ACUTE RENAL ISCHEMIA MODEL IN DOGS: EFFECTS OF METOPROLOL

Abstract

Introduction: To study the functional and hystological alterations in dog kidneys submitted to total ischemia for thirty minutes and the possible metoprolol protective action. Material and methods: Sixteen dogs anesthetized with sodium pentobarbital (SP) were studied and divided into two groups: G1–8 dogs submitted to left nephrectomy and right renal artery clamping for thirty minutes, and G2–8 dogs submitted to the same procedures of G1 and to the administration of 0.5 mg.kg⁻¹ metoprolol before ischemia. Attributes of renal function were studied. Results: There was acute tubular necrosis and a decrease ofrenal blood flow and glomerular filtration, and a increase of renal vascular resistance in both groups. Conclusion: The thirty minute renal ischemia appears to have determined the alterations found in the renal function and hystology in both groups. Metoprolol, used in G2, as to the time and dose applied didn't protect the kidney from the ischemic episode.

• Experimental renal ischemia
• Renal protection
• Metoprolol

INTRODUCTION

The acute renal failure, usually referred to as acute tubular necrosis, can be defined as an abrupt and sustained decline in glomerular filtration, caused by acute ischemic or toxic insults.

During the anesthesia procedure, there are many clinical situations that promote low renal perfusion, such as the diminished arterial pressure and the surgical technique, when it imposes not only low renal perfusion but also complete absence of that perfusion.

We created an experimental model of acute renal ischemia to simulate those clinical situations and to study the physiological and hystological changes of kidneys submitted to 30 minutes of total ischemia. We also administered metoprolol in this experimental model of acute ischemia. Metoprolol blocks β₁ receptors which can be involved with renin release [1-5]. The renin release by the ischemia would be one of the responsible factors of the postischemia acute renal failure development.

MATERIAL AND METHODS

We had the approval of the Ethics Committee of Animal Research of our institution. Anesthesia was induced in sixteen healthy mongrel dogs (9 to 18 kg) with SP 30 mg.kg⁻¹ and maintained with 5 mg.kg⁻¹. The animals were randomly divided in two groups of eight each.

Group 1 (G1): median laparotomy, right nephrectomy, and left renal artery clamping during 30 minutes.

Group 2 (G2): same G1 procedures and venous administration of 0.5 mg.kg⁻¹ metoprolol just before the left renal artery clamping.

A cuffed endotracheal tube was inserted, and animals received pancuronium bromide (0.08 mg.kg⁻¹ in the first dose and 0.03 mg.kg⁻¹ for maintenance). Ventilation was controlled by a K. Takaoka model 660 apparatus with air. Left and right femoral veins were catheterized for fluid infusiom, drug administration, blood collection, and inferior vena cava pressure (ICP) measurement. Catheterization of the left femoral artery was carried out for mean arterial pressure (MAP) measurements (Hg manometer). Through a median laparotomy, right nephrectomy was performed and the left renal artery was identified and individualized for posterior clamping. The left ureteral catheterization was performed.

The animals received Ringer's solution (0.4 mL.kg⁻¹.min⁻¹—extracellular volumeexpansion) for 30 minutes, followed by a prime dose of 30 mg.kg⁻¹ creatinine and 4 mg.kg⁻¹ para-aminohippuric acid (PAH). Immediately afterward, a solution of 0.15% creatinine and 0.06% PAH in Ringer's (0.6 mg.kg⁻¹.min⁻¹ creatinine and 0.24 mg.kg⁻¹.min⁻¹ PAH) was administered by continuous drip infusion until the end of the experiment. There were four clearance periods, each lasting 15 minutes.

Thirty minutes after prime dose, the first clearance period began with urinary output (V) measurement, venous blood collection, and determinations of MAP, ICP, and heart rate (HR). After the first clearance period end, 0.5 mg.kg⁻¹ metoprolol was administered in G2. The left renal artery clamping was carried out during 30 minutes: in G1 immediately after the first clearance period, and in G2 immediately after metoprolol dose. After the renal artery unclamping, the second clearance period started. The third clearance period began immediately after the end of the second, and the fourth started immediately after the end of the third. The urine was collected during all the clearance periods. Blood samples were collected in the middle of each period. The mean values of the readings (HR, MAP, ICP) were calculated from those obtained at the beginning and end of each clearance period. The following attributes were recorded:

• Hemodynamics: HR, MAP, ICP, PAH clearance (C_PAH), renal blood flow (RBF), and renal vascular resistance (RVR)

• Blood: hematocrit (Ht) and plasma osmolality (P_osm)

• Renal function: creatinine clearance (C_cr), filtration fraction (FF), V, urinary osmolality (U_osm), osmolar clearance (C_osm), free water clearance (C_H2O), sodium (Na) and potassium (K) clearance (C_Na and C_K), urinary excretion (U_NaV and U_KV), and fractional excretion (FE_Na and FE_K)

• Rectal temperature (T)

• Left kidney hystological study

The attributes were studied at the following times:

• M1, control time for each animal obtained 30 minutes after the prime dose of creatinine and PAH

• M2, M3, and M4, obtained immediately, 15, and 30 minutes after the left renal artery unclamping

In G2, M2, M3, and M4 were obtained thirty, fourty-five, and sixty minutes after metoprolol administration.

