Glibenclamide Effects on Renal Function and Histology after Acute Hemorrhage in Rats under Sevoflurane Anesthesia

Abstract

Introduction. Hypovolemia from hemorrhage evokes protective compensatory reactions, such as the renin-angiotensin system, which interferes in the clearance function and can lead to ischemia. This study was designed to evaluate the effects of glibenclamide, a K⁺_ATP channel blocker, on renal function and histology in rats in a state of hemorrhagic shock under sevoflurane anesthesia. Material and Methods. Twenty Wistar rats were randomized into two groups of 10 animals each (G1 and G2), only one of which (G2) received intravenous glibenclamide (1 μg.g⁻¹), 60 min before bleeding was begun. Both groups were anesthetized with sevoflurane and kept on spontaneous respiration with oxygen-air, while being bled of 30% of volemia in three stages with 10 min intervals. There was an evaluation of renal function—sodium para-aminohippurate and iothalamate clearances, filtration fraction, renal blood flow, renal vascular resistance—and renal histology. Renal function attributes were evaluated at three moments: M1 and M2, coinciding with the first and third stages of bleeding; and M3, 30 min after M2, when the animals were subjected to bilateral nephrectomy before being sacrificed. Results. Significant differences were found in para-aminohippurate clearance, G1 < G2, and higher renal vascular resistance values were observed in G1. Histological examination showed the greater vulnerability of kidneys exposed to sevoflurane alone (G1) with higher scores of vascular and tubular dilatation. There were vascular congestion and tubular vacuolization only in G1. Necrosis and signs of tubular regeneration did not differ in both groups. Conclusion. Treatment with glibenclamide attenuated acutely the renal histological changes after hemorrhage in rats under sevoflurane anesthesia.

Keywords:

• glyburide
• sevoflurane
• hemorrhage
• kidney
• rats

INTRODUCTION

Glibenclamide has been used for type II diabetes due to easy administration by once-daily oral dose and its proven efficacy in recently diagnosed patients. Nevertheless, scientific debate has been rekindled on its pertinence in clinical practice due to adverse cardiovascular effects.[1] Such questioning, already begun in the 1970s,[2] has gained momentum after recent studies on ischemic preconditioning (IPC) and the importance of K⁺_ATP channels in the sarcolemma and in cardiomyocyte mitochondria.[3] Glibenclamide, an antagonist of these channels, could contra-benefit IPC; even so, some laboratory studies have shown a possible relationship between its actions on K_ATP channels and effects on the inflammatory process.[4],[5] These channels can also be identified with specific properties in renal epithelial cells[6] justifying studies related to possible renal actions of different agents, including sevoflurane.[7],[8] An experimental rat model of ischemia in myocytes and K_ATP channel blockers in the sarcolemma and mitochondria (5-hydroxidecanoate and HMR-1098, respectively) concluded that sevoflurane acts on mitochondrial K_ATP channels. Also in rats, the main sevoflurane degradation product causes important fast changes at genetic expression, which could clarify the renal toxicity mechanism of these products.[9]

The renal mechanisms of the renin-angiotensin and sympathetic systems, which compensate hemorrhage, are responsible for the hormones and neurotransmitters that determine vasoconstriction and can lead to renal ischemia. The kidney has its own protector mechanisms, especially prostaglandins, which modulate vasoconstrictor effects of angiotensin II and noradrenaline.[10] Renal function alterations are more frequently due to the combination of several different small injuries to the organ than from a single agent. Thus, any preventive measures that add to and together provide a “protected” milieu should be stimulated and studied. Understanding already gained on preconditioning leads to more audacious experimental investigation designs, because the combination of different stress factors in the same experiment can help elucidate this extremely important mechanism of endogenous adaptation.[11]

The objective of this study was to observe possible alterations in renal function and histology considering two well-defined processes of organic aggression—acute hemorrhage and unfavorable genetic profile to sevoflurane—to study the effect of pre-treatment with glibenclamide.

