glomeruli.

The effects of amiloride on ANF binding and ANF stimulation of cGMP were evaluated in rat glomeruli. Amiloride increased ANF binding to whole glomeruli and to glomerular membrane preparations. In contrast, amiloride enhanced ANF-stimulated cGMP accumulation only at 37 degrees C in whole glomeruli, but not at 4 degrees C in whole glomeruli or at 37 degrees C in membrane preparations. These data suggest that under physiological conditions amiloride augments ANF-stimulated intracellular cGMP accumulation. The discrepancies between amiloride augmentation of ANF binding and failure to increase ANF-stimulated cGMP accumulation may result from ANF receptor heterogeneity. This is the first report of an amiloride augmentation of ANF-stimulated cGMP accumulation in renal tissue.

Effect of ATP and amiloride on ANF binding and stimulation of cyclic GMP accumulation in rat glomerular membranes.

We investigated ANF binding and stimulation of cGMP accumulation in isolated rat glomerular membranes in the presence and absence of amiloride and ATP. Amiloride enhanced high affinity binding of ANF without affecting its stimulation of cGMP. In contrast ATP decreased binding and decreased basal cGMP accumulation without affecting the ability of ANF to stimulate cGMP. These data indicate that ANF binding and stimulation of cGMP accumulation can be regulated independently supporting further the concept of receptor heterogeneity in renal glomerular membranes.

Vasorelaxant and natriuretic activities of synthetic atrial peptides.

The vasorelaxant and natriuretic activities of synthetic atrial natriuretic peptides (ANF) were compared by measuring relaxation of histamine-contracted rabbit aortic rings and changes in urinary sodium excretion in anesthetized rats. The peptides studied and their relation to pro-ANF were: atriopeptins (AP) I, II and III (ANF 103-123, 103-125, 103-126), cardionatrin (CN) (ANF 99-126), alpha-human atrial natriuretic polypeptide (alpha-hANP) (human 99-126) and a fragment of the last (hFrag) (human ANF 105-126). The concentration causing 50% relaxation (EC50) in the vasorelaxant assay for API was greater than that of the other peptides (p less than 0.05). The EC50 for hFrag was 20 times greater than that of alpha-hANP, but this difference was not statistically significant. API caused less natriuresis compared with the other peptides (p less than 0.05). The natriuresis induced by CN was significantly greater than that of the other peptides at the 485 pmol dose. These results indicated that, except for API, the peptides studied has similar vasorelaxant and natriuretic activities.

Association of the atrial natriuretic factor receptor with guanylate cyclase in solubilized rat glomerular membranes.

The elution profile of solubilized rat glomerular membranes from a gel filtration column showed two peaks of 125I-ANF (atrial natriuretic factor) binding (367 +/- 21, 156 +/- 12 KDa). Over 85% of the total binding for the extract was in the 367 KDa peak. Guanylate cyclase activity was correlated with 125I-ANF specific binding. ANF activation of guanylate cyclase was also observed. As observed previously with particulate membrane, Scatchard-analysis of ANF binding data with the solubilized extract was consistent with a two-site model. Both affinities (Kd's), 4 pM and 1 nM, are within the range of blood concentrations reported for ANF. These observations suggest that most rat glomerular ANF receptors are large molecular complexes coupled with guanylate cyclase in the 300-350 KDa size range.

Atrial natriuretic peptide binding properties of purified rat glomerular membranes.

125I-ANP (3-[125I] iodotyrosyl28) binding studies with purified rat glomerular membranes indicate two types of physiologically relevant hormonal receptors, Types I and II, Kd approximately 5 pM and approximately 2.5 nM, respectively. All preparations were essentially free of capsular and tubular contamination. Binding data indicated that Type I receptors were three times more concentrated than Type II receptors in purified membrane fractions. When purified membranes were cross-linked with 125I-rANP, using disuccinimidyl suberate and separated by SDS-PAGE, approximately 75- and approximately 140-kDa proteins were specifically labeled in a ratio of approximately 3:1, respectively. Thus, in purified renal glomerular membranes, Type I receptors with molecular weight of approximately 75-kDa appeared to predominate and would be detectably saturated at circulating ANP concentrations as low as 15 pg/ml. These findings could account for the exquisite sensitivity of natriuretic response to ANP.