All serum and urine samples were later assayed for creatinine and PAH concentrations using colorimetric techniques. Osmolality was obtained through cryoscopic lowering. Na and K levels were determined using a flame photometer with oxyacetylene.

From this information, the following were derived: clearance (C) = U.V/P, being U and P the urine and plasma substance concentration (mg.mL⁻¹); FF = C_cr/C_PAH; C_osm = U_osm.V/P_osm; C_H2O = V − C_osm; U_{Na or K} V = U_{Na or K.} V (μEq.min⁻¹); FE = C_{Na or K}/C_cr.100 (%); RBF = C_PAH/(1−Ht) (mg.mL⁻¹); and RVR = MAP/RBF (mmHg.mL⁻¹.min⁻¹).

Statistical analysis was carried out using the profile analysis. A value of p < 0.05 was considered statistically significant. The prefixed ∝ was 0.05.

For a brief description of the variables, the following were elaborated: table with mean (x) and standard deviation (S), and variation coeficient (VC) in each group and in the different moments in which the measurements were done.

The Profile Analysis was utilized for each variable with the hypothesis tests:

• HO₁—interaction between groups and moments. Similarity of the groups are verified.

• HO₂—difference between profiles. In case of group similarities the moment differences are verified.

• HO₃—difference among moments. In case of group similarities the moment mean differences for both groups are verified.

• HO₄—difference between groups in each moment separately.

• HO₅—difference among moments within each group separately.

The statistics were significant when p < 0.05. The prefixed ∝ was 0.05.

RESULTS

The kidney histological study of the animals of both groups, G1 and G2, showed acute tubular necrosis (ATN) with changeable intensity. The ATN was characterized by tubular apical brush border fragmentation with disappearance of cells structure and with cell detachment. There was proteic and hyaline intratubular material. Thetubular cells presented coagulated and necrotic cytoplasm with nuclear pyknosis and karyorrhexis. Degenerative phenomenon was observed in some tubules, which were characterized by cytoplasmatic vacuolization, and showing luminal impaction by numerous spherical and ovoid blebs with hyaline degeneration.

There was homogeneity among the results of HR, MAP, ICP, Ht, T, V, P_osm, C_Na, C_K, FF, Na and K UV, and FE of both groups. C_PAH, RBF, C_cr, U_osm, and C_osm presented the highest values in M1 before the renal artery clamping. RVR and C_H2O presented the lowest values in M1.

DISCUSSION

1. Experimental model

   If the renal function study is the aim of an experimental research study the dog is a good choice because this animal has similar behavior to that of human beings concerning this function. Otherwise, it is supposed that β₁ is the renin release receptor, both in humans and in dogs [[2]], [[4]], [[6]].

   Sodium pentobarbital was utilized for the anesthesia of both groups because some investigators have reported that with this barbiturate, cardiovascular conditions remain unchanged, as well as MAP and venous pressure [7-9]. For the renal ischemia experimental model, the hemodynamic stability is quite important, and we have obtained that in our study. The ICP and MAP remained unchanged during the experiment.

   Many investigators observed a slight increase in HR [[7]], [[9]] with SP use, which could be explained by a parasympatholytic [[10]] or sympathomimetic action [[11]], or by an arterial baroreceptor-mediated reflex origin [[12]].

   Initially, all dogs presented high HR, probably because of the SP action. In G2, where metoprolol was administered, there was a more accentuated HR decline.

   The heart has predominantly β₁ adrenergic receptors. The blocker effect of those receptors is mainly depressive—they diminish the formation of the sino-atrial node impulse—and therefore presents a negative chronotropic effect, reducing the HR. The metoprolol in G2 blocked the β₁ receptors and antagonized the amina action which acts on these receptors causing lower HR [[1]], [[6]].

2. Unilateral nephrectomy

   The unilateral nephrectomy of this experimental model of post-ischemia ARF was due to observation of the literature data, which indicate that unilateral nephrectomy determines faster recuperation of the renal failure and less ischemic injury. It is necessary a non-oliguric model if the objective is to study the renal function, and such procedure leads to non-oliguric ARF.

   In the literature, some works reported non-oliguric ARF in rats previously nephrectomized. The authors also demonstrated an early recuperation of the organ functionand less tubular injury. Oligury occurred in rats in which the contralateral healthy kidney was preserved [12-15].

