A temporal study of gene expression in rat lung following fixed-volume hemorrhage.

Previous studies have indicated that hemorrhage may predispose the lung to respiratory distress syndrome. Gene expression profiling with oligonucleotide microarrays was used to evaluate the genetic responses of the lung to hemorrhage. Conscious rats, chronically instrumented with a catheter and telemetry device to record blood pressure, heart rate, and temperature, had 40% of their estimated blood volume removed at a rate of 1 ml/min over 7-10 min. Groups of three or more rats were euthanized at 1, 3, 6, 16, 24, 48, or 72 h following hemorrhage. Two additional groups were unmanipulated controls and instrumented animals with sham hemorrhage. Total RNA was isolated from lung, reverse-transcribed to cDNA, fluorescently labeled, and hybridized to oligonucleotide microarrays probing 5,671 rat genes. After hemorrhage, statistically detectable alteration of expression was seen in approximately 0.8% of the genes at some time during the 72-h test period (vs. sham hemorrhage) as determined by false discovery rate statistics in the statistical analysis of microarrays program. A subset was confirmed by RT-PCR analysis. Hemorrhage influenced genes that regulate intracellular signaling and structure, growth factors, and hormonal receptors. There also appeared to be increased expression of genes that may mediate sequestration of neutrophils and mononuclear cells from the circulation. This hemorrhage model, although producing severe hemodynamic alterations, avoided mortality and histological evidence of lung damage, a feature intended to help ensure reliable evaluation of gene expression. These results indicate that gene expression profiling with microarrays provides a new tool for exploring the response of a tissue to systemic blood loss.

Hemorrhage and renal ischemia-reperfusion upregulates the epidermal growth factor receptor in rabbit duodenum.

To study the role of EGF-R in small intestinal adaptation to hemorrhage and I/R, anesthetized rabbits were implanted aseptically with arterial and venous catheters and bilateral renal artery Doppler flow probes and silastic occluders and allowed to recover. Rabbits were then randomly assigned to one of six groups: time control; hemorrhage (22.5 mL/kg) and 2.5 hours of renal occlusion (hemorrhage plus I/R); hemorrhage plus I/R and 2:1 LRS resuscitation; hemorrhage plus I/R and 3:1 LRS resuscitation; hemorrhage alone; or I/R alone. Rabbits were killed 48 hours after hemorrhage, and a section of duodenum was collected for analysis. Hemorrhage plus I/R induced a 2.5-fold increase in EGF-R tyrosine kinase activity compared with that found in the control group (P < .05), and this effect was not modified by either LRS resuscitation regimen. This increased activity was associated with similar Increases in EGF-R protein concentrations and approximately a 50% increase in EGF-R messenger (m)RNA levels compared with levels found in the control group. Further analysis of possible regulatory mechanisms for the increased EGF-R expression after hemorrhage plus I/R detected higher levels of EGF-R phosphorylation compared with those found in the control group but no significant increases in transforming growth factor-alpha mRNA levels. These data, coupled with a significant increase in duodenal thlobarbituric acid-reactive substance concentrations from rabbits in the hemorrhage plus I/R group, support the hypothesis that tyrosine kinase signal transduction pathways involving the EGF-R are activated in the small intestine after hemorrhage, renal I/R, or both, and this process may be mediated, at least in part, by oxidant stress.

The renal effects of 7.5% NaCl-6% dextran-70 versus lactated Ringer's resuscitation of hemorrhage in dehydrated sheep.

A small volume of 7.5% NaCl/6% Dextran-70 (HSD) can rapidly expand the plasma volume, but concerns exist regarding its adverse effects on renal function in the dehydrated state. Sheep were thirsted for 4 days (13% plasma volume contraction), and subjected to a fixed-pressure shock model (mean arterial pressure of 50 mmHg for 2 h), followed by resuscitation with either HSD (4 mL/kg) or lactated Ringer's solution (LR; 37 mL/kg). Mean arterial pressure was restored to 90%, cardiac output to 125% and 120%, and plasma volume to 78% and 72% of baseline in LR and HSD groups, respectively. Glomerular filtration rate improved to 100% of baseline following HSD compared with 82% following LR. No significant urinary 70,000 molecular weight dextran was observed, suggesting an intact renal glomerular membrane. These data suggest that small volume HSD resuscitation is effective, even with pre-existing dehydration. In addition, renal function is not compromised by HSD resuscitation of hemorrhaged, dehydrated animals.

Comparison of 7.5% NaCl/6% dextran-70 resuscitation of hemorrhage between euhydrated and dehydrated sheep.