Atrial natriuretic peptide decreases circulatory capacitance in areflexic rats.

The short-term hemodynamic response to atrial natriuretic peptide appears to be partly mediated by decreased venous return, which could result from increased circulatory capacitance or decreased blood volume. To determine if rat atrial natriuretic peptide 99-126 (0.5 microgram/kg/min IV for 30-70 minutes) dilated capacitance vessels or decreased blood volume, mean circulatory filling pressure (measured during brief circulatory arrest by inflating an intraatrial balloon) and blood volume (51Cr-erythrocytes) were measured in anesthetized rats. Mean circulatory filling pressure, central venous pressure, and blood volume decreased by 0.4 mm Hg, 0.5 mm Hg, and 3.4 ml/kg, respectively. To determine the total circulatory pressure-volume relationship without influence from autonomic reflexes, mean circulatory filling pressure and blood volume were measured in spinal-cord-transected rats before and immediately after infusing or withdrawing 5 ml blood. Atrial natriuretic peptide decreased mean circulatory filling pressure, central venous pressure, and blood volume by 0.9 mm Hg, 1.7 mm Hg, and 8.0 ml/kg, respectively, and displaced the pressure-volume relationship toward the pressure axis by decreasing extrapolated unstressed volume. Similar results were obtained in spinal-cord-transected rats that had initial vascular tone restored to a greater level by norepinephrine infusion. In anephric rats, atrial natriuretic peptide decreased central venous pressure by 0.3 mm Hg and blood volume by 1.6 ml/kg. The results indicate that short-term infusion of atrial natriuretic peptide reduced circulatory capacitance in rats and suggest that this reduction resulted from diminished blood volume due to urinary fluid loss followed by passive vascular recoil and active venoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of an attenuated strain of chikungunya virus for use in vaccine production.

An attenuated chikungunya (CHIK) virus clone was developed for production of a live vaccine for human use. CHIK strain 15561 was subjected to 18 plaque-to-plaque passages in MRC-5 cultures before CHIK 181/clone 25 was selected as vaccine seed based on homogeneous small plaque size, suckling mouse avirulence, reduced monkey viraemia and genetic stability. Oligonucleotide mapping demonstrated differences between parent and clone. Vaccine (pilot-lot production) elicited neutralizing antibody and protected mice and rhesus monkeys against challenge. After challenge, viraemias were absent in vaccinated monkeys. Vaccine was then produced and tested in accordance with governmental regulatory requirements of human use.

Hemodynamic effects of atrial natriuretic hormone.

The atrial natriuretic hormone (ANH) alters cardiovascular function independent of changes in body fluid volume. Most investigators agree that ANH decreases mean arterial pressure (MAP). However, although some investigators have observed a decrease in total peripheral resistance in association with the decrease in MAP, a more frequent observation has been decreased cardiac output (CO). The mechanism whereby ANH decreases CO is unknown, but does not appear to be the result of direct myocardial depression, reductions in intravascular or cardiopulmonary volumes, or venodilation. Alterations in skeletal muscle and splanchnic blood flow have been reported by some but not all investigators. Although increases in renal blood flow have been reported, they are transitory and have not been consistently observed by all researchers. The cardiovascular effects of ANH appear to be influenced not only by the dose, but also by the cardiovascular control mechanisms that operate at the time of ANH administration. Non-renin-dependent hypertensive models exhibit a decrease in MAP associated with decreased CO, whereas in renin-dependent animals this hypotension is associated with a decrease in total peripheral resistance.