   The humoral factors production, which stimulates the compensatory renal growth, is enhanced in nephrectomized rats, and accelerates the repair of the damaged cells. There was a higher RVR demonstrated in non-uninephrectomized rats [[13]].

   Some authors demonstrated that the unilateral nephrectomy results in natriuretic peptide release and diminished concentration of A2 tromboxane. Therefore, the established vasoconstriction is less intense, promoting the diuresis appearance, and consequently, facilitating the renal function study in those ischemic kidneys [16-18].

3. Hydration

   The liquids administration, in this experiment 0.4 mL.kg⁻¹.min⁻¹, propitiated the diuresis appearance. With the volume overload, there is an attenuation of the glomerular filtraton rate decline and less formation of detached cells, due to the imposed ischemia tothe dogs, which would obstruct the tubular lumen [[15]].

   The SP doesn't interfere in the renal function and the administration of the isotonic solution for hydration during the experiment determines the urinary output and sodium fractional excretion with increased values. In this study these results weren't obtained, probably due to imposed ischemiawhich determined stable values for these parameters [19-20].

4. Ischemia

   The RBF temporary interruption results in ARF the severity of which depends on the ischemia period imposed on the kidney. After five minutes of ischemia, the sphingomyelin and cholesterol contents diminish and enhance that of the phosphatidylcholine and phosphatidylinositol in the apical membrane [21-25].

   The enzyme Na⁺, K⁺-ATPase redistribution to the apical membrane occurs within ten minutes of ischemia. The Na⁺, K⁺ pump and ATPase enzyme localization normally is only on the basolateral membrane, which propitiates absorption of ions, solutes, and water from the lumen to the blood [21-22], [[26]].

   The redistribution that occurs during ischemia—the enzyme on the apical and basolateral membrane—leads to diminished efficacy of Na⁺ reabsorption, resulting in high natriuresis. These changes likely account for the disconnection of the ATP utilization and Na⁺ transport, resulting in ineffective use of the limited cell energy. In 15 minutes, all the proximal tubule shows these changes, and the tubular cell loses its polarity [21-25].

   When Na⁺, K⁺-ATPase becomes inactive, cells depolarize and there is increased intracellular sodium and chloride content. The cell takes up water, its cytoskeleton is disrupted and there is cell detachment. Therefore, according to the literature, we can assume that in 30 minutes of ischemia—the time we submitted our animals to—those changes could be found in both groups. Cell detachment contributes to obstruction of the tubular lumen and compromises the blood supply even more [[27]].

   The tubular obstruction increases the renal tubule pressure and the filtrate leaks out to the renal interstice [21-22]. Many authors have reported these changes [24-26], [28-34].

   The histopathological result of our study—30 minutes of total ischemia period—showed alterations similar to that reported in the literature, both in G1 and G2.

The renal physiologic alterations (increased RVR and decreased RBF and C_cr) found in this experimental work may be the result of the ischemia. The renin-angiotensin-aldosterone system would be activated. Renin and angiotensin, being potent vasoconstrictors, together with the release of other vasoconstrictors, would compromise the organ function even more.

The urinary output and sodium fractional excretion didn't change. In case of extracellular volume expansion, as it was done in this experiment, the kidneys increase the volume elimination, placing a multifactorial complex system. Pressoric natriuresis occurs due to the transmission of the systemic arterial pressure to the peritubular capillaries, increasing the capillary hydrostatic pressure. The oncotic pressure reduction associated with the hidrostatic capillary increase limits the fluid passage from the interstitial space to the capillary lumen, causing a higher fluid return to the tubular lumen with a consequent reduction of the sodium tubular reabsorption [[35]].

The atrial distension by the extracellular fluid expansion releases the atrial natriuretic factor, which increases the RBF, glomerular filtration rate (GFR), and FF. The inhibition of the antidiuretic hormone release and renin-angiotensin-aldeste-rone system, and some of the vasodilator prostaglandins by the extracellular volume expansion, determine the GFR increase, producing lower tubular reabsorption of sodium and water.

The total and temporary ischemia imposed on these kidneys commited the energetic offer to the tubular cell, preventing these physiological answers.

The activation of renal β₁ receptor is one of the mechanims proposed for renin release, both in humans and dogs [2-5]. The use of metoprolol in G2 would have blocked β₁ receptors and renin release. However, we did not observe any differences in the behavior of both experimental groups, probably due to the fact that renin release is also associated with other factors. The involvement of these vasoconstrictors, such as endothelin, prostaglandin PG₂∝, and tromboxane, is also important to the development of this syndrome.

