Organic mercurial diuresis: inhibition of glutamine utilization in the acidotic rat.

Organic mercurials inhibit mitochondrial glutamine metabolism in vitro while metabolic acidosis, a condition in which the predominant renal fuel is glutamine, potentiates mercurial diuresis. The following studies were undertaken to determine whether potentiation of diuresis reflects mercurial inhibition of glutamine utilization. (1) All three mercurials employed (mersalyl, chlormerodrin, and p-chloromercuribenzoate) are diuretics in the rat and this effect was potentiated by NH4Cl. (2) Despite reabsorbing less sodium, mercurial-treated rats had lower kidney ATP content (4.35 +/- 0.26 and 3.84 +/- 0.43 mumol/g dry weight (mercurial plus NH4Cl) than did controls (4.95 +/- 0.31 and 4.87 +/- 0.39 mumol/g dry weight (NH4Cl). (3) Isolated kidneys from NH4Cl and NH4Cl plus mercurial treated rats were perfused with 1 mM L-[U-14C]glutamine to determine rates of extraction and oxidation. Mercurial-treated acidotic rat kidneys had a reduced rate of glutamine uptake (40.8 +/- 7.4 vs. 64.8 +/- 5.8 mumol/h per kidney), a diminished rate of glutamine conversion to CO2 (14.8 +/- 3.6 vs. 26.4 +/- 5.2 mumol/h per kidney), and a reduction in glucose production (16 +/- 5 vs. 27 +/- 4 mumol/h per kidney). These results are consistent with an effect of organic mercurials upon glutamine utilization, limiting ATP availability, and thereby reducing tubular active sodium reabsorption.     

Supersensitivity to norepinephrine in chronically denervated kidneys: evidence for a postsynaptic effect

Denervation supersensitivity in chronically denervated kidneys increases renal responsiveness to increased plasma levels of norepinephrine. To determine whether this effect is caused by presynaptic (i.e., loss of uptake) or postsynaptic changes, we studied the effect of continuous infusion of norepinephrine (330 ng/min, i.v.) and methoxamine (4 micrograms/min, i.v.), an alpha 1-adrenergic agonist that is not taken up by nerve terminals, on renal function of innervated and denervated kidneys. Ganglionic blockade was used to eliminate reflex adjustments in the innervated kidney and mean arterial pressure was maintained at preganglionic blockade levels by an infusion of arginine vasopressin. With renal perfusion pressure controlled there was a significantly greater decrease in renal blood flow (-67 +/- 9 vs. -33 +/- 8%), glomerular filtration rate (-60 +/- 9 vs. -7 +/- 20%), urine flow (-61 +/- 7 vs. -24 +/- 11%), sodium excretion (-51 +/- 15 vs. -32 +/- 21%), and fractional excretion of sodium (-50 +/- 9 vs. -25 +/- 15%) from the denervated kidneys compared with the innervated kidneys during the infusion of norepinephrine. During the infusion of methoxamine there was a significantly greater decrease from the denervated compared with the innervated kidneys in renal blood flow (-54 +/- 10 vs. -30 +/- 14%), glomerular filtration rate (-51 +/- 11 vs. -19 +/- 17%), urine flow (-55 +/- 10 vs. -39 +/- 10%), sodium excretion (-70 +/- 9 vs. -59 +/- 11%), and fractional excretion of sodium (-53 +/- 10 vs. -41 +/- 10%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine production by the isolated perfused rat kidney

We used isolated perfused rat kidneys to examine dopamine (DA) production and its relation to renal function. Both innervated and chronically surgically denervated kidneys perfused with a solution containing neither albumin nor tyrosine, excreted 0.2 +/- 0.1 ng DA X min-1 X g wet weight-1 during the 10-min collection period between 30 and 40 min after starting perfusion. When perfused with 6.7% albumin, without tyrosine, innervated kidneys excreted 1.0 +/- 0.06 ng DA X min-1 X g-1 and denervated kidneys excreted 1.0 +/- 0.07 DA X min-1 X g-1. When 0.03 mM tyrosine was included in the albumin perfusate, innervated kidneys excreted 1.2 +/- 0.1 ng DA X min-1 X g-1 (p less than 0.1). Under these conditions DA excretion continued for at least 100 min at which time it was 0.6 ng X min-1 X g-1 and 86 ng/g kidney weight had been excreted. Denervated kidneys perfused with albumin + tyrosine excreted 0.9 +/- 0.13 ng DA X min-1 X g-1. Renal stores of free DA, conjugated DA, and dihydroxyphenylalanine (DOPA) could have provided at the most 30 ng/g of DA. Carbidopa inhibited DA excretion completely. DA excretion did not correlate with renal vascular resistance, inulin clearance, or fractional sodium excretion. In summary, nonneural tissue in isolated perfused kidneys produced DA at the same rate as denervated kidneys in vivo. Less than one-third of the DA produced by isolated kidneys could have come from intrarenal stores of DOPA, free DA, and conjugated DA; the rest was synthesized from unknown precursors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature modulates P2X receptor-mediated cardiovascular responses to muscle afferent activation