7.5% NaCl/6% dextran-70 (HSD) has been shown to be an effective, small volume resuscitation fluid following hemorrhage (HEM) in euhydrated (E) sheep. However, there is controversy whether hypertonic solutions would be effective in dehydrated (D) animals. Therefore, we used two groups (E and 4 days D) of chronically instrumented ewes to evaluate the responses to HSD following HEM. All sheep were bled and maintained at 50 mmHg mean arterial pressure (MAP) for 2 h, then resuscitated with a 4 mL/kg bolus of HSD. Dehydration did not affect baseline MAP, heart rate (HR), or total peripheral resistance (TPR), whereas cardiac output (CO: E, 5.28 +/- .31; D, 4.00 +/- .31 L/min), stroke volume (SV: E, 61 +/- 4; D, 44 +/- 4 mL/beat), urine flow rate (V: E, .51 +/- .11; D, .34 +/- .07 mL/min), and sodium excretion (UNa V: E, 22 +/- 8; D, 6 +/- 2 microEq/min) were reduced and plasma sodium (PNa: E, 150 +/- 3; D, 157 +/- 2 mEq/L) and protein (PTP E, 7.8 +/- .1; D, 8.8 +/- .6 g/dL) were elevated. The only difference between groups during HEM were HR (E, 98 +/- 8; D, 150 +/- 17 bpm), SV (E, 31 +/- 3; D, 14 +/- 2), and TPR (E, 23 +/- 2; D, 32 +/- 3). Resuscitation with HSD restored MAP (E, 92 +/- 3; D, 92 +/- 2), CO (E, 6.2 +/- .3; D, 4.2 +/- .2), and TPR (E, 15 +/- 1; D, 23 +/- 1) to baseline values. SV was increased above hemorrhage values but was not restored to baseline values in either group (E, 50 +/- 7; D, 27 +/- 3). HR increased further following HSD (E, 143 +/- 11; D, 158 +/- 5). PNa was raised 10 and 16 mEq/L in the E and D sheep, respectively, following HSD infusion, but no adverse effects associated with elevated PNa were observed in either group. Thus, HSD was effective in restoring MAP, CO, and TPR to baseline values in D sheep but it was at the expense of a lower SV and a higher HR than in E sheep.

Measurement of glomerular filtration rate in ICU patients using 99mTc-DTPA and inulin.

Improved and reliable methods for assessing glomerular filtration rate (GFR) in intensive care patients are needed in light of known deficiencies using creatinine clearance. We compared simultaneous two-hour clearances of inulin (CIn), creatinine (CCr), and 99mTc-diethylenetriaminepentaacetic acid (CDTPA) in 18 medical or surgical intensive care patients (range, 49 to 92 years old) with blood urea nitrogen (BUN) levels greater than 17.9 mmol/liter (0.5 mg/ml), serum creatinine levels greater than 150 mumol/liter (0.02 mg/ml), or estimated Cockcroft clearance less than 60 ml/min. Patients had severe renal dysfunction with average GFR of 35 ml/min (range, 2 to 69 ml/min). CDTPA and CCr correlated significantly with CIn, although CDTPA tended to provide a closer approximation. Cockcroft clearance (32 +/- 4 ml/min) was grossly similar to CDTPA and CIn and correlated significantly, especially when weight was calculated using actual as opposed to ideal body weight. In a subset of 13 patients with CIn less than 30 ml/min, only CDTPA was significantly correlated with CIn. In patients in the intensive care unit, CDTPA provides a rapid, accurate, and inexpensive clinical assessment of GFR, even at very low GFRs.

Hypertonic saline/dextran improves renal function after hemorrhage in conscious swine.

This study was performed to determine whether resuscitation with a single bolus of 7.5% NaCl/6% Dextran 70 (hypertonic saline/Dextran, HSD) could restore renal function following hemorrhage. Chronically instrumented, conscious pigs were hemorrhaged 28 ml/kg. This level of hemorrhage reduced mean arterial pressure (MAP) and cardiac output (CO) to nearly half, renal blood flow (RBF) to approximately 25%, and glomerular filtration rate (GFR) and urine flow (V) to less than 10% of their initial values. A single, 4 ml/kg bolus injection of HSD increased MAP and RBF to approximately 80% of baseline values and restored CO and GFR to levels which were significantly different from control values. These improvements were sustained for 2 h with no further treatment. Urine flow transiently increased although not to pre-hemorrhage values, and then subsided. Plasma osmolality increased from 275 to 282 mOsm/kg H2O, and plasma sodium increased from 141 to 149 mEq/l. Recovery following administration of an equal volume of normal saline was significantly less for all variables. Euvolemic animals showed no response in MAP, CO, RBF, or GFR when treated with HSD although V, osmotic and sodium excretion increased. These results demonstrate that resuscitation with HSD following hemorrhage not only restores MAP and CO, but maintains renal function as well.

