Ischemia-Reperfusion Compensatory renal growth and mitochondrial function : the influence of warm ischemia and reperfusion

Purpose: To evaluate the influence of ischemia/reperfusion injury on renal compensatory growth (CGR) and mitochondrial function. Methods: Forty five Wistar rats were divided in 3 groups: Control Group (GC) – 21 rats were submitted to a sham laparotomy and sacrificed at 1 (6 rats) and 7 (15 rats) postoperative days to evaluate the dry weight of both kidneys and their growth during 1 week (6 rats) and to quantify mitochondrial respiration (9 rats); Group 1 (G1) 12 rats underwent right nephrectomy and were sacrificed 7 days later for analysis of renal mitochondrial function (6 rats) and dry weight (6 rats). Group 2 (G2) – renal warm ischemia for 60 minutes followed by right nephrectomy was performed in 12 rats; they were sacrificed 7 days later to evaluate renal mitochondrial function (6 rats) and dry weight (6 rats). Results: Dry weight (mg) of left kidneys at 7 day: GC 219±18, G1 281±23 and G2 338±39 (GCxG1 p<0.01; GCxG2 p<0.001; G1xG2 p<0.01). State 4 mitochondrial respiration rate and respiratory control ratio (RCR) were similar in all groups (p>0.05). State 3 respirations (mM/min/mg) in GC, G1 and G2 was respectively: 99±23, 132±22 and 82±44 (p<0.02; the only statistical difference noted was between groups G1xG2 – p<0.05). Conclusions: Following unilateral nephrectomy CRG is associated with an increase in state 3 of mitochondrial respiration. Renal ischemia/reperfusion injury enhances the CRG provoked by unilateral nephrectomy but such enhancement seems independent on mitochondrial respiration.


Introduction
3][4][5] The CRG following unilateral nephrectomy of the adult animal does not involve formation of new nephrons while CRG resulting from unilateral nephrectomy of fetuses during the period of neprogenesis seems to be associated with nephron endowment. 65 The beginning of renal transplantation programs arouses a new opportunity for studying CRG in association with renal ischemia/reperfusion injury.7][18][19] Subsequently, multiple enzyme systems including proteases, nitric oxide synthases, phospholipases and endonuclease are activated and responsible for cytoskeleton disruption, lipid peroxidation, membrane damage, and DNA degradation, and eventually cell death.][22][23][24] The aim of this research was to evaluate the influence of ischemia/reperfusion injury on renal compensatory growth (CGR) and mitochondrial function.

Methods
Forty five male Wistar rats, weighing 180-200g, were divided at random in 3 groups: Control group (GC) (renal weight) -21 rats were submitted to a sham laparotomy and sacrificed at 1 st (6 rats) and 7 th (15 rats) postoperative days to evaluate the dry weight of both kidneys and their normal growth during 1 week (6 rats) and to quantify mitochondrial function (9 rats).Group 1 -12 rats underwent right nephrectomy and sacrifice 7 days later.Mitochondrial function was studied in the left kidneys of 6 animals and the left renal dry weight was determined in the remaining 6 rats.Group 2the left renal artery of 12 rats was clamped to promote left renal warm ischemia for 60 minutes followed by right nephrectomy.At the 7 th postoperative day they were sacrificed to harvest the left kidney to evaluate mitochondrial function (6 rats) and the dry weight (6 rats).
For surgical procedures all rats were anesthetized by an intraperitoneal injection of thionembutal (50mg/kg).After harvesting the kidneys all animals were sacrificed by an overdose of anesthetic.
To determine the dry weight the kidneys were placed on a piece of aluminum foil previously weighed, and kept within a stove at 58ºC.The kidneys were weighed every 24 hours for the time required to achieving a steady weight, and afterward they were cooled at room temperature in a dryer vacuum chamber.To determine the final weight we subtracted the weight of the aluminum foil.
To extract renal mitochondria the kidneys were washed with cool saline (0.15M NaCl) in a Becker recipient right after the nephrectomy.The kidneys were homogenized (Potter-Ehlvejem homogenizer), 3 cycles of 3 minutes with an interval of 1 minute between the cycles, in 10ml of a solution containing sucrose (250mM), EGTA (1mM), BSA (0.2% of volume) and Tris-HCl (10mM), pH 7.2.The homogenate was centrifuged at 750G for 3 minutes.The supernatant containing the mitochondria was aspirated and centrifuged at 15,000G for 10 minutes.The pellet was suspended in the above mentioned buffer without EGTA, and centrifuged at 15,000G for 10 minutes.The pellet was suspended in 0.5ml of buffer without EGTA.All steps were carried out at 4ºC.The protein content of mitochondria suspension was quantified by the biuret method with addition of 1% collate. 24he mitochondrial respiratory function was measured immediately after isolation by polarographic analysis at 30ºC.Briefly, mitochondria (1 mg) were added to 1.4 ml of solution containing 250mM sucrose, 8.5mM KH 2 PO 4 , 10mM EDTA, 2mg/ml BSA and 10mM Tris-HCl, pH 7.4.Mitochondrial respiration was initiated by addition of succinate (5 mM final concentration) plus rotenone (2 µM), and oxidative phosphorylation was initiated by addition of 400 mM ADP. O 2 consumption recordings allowed the calculation of V3 [rate of state 3 (ADPstimulated) respiration], of V4 [rate of state 4 (non-ADPstimulated) respiration] and of the respiratory control ratio (RCR = V3/V4).The oxygen uptake of V3 and V4 was expressed in nmol oxygen/min/mg mitochondrial protein.
As controls of mitochondrial respiratory function we used the kidneys of 9 rats of the control group.
Data were analyzed by the software program GraphPad Prism 4. Continuous variables were compared by the two-tail one-way ANOVA and by the Tukey-Kramer multiple comparisons test.A p value <0.05 was set as significant.

Results
The dry weight of kidneys of normal rats from Control Group rose in the period of 7 days and there was no difference between right and left kidneys (Table 1).
The results of the dry weight of the left kidney at 7 th day following the surgery are displayed in the Figure 1.
The results of mitochondrial respiration rate are shown in Table 2.The only significant difference was in state 3 respiration rates.

Discussion
5]25,26 But, our demonstration that renal warm ischemia enhances the CRG due to unilateral nephrectomy was not reported previously.It is not clear how the ischemia/reperfusion injury stimulates the CRG or how and when such challenge influences the release of growth factors.
During the first days of CRG there is an increase in the number and or volume of mitochondria 27,28 which suggest that the kidney undergoing CRG requires a higher demand of energy. 29Thus, in such condition one could expect that mitochondria would consume more oxygen.This was confirmed by the increase of the mitochondrial state 3 respiration rates that we observed in Group 1 (unilateral nephrectomy).However, in Group 2 (unilateral nephrectomy plus ischemia/reperfusion of the remaining kidney) the changes of states 3 and 4 of mitochondrial respiration rates, as well as the RCR, did not reach statistical significance in comparison with the Control Group although they showed a tendency to be lower.The mitochondria from left kidneys of animals from Group 2 were challenged in two ways: 1 st ) renal ischemia/reperfusion injury that should decreases mitochondrial ATP generation and oxygen consume as reported elsewhere [16][17][18][19] , and 2 nd ) stimuli for undergoing CRG that should increase mitochondrial ATP generation and oxygen consume. 29As a net result at the 7 th day, Group 2 exhibited an enhancement of the renal growth that was not associated with an increase in mitochondria respiration.Thus, in Group 2 the CRG seemed to be not associated with higher mitochondrial respiration rates, which possibly means that a higher demand of energy or more oxygen consume does not take a central role on CRG.