Static muscle contraction increases ATP release into the muscle interstitial space. Elevated ATP in muscle stimulates thin fiber muscle afferents and increases blood pressure via engagement of purinergic P2X receptors. In addition, ATP activates P2X receptors and enhances cardiovascular responses induced by stimulation of muscle mechanoreceptors. In this study, we examined whether elevated muscle temperature would attenuate and whether reduced temperature would potentiate P2X effects on reflex muscle responses. alpha,beta-Methylene ATP (alpha,beta-MeATP) was injected into the arterial blood supply of hindlimb muscle to stimulate P2X receptors, and muscle stretch was induced to activate mechanically sensitive muscle afferents as alpha,beta-MeATP was injected in 10 anesthetized cats. Femoral arterial injection of alpha,beta-MeATP (1.0 mM) increased mean arterial pressure (MAP) by 35+/-5 (35 degrees C), 26+/-3 (37 degrees C), and 19+/-3 mmHg (39 degrees C; P<0.05 vs. 35 degrees C), respectively. Muscle stretch (2 kg) elevated MAP. The MAP response was significantly enhanced 34% and 36% when alpha,beta-MeATP (0.2 mM) was arterially infused 5 min before muscle stretch at 35 degrees and 37 degrees C, respectively. However, as muscle temperature reached 39 degrees C, the stretch-evoked response was augmented only 6% by alpha,beta-MeATP injection, and the response was significantly attenuated compared with the response with muscle temperature of 35 degrees and 37 degrees C. In addition, we also examined effects of muscle temperature on alpha,beta-MeATP enhancement of the cardiovascular responses to static muscle contraction while the muscles were freely perfused and the circulation to the muscles was occluded. Because muscle temperature was 37 degrees C, arterial injections of alpha,beta-MeATP significantly augmented contraction-evoked MAP response by 49% (freely perfused) and 53% (ischemic condition), respectively. It is noted that this effect was significantly attenuated at a muscle temperature of 39 degrees C. These data indicate that the effect of P2X receptor on reflex muscle response is sensitive to alternations of muscle temperature and that elevated temperature attenuates the response.     

Effect of potassium concentration and ouabain on the renal adaptation to potassium depletion in isolated perfused rat kidney

Renal adaptation for potassium (K) conservation has been demonstrated in isolated perfused kidneys from rats within 3 days of K depletion and appears to be independent of aldosterone and sodium excretion. This study was designed to investigate whether the renal adaptation for K conservation is independent of ambient [K] and renal tissue levels of K and whether ouabain may have effects on K excretion, which are in contrast to the effects on K excretion in normal animals. In the first study, rats K depleted for 3 days received 2500 mu equiv. KCI intraperitoneally, while other K-depleted rats and a group of control diet animals received intraperitoneal H2O alone to determine whether simple restoration of K deficits would reverse the renal adaptation for K conservation. Intraperitoneal KCI increased plasma [K] and kidney tissue K significantly within 3 h in the K-repleted group compared with the K-depleted rats. Isolated Kidneys were perfused from the three groups of rats 3 h after intraperitoneal injection. Despite K repletion in vivo, perfused kidneys from the K-repleted group still had significantly decreased K excretion (1.28 +/- 0.085 mu equiv./min) compared with controls (2.05 +/- 0.291 mu equiv./min), and K excretion was still not different from the K-depleted group (0.57 +/- 0.134 mu equiv./min). However, fractional K excretion by the kidneys from K-repleted rats was increased above K-depleted kidneys (0.48 +/- 0.051 vs. 0.18 +/- 0.034, p less than 0.01). Despite the increased renal tissue K in K-repleted kidneys at the start of perfusion (285 +/- 5.1 vs. 257 +/- 5.4 mu equiv./g), by the end of the perfusion tissue K in perfused kidneys was identical in all three groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal adjustments to nonexertional heat stress in mature and senescent Fischer 344 rats