Effect of hydration state on renal responses to head-out water immersion in conscious dogs.

Renal responses to head-out water immersion (WI) (37 degrees C, WI 100 min) were studied in conscious, instrumented dogs during volume repletion (R), when all blood and urine losses were replaced with 0.9% NaCl, or without volume repletion (NR), to determine the influence of hydration state. The lithium clearance method was used to estimate the locus of the renal tubular fractional sodium excretion (FENa) responses. WI in the R condition increased urine flow (V) from 0.9 (+/- 0.1 SE) to 4.2 (+/- 0.6) ml/min and FENa from 0.7 (+/- 0.1) to 3.2 (+/- 0.8)%. Fractional proximal sodium reabsorption (FPRNa) decreased from 0.82 (+/- 0.03) to 0.69 (+/- 0.1)% and fractional distal sodium reabsorption decreased from 0.96 (+/- 0.01) to 0.88 (+/- 0.04)%. By comparison, WI in the NR condition elicited smaller increments in V and FENa, no change in FPRNa and a significant decrease of FDRNa from 0.97 (+/- 0.01) to 0.93 (+/- 0.01). Although there were quantitative differences in the renal responses in the R and NR conditions, there were similar increments in both arterial and atrial pressures as well as plasma atrial natriuretic peptide concentration; plasma arginine vasopressin was unaltered in either situation, and plasma renin activity was depressed in both conditions. Since plasma protein concentration was significantly lower during the R condition, the differing renal responses are probably related to differing levels of volume expansion in the R vs. NR condition.

Renal responses to graded hemorrhage in conscious pig.

We developed a conscious pig model with a chronically instrumented kidney to measure renal blood flow (RBF), glomerular filtration rate (GFR), and excretory functions during hemorrhage. Seven to 10 days before experimentation, pigs were splenectomized, arterial and venous catheters were implanted, an ultrasonic flow probe was placed on the renal artery, and a pyelostomy was performed for nonocclusively placing a ureteral catheter. Measurements were taken before hemorrhage, and at hemorrhage volumes of 7, 14, 21, and 28 ml/kg (equivalent to 10.5, 21, 31, and 42% of the estimated blood volume), or at corresponding time points for controls. RBF was decreased by 30% when 21% of the blood (14 mg/kg) was removed, before arterial pressure, GFR, or urine flow or excretion was changed. At volumes of hemorrhage greater than 14 ml/kg, there were progressive decreases in RBF, GFR, urine flow rate, osmotic and electrolyte excretion, and arterial pressure. Thus pigs, like humans, respond to hypovolemia with an early redistribution of blood flow away from the kidney.

Clearance and urinary excretion of vasopressin in conscious dogs.

The purpose of this study was to determine the relationship between plasma arginine vasopressin (PAVP) levels and the urinary clearance rate, the nonurinary plasma clearance rate, and the urinary excretion rate of AVP. Female conscious dogs were given an intravenous infusion of p-aminohippuric acid and inulin, and, after a 90-min control period, the infusion was either continued or AVP was added to the infusate to produce doses of 0, 40, 100, and 200 microU.kg-1.min-1 for the last 90 min of the experiment. The resulting PAVP levels were 1.2, 8.2, 11.4, and 21.6 microU/ml, respectively. The urinary clearance rate of AVP nearly doubled between the infusion rates of 0 and 100 microU.kg-1.min-1. Likewise, the nonurinary plasma clearance rate of AVP also nearly doubled between the infusion rates of 40 and 100 microU.kg-1.min-1. However, at the rate of infusion of 200 microU.kg-1.min-1, both the urinary and nonurinary clearance rates were unchanged compared with the 100-microU.kg-1.min-1 rate. The largest incremental rise in PAVP was observed between the doses of 100 and 200 microU.kg-1.min-1, when the clearance rates were apparently plateaued. The urinary excretion rate of AVP was linearly correlated with PAVP (R = 0.91) under the conditions of this study. The results suggest that the measurement of the urinary excretion rate of AVP is a reliable method of assessing PAVP and that the clearance of AVP increases when PAVP is elevated.