MATERIAL AND METHODS

After approval from the Animal Experimentation Ethics Commission, College of Medicine of Botucatu, UNESP, twenty >250g Wistar rats were randomized into two groups of ten: G1 (i.e., rats anesthetized with sevoflurane and submitted to hemorrhage of 30% of the volemia) and G2 (i.e., rats anesthetized with sevoflurane, treated with glibenclamide by venous route, and also submitted to hemorrhage of 30% of the volemia). Rats were housed in a bell jar suitable for small animal inhalation anesthesia. Anesthesia was started with 4% sevoflurane (vaporizer, Ohmeda) with a total flow of 1L.min⁻¹ oxygen and 1L.min⁻¹ air. Once the animal could be manipulated, the sevoflurane concentration was reduced to 2.5% and administered by a mask non-rebreathing system under spontaneous respiration. Rectal temperature (T) was monitored by a rectal thermometer and maintained between 35.5°C and 37.5°C with a thermal blanket. The internal right jugular vein was dissected and cannulated with 24 GA venocath to maintain infusion of Ringer lactate solution (RL) (5mL. kg⁻¹ h⁻¹)[12] and administer sodium para-aminohippurate (PAH), sodium iothalamate (IOT),[13] and glibenclamide. Shortly after, the left carotid artery was dissected and cannulated with a 24 GA venocath to monitor mean arterial pressure (MAP) through the transducer of a Datex Engstron recorder (Finland). Immediately after internal jugular vein catheterization, both groups received a 1mg prime solution of PAH (20%, Sigma) and 0.5 mg IOT (70%, Mallinckrodt) in 0.5mL 0.9% NaCl solution infused in one minute. Then, continuous infusion was initiated for both agents: 1mg. h⁻¹ PAH and 0.25mg. h⁻¹ IOT in 0.9% NaCI solution till the end of the experiment using an Anne^® infusion pump (Abbott). Immediately after prime solution injection, G2 received glibenclamide (Glyburide^®, Sygma, 0.1%), 1 μg.g⁻¹. A sixty minute period of IOT and PAH infusion was allowed for both agents to reach plasmatic equilibrium. At this moment, bleeding was initiated in both G1 and G2 via carotid artery in three steps, 10 minutes apart, which corresponded to a total blood removal of 30% of the volemia. Volemia was estimated as 6% total animal body weight[14] (see Figure 1). The arterial blood collected underwent laboratory analysis for hematocrit (Ht) and for plasmatic PAH and IOT concentrations, by high performance liquid chromatography (HPLC).

Figure 1. Experimental sequence. Abbreviations: SEVO = start of the experiment with 4% sevoflurane, JC/CC = jugular vein/carotid artery cannulation, RL = Ringer lactate solution, PAH/IOT = PAH and IOT loading solution followed by continuous infusion, GLIB = glibenclamide in G2.

After each hemorrhagic episode, if animal's MAP dropped below 80mmHg, blood was replaced by RL at 1.6 mL kg⁻¹. After the last bleeding, the animals remained under anesthesia for another 30 minutes, then a blood sample was collected for laboratory analysis and they underwent bilateral nephrectomy and sacrifice with sodium pentobarbital. Both removed kidneys were longitudinally sectioned along their major axis and stored for histological analysis in Dubosque-Brazil solution (120 mL formol, 30 mL acetic acid, and 2g picric acid), where they remained for between 12h and 24h. Kidneys were then identified by code numbers so that the pathologist responsible for histological analysis was unable to relate the sample examined to the experimental animal.

Slices with fragments from both kidneys were prepared by fixing in paraffin and then staining with hematoxylin/eosin. Histology was evaluated and based on the following criteria: tubular dilation, vascular dilation, vascular congestion, tubular vacuolization, necrosis, and evidence of tubular regeneration. Tubular necrosis was diagnosed by identifying nuclear necrosis and cytoplasmatic debris. Findings were attributed scores corresponding to the lesion importance level: zero (0) absence of lesion, one (1) discreet lesions, two (2) moderate lesions, and three (3) intense lesions.

The experimental model followed methodology adequate for measuring renal clearance in small rodents with reduced urine volume after hemorrhage.[13] IOT and PAH concentrations (mg.mL⁻¹) were obtained from HPLC and clearances (C) for IOT and PAH (C_IOT and C_PAH), according to the Fick principle:

C_PAH estimated the effective renal plasma flow (ERFP), and C_IOT estimated the glomerular filtration rate (GFR). Filtration fraction (FF) was defined as GFR/ERFP (C_IOT/ C_PAH), renal blood flow (RBF) as ERFP/(1 – Ht), and renal vascular resistance (RVR) as MAP/RBF.