The purpose of this study was to test the hypothesis that the rise in colonic temperature (Tc) during nonexertional heat stress is exaggerated in senescent (SEN, 24 mo, n = 12) vs. mature (MAT, 12 mo, n = 15) conscious unrestrained Fischer 344 rats. On 2 separate days (48 h apart) each SEN and MAT animal was exposed to an ambient temperature (Ta) of 42 degrees C (relative humidity 20%) until a Tc of 41 degrees C was attained and then cooled at a Ta of 26 degrees C until Tc returned to the initial control level. Control Tc was similar in the two groups for both trials. The rate of Tc change during heating was 63% greater (0.070 +/- 0.005 vs. 0.043 +/- 0.004 degrees C/min, P less than 0.05) and the time to 41 degrees C reduced by 36% (54 +/- 6 vs. 85 +/- 10 min, P less than 0.05) in MAT vs. SEN animals during the first exposure, although the cooling rate was slower in the MAT (0.048 +/- 0.004 degrees C/min) vs. SEN (0.062 +/- 0.006 degrees C/min) animals (P less than 0.05). The heating rate was unchanged in MAT animals between trials 1 and 2. However, SEN animals had a 95% increase in heating rate in trial 2 compared with trial 1 (P less than 0.05), and the corresponding time to 41 degrees C was decreased by 44% (P less than 0.05). As a result, rate of heating and time to 41 degrees C were similar in the two groups during trial 2. The cooling rate was similar between trials within each group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preservation of renal function by adenosine-stimulated ATP synthesis in hypothermically perfused dog kidneys

Dog kidneys were hypothermically perfused for 1 to 5 days in the presence or absence of adenosine (5 mM). Following 1, 3, and 5 days, kidneys were reperfused at normothermia in an isolated perfusion system using a bovine serum albumin containing perfusate and renal function was determined. At the end of normothermic perfusion, kidney cortical slices were removed for biochemical analysis. Kidneys preserved in the presence of adenosine generated much higher concentrations of ATP during normothermic perfusion than kidneys preserved in the absence of adenosine at all time periods studied. In kidneys reperfused (37 degrees C) after 3 days of preservation, the ATP concentration averaged 9.15 mumol/g dry wt (+adenosine) vs. 4.75 mumol/g dry wt (-adenosine). After 5 days, the average was 12.65 mumol/g dry wt (+adenosine) vs. 4.00 mumol/g dry wt (-adenosine). The tissue concentration of K+ was higher in kidneys perfused in the presence of adenosine for all time periods studied. The presence of adenosine had little effect on the GFR (creatinine clearance) which was reduced by about 90% from control values at both 3 and 5 days of preservation. The primary effect of adenosine on renal function was a greater preservation of the capability of the isolated perfused kidney to reabsorb Na+ from the glomerular filtrate. In the absence of adenosine Na+ reabsorption was reduced from 97 to 50% whereas in the presence of adenosine was reduced to only 80% after 3 days of preservation. After 5 days of perfusion Na+ reabsorption was unaffected by the presence of adenosine and the amount resorbed was only 25-30% of the amount filtered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal instability of rat muscle sarcoplasmic reticulum Ca(2+)-ATPase function

To examine the thermal instability and the role of sulfhydryl (SH) oxidation on sarcoplasmic reticulum (SR) Ca(2+)-ATPase function, crude homogenates were prepared from the white portion of the gastrocnemius (WG) adult rat muscles (n = 9) and incubated in vitro for < or =60 min either at a normal resting body temperature (37 degrees C) or at a temperature indicative of exercise-induced hyperthermia (41 degrees C) with DTT and without DTT (CON). In general, treatment with DTT resulted in higher Ca(2+)-ATPase and Ca(2+) uptake values (nmol. mg protein(-1). min(-1), P < 0.05), an effect that was not specific to time of incubation. Incubations at 41 degrees C resulted in lower (P < 0.05) Ca(2+) uptake rates (156 +/- 18 and 35.9 +/- 3.3) compared with 37 degrees C (570 +/- 54 and 364 +/- 26) at 30 and 60 min, respectively. At 37 degrees C, ryanodine (300 microM), which was used to block Ca(2+) release from the calcium release channel, prevented the time-dependent decrease in Ca(2+) uptake. A general inactivation (P < 0.05) of maximal Ca(2+)-ATPase activity (V(max)) in CON was observed with incubation time (0 > 30 > 60 min), with the effect being more pronounced (P < 0.05) at 41 degrees C compared with 37 degrees C. The Hill slope, a measure of co-operativity, and the pCa(50), the cytosolic Ca(2+) concentration required for half-maximal activation of Ca(2+)-ATPase activity, decreased (P < 0.05) at 41 degrees C only. Treatment with DTT attenuated the alterations in enzyme kinetics. The increase in V(max) with the Ca(2+) ionophore A-23187 was less pronounced at 41 degrees C compared with 37 degrees C. It is concluded that exposure of homogenates to a temperature typically experienced in exercise results in a reduction in the coupling ratio, which is mediated primarily by lower Ca(2+) uptake and occurs as a result of increases in membrane permeability to Ca(2+). Moreover, the decreases in Ca(2+)-ATPase kinetics in WG with sustained heat stress result from SH oxidation.     