CONCLUSION

The alterations in the renal hemodynamic (diminished RBF, C_PAH, and C_cr, and enhanced RVR) diuresis, FE_Na, and renal histology observed in the dog kidneys in this study were due to temporary and total renal ischemia. It probably caused a more accentuated action of the renal vasoconstrictor substances released during the ischemic episode.

Metoprolol, as utilized, did not significantly protect the function of the kidneys submitted to 30 minutes of total ischemia.

Figure 1. Experimental Sequence.

Table 1. Hemodynamic and Renal Function Attributes in G1 (control) and G2 (metoprolol)

	Moments	M1	M2	M3	M4	Commentary
CPAH	G1	3.51 ± 2.65	2.69 ± 2.50	1.69 ± 0.82	1.49 ± 1.18	G1: M1 ≥ M3 = M4
						M2 is interm 0.05 < p < 0.10
(mL.min^−1.kg^−1)	G2	4.36 ± 2.64	2.72 ± 1.14	2.25 ± 0.59	2.78 ± 1.43	G2: M1 > (M3 = M4)
						M2 is interm p < 0.05
FSR	G1	4.91 ± 3.60	3.74 ± 3.51	2.32 ± 1.13	2.04 ± 1.65	G1: M1 ≥ M3 = M4
						M2 is interm 0.05 < p < 0.10
(mL.min^−1.kg^−1)	G2	6.18 ± 4.02	3.78 ± 1.68	3.10 ± 0.91	3.84 ± 2.03	G2: M1 > (M3 = M4)
						M2 is interm p < 0.05
RVR	G1	3.36 ± 4.29	3.90 ± 4.27	4.41 ± 4.02	9.65 ± 11.39	G1: M1 < M2 < M3 < M4, p < 0.05
(mmHg.mL.min^−1)	G2	2.62 ± 1.61	3.05 ± 1.17	3.61 ± 1.30	3.80 ± 3.08	G2: M1 < M2 < M3 < M4, p < 0.10
Ccr	G1	1.28 ± 0.83	0.51 ± 0.43	0.45 ± 0.34	0.55 ± 0.46	G1: M1 = M2 = M3 = M4, p < 0.50
(mL.min^−1.kg^−1)	G2	2.90 ± 3.99	0.75 ± 0.76	0.62 ± 0.37	0.79 ± 0.55	G1: M1 ≥ (M2 = M3 = M4).
						0.05 < p < 0.10
Uosm	G1	435.25 ± 261.50	274.37 ± 115.14	222.12 ± 87.00	265.25 ± 81.00	G1: M1 > M2 = M3 = M4, p < 0.05
(mOsm.kgH2O^−1)	G2	453.37 ± 190.63	258.62 ± 35.13	275.75 ± 63.38	267.25 ± 62.17	G2: M1 > (M2 = M3 = M4), p < 0.05
Posm	G1	270.25 ± 17.94	266.37 ± 14.17	271.75 ± 14.20	270.12 ± 12.04	G1: M1 = M2 = M3 = M4, p > 0.10
(mOsm.kgH2O^−1)	G2	270.12 ± 12.04	277.62 ± 5.20	280.00 ± 6.34	289.00 ± 44.74	G2: M1 = M2 = M3 = M4, p > 0.10
Cosm	G1	0.068 ± 0.032	0.005 ± 0.049	0.047 ± 0.034	0.058 ± 0.045	G1: M1 = M2 = M3 = M4, p > 0.10
(mL.min^−1.kg^−1)	G2	012 ± 0.096	0.049 ± 0.043	0.058 ± 0.036	0.059 ± 0.036	G2: M1 > (M2 = M3 = M4), p < 0.05
CH2O	G1	−0.009 ± 0.047	0.0018 ± 0.027	0.0018 ± 0.027	0.021 ± 0.038	G1: (M1 = M2) < (M3 = M4), p < 0.05
(mL.min^−1.kg^−1)	G2	−0.046 ± 0.070	0.0095 ± 0.014	0.0095 ± 0.019	0.0062 ± 0.019	G2: M1 < (M2 = M3 = M4), p < 0.05
V	G1	0.053 ± 0.043	0.055 ± 0.043	0.066 ± 0.040	0.069 ± 0.040	G1: M1 = M2 = M3 = M4, P > 0.50
(mL.min^−1.kg^−1)	G2	0.074 ± 0.048	0.058 ± 0.054	0.067 ± 0.045	0.069 ± 0.049	G2: M1 = M2 = M3 = M4, p < 0.50
						p < 0.50
FENa	G1	6.37 ± 7.81	37.32 ± 68.27	48.24 ± 102.64	42.56 ± 84.27	G1: M1 = M2 = M3 = M4, p > 0.10
%	G2	7.12 ± 9.43	28.46 ± 62.85	14.22 ± 14.02	15.28 ± 18.65	G2: M1 = M2 = M3 = M4, p > 0.50.
						p < 0.10 p < 0.10