The attributes studied were hematocrit, mean arterial pressure, IOT clearance, PAH clearance, filtration fraction, renal blood flow, renal vascular resistance, and histological analysis of both kidneys. Studied moments were: M1, control and coinciding with first hemorrhage; M2, obtained 20 minutes after first hemorrhage and 10 minutes after the second, and coinciding with the third hemorrhage; and M3, immediately before nephrectomy and animal sacrifice.

Weight was analyzed by the Student's t test. Attributes in the animals evaluated over time (M1, M2, and M3) were analyzed by Profile Analysis. Histological variable values (0–3) in each kidney and comparison between the two groups were analyzed by the non-parametric Mann-Whitney test for independent groups. In all analyses, statistics were considered significant when p < 0.05.

RESULTS

Both groups were similar for weight (p > 0.50; see Table 1). G1 animals presented higher hematocrit values in M1, but over the experiment, both groups showed similar significant reductions (p < 0.05; see Table 1). Mean arterial pressure did not significantly differ between groups (p > 0.10), but there was a gradual drop in their values in M2 and M3 (p < 0.05; see Table 1). PAH clearance showed results significantly higher in G2 than in G1 in M2 and M3, diminished in M3 of G1, and increased through the experiment in G2 (p < 0.05; see Table 2). IOT clearance did not present differences between groups and between moments in G2, but there was a significant reduction in G1 in M3 (p < 0.05; see Table 2). Filtration fraction increased in the control group (p < 0.05 in M3) and was higher than G2 from M2 onwards (p < 0.05; see Table 2). Renal blood flow was not significantly different between groups, and moments and renal vascular resistance did not change between moments, but G1 was higher than G2 in M2 and M3 (p < 0.05; see Table 2).

Table 1 Baseline values (M1, M2, and M3) in control (G1) and glibenclamide (G2) groups

Parameter	Control (n = 10)	Glibenclamide (n = 10)
W (g)	346 ± 81	338 ± 62
MAP (mmHg)	96 ± 25	89 ± 20
	89 ± 20	79 ± 21
	75 ± 20	73 ± 15
Ht (%)	41 ± 2	37 ± 5
	36 ± 3	34 ± 4
	33 ± 3	31 ± 3

Values are means ± SE; n is number of animals.

Abbreviations: W = weight, MAP = mean arterial pressure, Ht = hematocrit.

*p < 0.05 vs M1.

Table 2 Renal function values (M1, M2, and M3) in control (G1) and glibenclamide (G2) groups

Parameter	Control (n = 10)	Glibenclamide (n = 10)
CIOT (mL.min^−1)	0.992 ± 0.050	1.020 ± 0.008
	1.002 ± 0.042	1.020 ± 0.050
	0.924 ± 0.169	1.000 ± 0.050
CPAH (mL.min^−1.g^−1)^†	0.068 ± 0.024	0.083 ± 0.024
	0.067 ± 0.026	0.096 ± 0.016
	0.054 ± 0.023	0.096 ± 0.016
FF^†	0.052 ± 0.030	0.042 ± 0.021
	0.053 ± 0.029	0.032 ± 0.003
	0.060 ± 0.030	0.032 ± 0.001
RBF (mL.min^−1.g^−1)	0.114 ± 0.040	0.132 ± 0.039
	0.106 ± 0.042	0.146 ± 0.024
	0.080 ± 0.035	0.140 ± 0.023
RVR (mmHg.min^−1.mL^−1)^‡	3.22 ± 2.57	2.27 ± 1.30
	3.10 ± 2.06	1.64 ± 0.40
	3.44 ± 2.25	1.58 ± 0.35

Values are means ± SE; n is number of animals.

Abbreviations: C_IOT = clearance of sodium iothalamate, C_PAH = clearance of sodium para-aminohippurate, FF = filtration fraction, RBF = renal blood flow, RVR = renal vascular resistance.

*p < 0.05 vs. M2;

^†p < 0.05 G1 < G2 in M2 and M3;

^‡p < 0.05 G1 > G2 in M2 and M3.