Cryopreservation of organs by vitrification: perspectives and recent advances

The cryopreservation of organs became an active area of research in the 1950s as a result of the rediscovery of the cryoprotective properties of glycerol by Polge, Smith, and Parkes in 1949. Over the ensuing four decades of research in this area, the advantages of vitrification, or ice-free cryopreservation, have become apparent. To date, experimental attempts to apply vitrification methods to vascularized whole organs have been confined almost entirely to the rabbit kidney. Using techniques available as of 1997, it was possible to vitrify blood vessels and smaller systems with reasonable success, but not whole organs. Beginning in 1998, a series of novel advances involving the control of cryoprotectant toxicity, nucleation, crystal growth, and chilling injury began to provide the tools needed to achieve success. Based on these new findings, we were first able to show that an 8.4M solution (VMP) designed to prevent chilling injury at -22 degrees C was entirely non-toxic to rabbit kidneys when perfused at -3 degrees C and permitted perfusion-cooling to -22 degrees C with only mild additional damage. We next investigated the ability of the kidney to tolerate a 9.3M solution known as M22, which does not devitrify when warmed from below -150 degrees C at 1 degrees C/min. When M22 was added and removed at -22 degrees C, it was sometimes [corrected] fatal, but when it was perfused for 25min at -22 degrees C and washed out simultaneously with warming, postoperative renal function recovered fully. When kidneys loaded with M22 at -22 degrees C were further cooled to an average intrarenal temperature of about -45 degrees C (about halfway through the putative temperature zone of increasing vulnerability to chilling injury), all kidneys supported life after transplantation and returned creatinine values to baseline, though after a higher transient creatinine peak. However, medullary, papillary, and pelvic biopsies taken from kidneys perfused with M22 for 25min at -22 degrees C were found to devitrify when vitrified and rewarmed at 20 degrees C/min in a differential scanning calorimeter. It remains to be determined whether this devitrification is seriously damaging and whether it can be suppressed by improving cryoprotectant distribution to more weakly perfused regions of the kidney or by rewarming at higher rates. In conclusion, although the goal of organ vitrification remains elusive, the prospects for success have never been more promising.     

Ultrastructural and hormonal metabolic studies of rat liver maintained in vitro by perfusion at 30 degrees C and 37 degrees C: a time course study by TEM, SEM and RIA

Isolated rat liver perfusion system has been extensively used for metabolic and functional studies. Results derived from the application of this system may reflect true biochemical changes but they may also be associated with some structural changes. This study was undertaken to correlate the cytological changes and functional integrity of isolated rat liver perfused in vitro at normal physiological temperature (37 degrees C) and 30 degrees C, using a non-recirculating system. The livers were perfused for 3 hours with modified Ham's F10 culture medium supplemented with thyroxine hormone (T4). The hepatocyte structural integrity was studied by light microscopy, transmission and scanning electron microscopy. The triiodothyronine (T3) and T4 hormones in the perfusion medium and the effluent fractions were assessed by radioimmunoassay. The livers perfused at 30 degrees C remained morphologically intact at the ultrastructural level for 3 hours whilst at 37 degrees C, hepatocytes in the centrilobular zone exhibited marked structural alterations. The percentage of T4 uptake was significantly higher (P less than 0.01) in livers perfused at 30 degrees C (50.8 +/- 7.7% vs 38 +/- 7.7%, 37 degrees C), but the net T3 output (3.16 +/- 1.04 micrograms) and the conversion of T4 to T3 (4 +/- 0.62%) were significantly higher (P less than 0.001) in livers perfused at 37 degrees C in comparison to livers perfused at 30 degrees C (1.61 +/- 0.84 micrograms and 1.68 +/- 0.76%, respectively). In conclusion, at 30 degrees C the hepatic T4 uptake is not inhibited, but the rate of T4 to T3 conversion has decreased, additionally the livers remain morphologically well preserved throughout the experimental period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preserved reflex cutaneous vasodilation in cystic fibrosis does not include an enhanced nitric oxide-dependent mechanism.

In humans, vasoactive intestinal peptide (VIP) may play a role in reflex cutaneous vasodilation during body heating. We tested the hypothesis that the nitric oxide (NO)-dependent contribution to active vasodilation is enhanced in the skin of subjects with cystic fibrosis (CF), compensating for sparse levels of VIP. In 2 parallel protocols, microdialysis fibers were placed in the skin of 11 subjects with CF and 12 controls. Lactated Ringer was perfused at one microdialysis site and NG-nitro-L-arginine methyl ester (2.7 mg/ml) was perfused at a second microdialysis site. Skin blood flow was monitored over each site with laser-Doppler flowmetry. In protocol 1, local skin temperature was increased 0.5 degrees C every 5 s to 42 degrees C, and then it maintained at 42 degrees C for approximately 45 min. In protocol 2, subjects wore a tube-lined suit perfused with water at 50 degrees C, sufficient to increase oral temperature (Tor) 0.8 degrees C. Cutaneous vascular conductance (CVC) was calculated (flux/mean arterial pressure) and scaled as percent maximal CVC (sodium nitroprusside; 8.3 mg/ml). Vasodilation to local heating was similar between groups. The change (Delta%CVCmax) in CVC with NO synthase inhibition on the peak (9+/-3 vs. 12+/-5%CVCmax; P=0.6) and the plateau (45+/-3 vs. 35+/-5%CVCmax; P=0.1) phase of the skin blood flow response to local heating was similar in CF subjects and controls, respectively. Reflex cutaneous vasodilation increased CVC in CF subjects (58+/-4%CVCmax) and controls (53+/-4%CVCmax; P=0.37) and NO synthase inhibition attenuated CVC in subjects with CF (37+/-6%CVCmax) and controls (35+/-5%CVCmax; P=0.8) to a similar degree. Thus the preservation of cutaneous active vasodilation in subjects with CF is not associated with an enhanced NO-dependent vasodilation.     

Enzymes of adenylate metabolism and their role in hibernation of the white-tailed prairie dog, Cynomys leucurus

AMP deaminase (AMPD) and adenylate kinase (AK) were purified from skeletal muscle of the white-tailed prairie dog, Cynomus leucurus, and enzyme properties were assayed at temperatures characteristic of euthermia (37 degrees C) and hibernation (5 degrees C) to analyze their role in adenylate metabolism during hibernation. Total adenylates decreased in muscle of torpid individuals from 6.97 +/- 0. 31 to 4.66 +/- 0.58 micromol/g of wet weight due to a significant drop in ATP but ADP, AMP, IMP, and energy charge were unchanged. The affinity of prairie dog AMPD for AMP was not affected by temperature and did not differ from that of rabbit muscle AMPD, used for comparison. However, both prairie dog and rabbit AMPD showed much stronger inhibition by ions and GTP at 5 degrees C, versus 37 degrees C, and inhibition by inorganic phosphate, NH(4)Cl, and (NH(4))(2)SO(4) was much stronger at 5 degrees C for the prairie dog enzyme. Furthermore, ATP and ADP, which activated AMPD at 37 degrees C, were strong inhibitors of prairie dog AMPD at 5 degrees C, with I(50) values of 1 and 14 microM, respectively. ATP also inhibited rabbit AMPD at 5 degrees C (I(50) = 103 microM). Strong inhibition of AMPD at 5 degrees C by several effectors suggests that enzyme function is specifically suppressed in muscle of hibernating animals. By contrast, AK showed properties that would maintain or even enhance its function at low temperature. K(m) values for substrates (ATP, ADP, AMP) decreased with decreasing temperature, the change in K(m) ATP paralleling the decrease in muscle ATP concentration. AK inhibition by ions was also reduced at 5 degrees C. The data suggest that adenylate degradation via AMPD is blocked during hibernation but that AK maintains its function in stabilizing energy charge.     

The effects of atrial natriuretic peptide on whole-kidney and proximal straight tubular function in the rabbit