Both groups showed statistical differences in four (p < 0.05; see Table 3) out of the six histological attributes and G2 had less repercussion. Only the presence of necrosis and tubular regeneration did not differ in both groups (p > 0.10). Despite the similar stress in G1 and G2, the presence of tubular dilation was discreet in the kidneys of G2 (p < 0.05) (see Figure 2). In the renal vasculature examination, G2 did not present histological changes, differentiating it from G1, which showed evidence of discreet and moderate vascular dilation and congestion. G2 showed signs of tubular vacuolization in only three animals (see Figure 3). Cellular necrosis intensity in G2 was discreet in one animal, moderate in two, and intense in only one. Also, two control group animals presented signs of intense necrosis; however, moderate and discreet necrotic lesions were detected in six out of the ten animals examined in this group. Evidence of tubular regeneration was low in both groups.

Figure 2. Dilated tubules in the renal cortex of rats submitted to hemorrhage of 30% of volemia.

Figure 3. Cytoplasmic vacuolization of tubules in the renal cortex of rats submitted to hemorrhage of 30% of volemia.

Table 3 Data of renal histological analysis of G1 and G2 groups with the sum of scores of left and right kidneys

G1							G2
Rat	DT	DV	VC	TV	N	R	Rat	DT	DV	VC	TV	N	R
1	4+	2+	2+	2+	1+	1+	1	1+	0	0	0	3+	1+
2	4+	2+	1+	2+	6+	1+	2	2+	0	0	0	2+	0
3	2+	2+	3+	0	2+	2+	3	2+	0	0	0	0	0
4	5+	3+	2+	2+	0	0	4	2+	0	0	0	0	0
5	3+	2+	0	0	0	0	5	2+	0	0	0	0	0
6	3+	3+	2+	1+	1+	0	6	2+	0	0	2+	5+	1+
7	3+	2+	2+	2+	3+	0	7	0	0	0	1+	0	0
8	2+	2+	0	2+	4+	0	8	1+	0	0	1+	3+	0
9	3+	1+	0	1+	3+	0	9	1+	0	0	0	0	0
10	4+	4+	0	2+	5+	1+	10	0	0	0	0	0	0

Scores (+) = 1−2, discreet; 3−4, moderate; 5−6, intense.

Abbreviations: DT = dilated tubules, DV = dilated vessels, VC = vascular congestion, TV = tubular vacuolization, N = necrosis, R = regeneration.

*p < 0.05 and G1 > G2.

DISCUSSION

Controlling animal body temperature avoided hypothermia, which could be a renal protection factor in reperfusion situations, and hyperthermia, which can help worsen renal lesion possibly under ischemia conditions by altering ATP availability and promoting an increase in oxygen free radicals.[15] Reduced hematocrit could be attributed to the amount of solution utilized.

Arterial hypotension seen throughout the experiment was caused by hemorrhage. The activation of K⁺_ATP channels in arterioles has been highlighted as an important mechanism in arterial hypotension in states of shock from vasodilation.[16] In pigs, improved systemic arterial pressure after intravenous bolus injection of glibenclamide (10 μg.g⁻¹) reflected improved renal blood flow and higher renal cortical ATP concentration.[17] The physiological antagonism between nitric oxide and angiotensin II, which has already been demonstrated in rats[18] and later in pigs, shows an association between activated K⁺_ATP channels and nitric oxide.[19] The recovery of mean arterial blood pressure in hemorrhagic shock treated with K⁺_ATP channel inhibitors has already been reported in rats[20] but was not seen under the acute conditions of this study.

The greater PAH clearance seen in G2, without significant alterations in renal blood flow, could reflect possible afferent arteriole dilatation and a discreet increase in efferent arteriole resistance. These responses could maintain glomerular filtration rate, leaving renal vascular resistance stable until the end of the procedure. G2 filtration fraction was lower, which was consistent with the increased effective renal blood flow being proportionally higher than glomerular filtration rate, compared to the same absolute values in the control group.

Research[21] with ischemic preparations in isolated rat kidney with glibenclamide and diazoxide (separately and simultaneously) added to perfusate showed a significant reduction in renal vascular resistance only with glibenclamide, without changing glomerular filtration rate in relation to control. Preparations in isolated organs present an important bias when neutrophils are not used in the perfusate, as one of the possible mechanisms of glibenclamide is in neutrophil K⁺ _ATP channels.