The mechanisms by which atrial natriuretic peptide (ANP) produces a diuresis and natriuresis remain unclear. It has been suggested that the major if not sole mediator of ANP's renal effects is a hemodynamically induced increase in glomerular filtration rate (GFR). Data from clearance studies in anesthetized rabbits demonstrate that ANP administration can produce a significant increase in absolute and percentage sodium excretion (42.0 +/- 5.9----64.6 +/- 10.2 mu eq/min, P less than 0.01, and 1.97 +/- 0.28----3.12 +/- 0.35%, P less than 0.001, respectively) without increasing GFR (16.8 +/- 2.1----16.1 +/- 2.5 cc/min, P greater than 0.30). The natriuresis occurred despite a fall in renal plasma flow (RPF) (56.7 +/- 7.0----44.5 +/- 9.4 cc/min, P less than 0.01), a rise in filtration fraction (0.33 +/- 0.01----0.46 +/- 0.05, P less than 0.01), and an unchanged filtered load of sodium (2.28 +/- 0.27----2.16 +/- 0.32 mu eq/min, P greater than 0.10). Isolated tubular microperfusion studies demonstrated that ANP, present as a 10(-9) M concentration in the solution bathing perfused proximal straight tubules (PST), did not affect fluid flux (Jv) (0.38 +/- 0.07----0.41 +/- 0.07 nl/mm/min, P greater than 0.30) or phosphate reabsorption (Jp) (1.50 +/- 0.5----1.38 +/- 0.36 pmole/mm/min, P greater than 0.50). When ANP was infused into rabbits prior to harvesting the PSTs for isolated tubular microperfusion and the results were compared to tubules taken from control animals, there was again no effect on Jv (0.37 +/- 0.05 vs 0.42 +/- 0.05 nl/mm/min, P greater than 0.50) or Jp (2.41 +/- 0.27 vs 2.42 +/- 0.44 pmole/mm/min, P greater than 0.90). These findings suggest that ANP can inhibit sodium transport without increasing whole-kidney GFR or RPF, but does not directly inhibit transport in the proximal straight tubule.     

Comparison of the effect of 3- and 5-day hypothermic perfusion of dog kidneys on metabolism of tissue slices

Hypothermic perfusion effectively preserves the viability of kidneys for 3 days. Long-term preservation (5 days or greater) has not been consistently obtained. In this study, the differences between kidneys perfused for 3 and 5 days were compared by determining the "integrated-metabolic" capabilities of tissue slices incubated in vitro at 30 degrees C. The "integrated-metabolic" parameters determined include (1) respiration rates, (2) cell volume regulation [total tissue water (TTW) and saccharide permeable space], (3) rate of reaccumulation of K+ and pumping of Na+, (4) maintenance of ATP concentrations, and (5) mitochondrial functions. Conditions that result in high and low concentrations of ATP following perfusion of kidneys for 5 days were also compared for effects on tissue slice metabolism. The results indicate that energy metabolism in tissue slices is well preserved under all conditions and times of perfusion of kidneys. This includes average respiration rates (315 +/- 50, 275 +/- 35, and 255 +/- 45 mumol O2/hr/g dry wt at 0, 3, and 5 days, respectively, mitochondrial function [respiratory control ratio (RCR) = 4.6, 4.0, and 4.1 for 0, 3, and 5 days, respectively], and steady-state concentration of ATP in slices after incubation (4.0 +/- 1.45, 3.9 +/- 1.28, and 3.3 +/- 0.81 mumol/g/dry wt, for 0, 3, and 5 days, respectively). The primary differences between 3- and 5-day perfused kidneys were the capability of the slices to regulate cell volume and reaccumulate K+. Slices from kidneys perfused for 3 days maintained the TTW at 3.8 kg/kg dry wt, a value similar to that of control tissue slices. However, slices from 5-day perfused kidneys remained swollen (TTW = 4.6 kg/kg dry wt). Also, slices from the 5-day perfused kidney pumped K+ at less than one-half the rate found in slices from control or 3-day preserved kidneys. No significant differences were apparent in the permeability properties of the tissue slices from kidneys perfused for 3 and 5 days to radiolabeled saccharides. The defects in membrane-linked transport functions, resulting from long-term kidney perfusion, were reduced in kidneys containing a high concentration of ATP. The results suggest that one factor which may limit successful preservation of kidneys is the increased membrane permeability (to electrolytes) which is partially prevented by maintaining elevated concentrations of tissue ATP during perfusion.     

Isolation and characteristics of scallop sarcoplasmic reticulum with calcium transport activity.