Oliguria could be the first intraoperative sign of renal hypoperfusion, as a compensatory mechanism for acute hypovolemia. About 60 minutes after its initiation, signs of acute tubular necrosis could be seen if the factors that triggered it persist, especially in patients with previous renal disease or with concomitant exposure to exogenous nephrotoxic agents. Signs of proximal tubule lesions occur much earlier in experimental models—after a few minutes of ischemia.[22]

One study[21] used ischemia preparations in isolated rat kidney with glibenclamide added to perfusate, quantified histological parameters, and compared them to controls (without ischemia or added drugs). The histological analysis revealed renal changes in preparations using glibenclamide, mainly in the ascending limb of Henle's loop, but these were modest.

Another study[5] with real ischemia (45 min) in Wistar rats reported that glibenclamide inhibited the increase of vascular permeability associated with reperfusion (renal and pulmonary) and led to the appearance of inflammation signs (neutrophil and TNF-α accumulation). The suppressor effect of hypotension normally occurring at the beginning of reperfusion was not seen, but renal architecture and cytology were preserved, along with discreet vascular congestion. Our histology exam showed more consistent results than the functional findings, although the glibenclamide pre-treated group had presented a renal function behavior more favorable towards protection. Engbersen et al.,[6] in epithelial cell preparations from proximal tubules, also concluded that glibenclamide reduces renal lesions induced by hypoxia. They highlighted, however, the importance of keeping to the correct dose (10μM, in isolated preparation).

A study of ischemia and pharmacological preconditioning in rats,[23] comparing control, sham, ischemia, and sevoflurane groups and analyzing renal function and histology, concluded that there was not pharmacological (sevoflurane) or ischemic preconditioning.[24] The lesions reported in preconditioning preparations were not related to the nephrotoxic action of sevoflurane because they did not use a CO₂ absorber or high concentrations of sevoflurane over a prolonged period (the rat hepatic enzyme system produces nephrotoxic metabolites).[9]

Research on pharmacological inflammation induction in rats demonstrated reduced neutrophil exudation and migration after glibenclamide, characterizing possible renal anti-inflammatory action.[4] Solez et al.[25] were the first to determine the presence of leukocyturia due to vascular congestion in renal medulla, showing its importance in triggering acute renal failure. Considering the complete absence of vascular congestion in the histological findings in G2 of this study, as well as the significant difference with control, it is evident that glibenclamide collaborates in the process, which reinforces the hypothesis on its predominant renal protector action as a polymorphonuclear migration blocker. One must not forget that leukocytes are activated locally by different factors, including cytokines and oxygen free radicals. Renal tubule cells can produce pro-inflammatory cytokines including TNF-α and IL-6.[7] The use of normal and genetically modified mice manipulated as non ICAM-1 producers,[26] an important endothelial adhesion molecule, can show that the absence of ICAM-1 promotes renal protection. This fact would suggest that ICAM-1 is one of the mediators of acute renal failure and potentializes interaction routes between neutrophils and the endothelium.

The abrupt reduction in glomerular filtration rate can determine renal dysfunction as a result of injury, either in the surrounding tubules, the glomerular vasculature, or in both simultaneously. Pre-glomerular vasomotor change and consequent renal lesion are well known.[27] The actual importance of this mechanism as the main cause of renal dysfunction states is questionable,[7] mainly when considering the distribution and consumption of oxygen in renal tissue (low medullar flow). Endothelial mechanisms, such as the expression of cellular adhesion, leukocyte activation, and other substances in renal tubules, can trigger and perpetuate blood flow obstruction with consequences for cellular metabolism, including their self-regulation of renal blood flow.[28]

In the final analysis of these results, considering the interaction between functional attributes and histology, one can suppose that other mechanisms exist in addition to pre-glomerular regulation of renal flow, which would help the histologically verified acute renal protection given by glibenclamide. There are many protection mechanisms interacting with each other. Glibenclamide, as an intervention agent, also works in other sites, antagonizing thromboxane A2 receptors and inhibiting Na+/K+ ATPase.[29] The understanding of the interactive nature of tissue protection mechanisms (cellular, inflammatory, endothelial, tubular, etc.) is still imprecise and of difficult distinction. In conclusion, after hemorrhage in rats under sevoflurane anesthesia, glibenclamide presented more acute signs of renal protection by histological than functional analysis. This action in renal protection processes could be by a still not well-established mechanism that requires further studies to be corroborated.

ACKNOWLEDGMENT

The authors are grateful to Maria Luiza Cassetari for the technical advice.

Research developed at Experimental Laboratory of Department of Anesthesiology, College of Medicine of Botucatu, UNESP—São Paulo State University, SP, Brazil.