Sarcoplasmic reticulum with calcium transport activity has been isolated from the cross-striated adductor muscle of the scallop, which lives in cold (< or = 20 degrees C) sea water, by using pH 7.0 buffer solution both to homogenize the tissue and to sediment the membrane fraction. The yield of the preparation was 60-100 mg protein from 100 g of the scallop muscle. Ca(2+)-activated ATPase protein of about 100 kDa accounted for 40-50% of the protein preparation. The maximum activities of ATP-dependent, oxalate-facilitated calcium accumulation and Ca(2+)-ATPase were observed at a pH of about 7.0 and temperature of 20-30 degrees C, and their values were about 2 mumol Ca2+/mg of protein/min and about 3 mumol ATP hydrolysis/mg of protein/min, respectively. At 0 degree C, 10-20% of these activities was maintained, while at 37 degrees C, the activities were irreversibly lost. The Ca(2+)-ATPase activity was half-maximally activated at about 0.3 microM [Ca2+]. The ATPase activity exhibited non-Michaelian behavior with respect to ATP, with two different Km values of approximately 10 microM and 0.1-0.3 mM. GTP, CTP, and ITP were also hydrolyzed by the preparation at a rate of 10-30% of that of ATP. The preparation was stored at -80 degrees C with retention of function for about a year.     

Active ion transport in the renal proximal tubule. I. Transport and metabolic studies

Various aspects of the interrelationship between ion transport and cellular metabolism were investigated using a suspension of rabbit cortical tubules that were mainly proximal in nature. Using the intact tubules, the compartmentation of K within the renal cell was studied by performing 42K uptake studies. The oxygen consumption (QO2) of the tubules was measured under similar conditions, as well as when the Na pump was stimulated by increasing Na+ entry with nystatin. In addition, the state 3 rate of respiration was measured when the mitochondria of digitonin-permeabilized tubules were stimulated by ADP. At 37 and 25 degrees C, a single-compartmental uptake of 42K was observed, which suggests that extracellular K+ communicates with a single compartment within the renal cell. Between 37 and 15 degrees C, the ouabain- sensitive QO2 and the initial 42K uptake rate were parallel in an Arrhenius-type plot, which indicated that active ion transport and oxidative phosphorylation remain tightly coupled within this temperature range. At all temperatures between 37 and 15 degrees C, nystatin stimulated the QO2, which demonstrates that the entry of Na+ into the renal cells was rate limiting for active Na+ transport throughout this temperature range. Between 37 and 20 degrees C, the nystatin-stimulated QO2 was nearly equal to the state 3 rate of respiration, which suggests that active ion transport may be limited by ATP availability under these conditions. At 15 degrees C, nystatin addition stimulated the QO2 well below the state 3 respiratory rate.     

Mechanism for pressor response to nonexertional heating in the conscious rat

The purpose of this study was to determine the systemic hemodynamic mechanism(s) underlying the pressor response to nonexertional heat stress in the unrestrained conscious rat. After a 60-min control period [ambient temperature (Ta) 24 degrees C], male Sprague-Dawley rats (260-340 g) were exposed to a Ta of 42 degrees C until a colonic temperature (Tc) of 41 degrees C was attained. As Tc rose from control levels (38.1 +/- 0.1 degrees C) to 41 degrees C, mean arterial blood pressure (carotid artery catheter, n = 33) increased from 124 +/- 2 to 151 +/- 2 mmHg (P less than 0.05). During this period, heart rate increased (395 +/- 5 to 430 +/- 6 beats/min, P less than 0.05) and stroke volume remained unchanged. As a result, ascending aorta blood flow velocity (Doppler flow probe, n = 8), used as an index of cardiac output, did not change from control levels during heating, but there was a progressive Tc-dependent increase in systemic vascular resistance (+30% at end heating, P less than 0.05). This systemic vasoconstrictor response was associated with decreases in blood flow (-31 +/- 9 and -21 +/- 5%) and increases in vascular resistance (94 +/- 16 and 53 +/- 8%; all P less than 0.05) in the superior mesenteric and renal arteries (n = 8 each) and increases in plasma norepinephrine (303 +/- 37 to 1,237 +/- 262 pg/ml) and epinephrine (148 +/- 28 to 708 +/- 145 pg/ml) concentrations (n = 12, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of atrial natriuretic peptide, urodilatin, guanylin and uroguanylin in the isolated perfused rat kidney

Escherichia coli heat-stable enterotoxin (STa), guanylin and uroguanylin are novel natriuretic and kaliuretic peptides that bind to and activate membrane guanylate cyclase (GC) receptors such as GC-C and OK-GC that are expressed in the kidney and intestine. Atrial natriuretic peptide (ANP) and its renal form (urodilatin, UROD) elicit natriuretic effects by activation of a different membrane guanylate cyclase, GC-A. Experiments were done in perfused rat kidneys to search for possible synergistic interactions between ANP, UROD, guanylin and uroguanylin on renal function. Pretreatment with ANP (0.03 nM) enhanced guanylin (0.19 microM) natriuretic activity (%ENa(+); from 18.5+/-4.25 to 31.5+/-1.69, P<0.05, 120 min) and its kaliuretic activity (%EK(+); from 24.5+/-4.43 to 50.6+/-3.84, P<0.05, 120 min). Furthermore, ANP increased the natriuretic (29.05+/-3.00 to 37.8+/-2.95, P<0.05, 120 min) and kaliuretic (from 33.2+/-3.52 to 42.83+/-2.45, P<0.05, 120 min) responses of perfused kidneys treated with low-dose (0.06 microM) uroguanylin. In contrast, ANP clearly inhibited the uroguanylin-induced (0.31 microM) increase in %ENa(+) (from 35.9+/-2.37 to 14.8+/-1.93, P<0.05, 120 min), and in %EK(+) (from 51.0+/-4.43 to 38.8+/-3.61, P<0.05, 120 min). UROD (0.03 nM) also enhanced the guanylin-induced natriuresis (to %ENa(+)=31.0+/-1.93, P<0.05, 120 min) and kaliuresis (to %EK(+)=54.2+/-3.61, P<0.05, 120 min), and inhibited the %ENa(+) of uroguanylin (0.31 microM) to 17.9+/-1.67 as well as its %EK(+) to 24.3+/-3.13 (both at 120 min, P<0.05). The synergism between ANP and UROD with either guanylin or uroguanylin at sub-threshold doses and the unexpected antagonism between ANP and UROD with uroguanylin at a pharmacological dose point to possible interactions between natriuretic peptide receptor (NPR) and uroguanylin/guanylin receptor signaling pathways. The interactions herein described may play a contributory role in the regulation of kidney function in many pathophysiological states, such as in the saliuresis following ingestion of salty meals.     

Mechanisms of the early and late response of the kidney to contralateral nephrectomy.

The immediate (1 day, D1) and late (90 days, D90) effects of unilateral nephrectomy on contralateral renal hemodynamics, and the renal handling of electrolytes and water were investigated in the whole animal. The immediate and late ability of the remnant kidney to autoregulate perfusate flow and glomerular filtration rate (GFR) was studied in the isolated perfused kidney of the rat. In the whole animal, in D1 rats as compared to controls, GFR calculated for a single kidney increased from 0.85 +/- 0.3 to 1.1 +/- 0.2 ml/min (p less than 0.05). In D90 rats GFR increased further and was similar to prenephrectomy GFR (1.4 +/- 0.5 vs. 1.7 +/- 0.5 ml/min, p NS). Urinary prostanoid excretion in 24 h, calculated for one kidney, increased by 50-500% in D1 rats, but returned to prenephrectomy values in D90 rats. In the isolated perfused kidney, decreasing perfusion pressure (PP) from 100 to 70 mmHg did not change the renal vascular resistance (RVR) in control and D90 kidneys, but in D1 kidneys RVR decreased from 8.6 +/- 1.3 to 7 +/- 1.3 mm Hg/ml/min (p less than 0.05). In D90 kidneys RVR was significantly lower as compared to control and D1 kidneys at all perfusion pressures. Decreasing PP from 100 to 70 mm Hg resulted in a significant decrease in perfusion flow in control, D1 and D90 kidneys, while with the increase in PP from 100 to 130 mm Hg the perfusion flow increased significantly in all three kidney groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermographic studies of phantom and canine kidneys thawed by microwave radiation

Whole organs, such as kidneys, must be thawed quickly and uniformly to prevent damage during thawing due to excessive heating. Electromagnetic heating with microwaves thaws the kidneys quickly but frequently produces "hot spots" with heat damage. To study heat damage, phantom gelatin kidneys with different dielectric constants and canine kidneys perfused with 12.5% glycerol, ethylene glycol, or dimethyl sulfoxide before freezing were microwave thawed, and the interior temperature was measured by thermography. Phantom kidneys were thawed free standing and canine kidneys were either free standing or packed in a gel mixture. Both phantom and canine kidneys were split symmetrically and separated with a sheet of Styrofoam to facilitate immediate separation and evaluation of the halves after thawing (approximately 3 sec). All the phantoms, regardless of dielectric properties, had areas less than 0 degrees C or greater than 37 degrees C after thawing. The free-standing canine kidneys and the gel-packed ethylene glycol-perfused kidneys had frozen areas (less than 0 degrees C) and hot spots (greater than 37 degrees C). However, glycerol- and dimethyl sulfoxide-perfused kidneys packed in gel before thawing had no areas less than 0 degrees C or greater than 37 degrees C. Altering the geometry from a "kidney shape" to a cylindrical shape with increased volume improved the uniformity of thawing and was more effective than altering the dielectric constant over the range evaluated.