Ligation of the left renal vein in epml-wistar rats : functional and morphologic alterations in the kidneys , testes and suprarenal glands

Objective: The Iigation of the left renal vein (LLVR) in man is a contraversial procedure in view of the risks of lesion to the renal parenchyma. With the objecUve of studying the morphologic and functional alterations caused by these lesions, we conducted experimental research with rats. Material and Methods: 64 male adult EPM1-WISTAR rats were used, divided into 8 groups 4 for . LLRV and four for control. Each LLRV group and corresponding control group were sacrificed progressively on the 7th, 15th, 30th and 60th day after the initial surgery. Results: We found morphofunctional alterations only in animais that underwent LLRV in the four periods of sacrifice. The proteinuria creatinine in serum, testosterone in serum and serum corticosterone in serum showed practically no alteration in relation to the normal values for rats. Statistically significant severe histological lesions were found in the kidneys and testes of the LLRV groups. Lesions in the suprarenal glands were also presentin these groups, but no sufficient to demonstrate statistical significance Conclusion: Based on these results we can conclude that the Iigation of the left renal vein is a procedure of high risk in these animais.


INTRODUCTION
P requently, the surgeon finds himself in a difficult situatión in relation to normal human anatomy.Due to the syntropy of the elements of the human body, a good surgical approach to a certain organ or segment .of the body can sometimes cause damage to another element with significant repercussions.
The left renal vein (LRV) due to its syntropy with the abdominal aorta, may hamper an ideal approach to that artery.l

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José Carlos Costa Baptista-Silva Rua Prof. Artur Ramos, 178, apto 123 Vega São Paulo/SP -Brasil-CEP 01454-904 As the LRV has many tributaries, many authors recommend its ligation or temporary section to facilitate the surgical procedure.2 -27 However, in the case of the right renal vein (RRV) which has no tributaries, its ligatation will certain1y result in severe renallesion, and consequently this procedure is forbidden.28 -32 The presence of multi pIe r~nal veins is most frequent on the right, which is shorter, drains into the cava and has no relationship to the aorta.The LRV is normal1y unique, longer, and crosses transversally in front of the aorta before draining into the inferior vena cava and generally receives three tributaries.28 -52 Many complications have been published after the ligation of the left renal vein (LLRV), including loss of the renal function requiring hemodialysis, retroperitoneal hemorrhage, increase in creatinine leveIs, congestion of the renal vein, renal necrosis, rupture of the left kidney, thus increasing the patient' s morbidity.2,9,10, 11,14,34,44,[53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70]   The rats were identified from 1 to 64. "INITIAL" referred to the first day of surgery and "SACRIFICE" to. the last day of the experience.(Table 1).
The animaIs were submitted to anesthesia by inhalation of ethyl ether.A medianus laparotomy of about 3 cm was performed, with deviation ofthe intestinalloops to the right, opening of the retroperitoneal and careful dissection of the structures and ligature of the left renal vein close to the inferior vena cava, with a 7-0 monofilament polypropylene thread, preserving the renal drainage to the suprarenal, testicular and renolumbar veins.Closure of the abdominal wall was performed using a 3-0 cotton thread.A laparotomy was performed in the controI group, as well as dissection of the LRV; threading and withdrawal of a monofilament polypropylene 7-0 thread under the.LRV was conducted without ligation.The anim~ls were weighed before surgery and after sacrifice and were always sacrificed by decapitation between 10 and 12 hrs.The animaIs were beheaded and the blood collected from the neck vessels placed in heparinized tubes and sent for biochemical analysis.After the beheading and collecting of blood, the animaIs were submitted to xiphopubic laparotomy, assessment of the abdominal condition,.accurate exam of the intra and retroperitoneal organs.The kidneys, testes and suprarenal glands were examined, withdrawn and weighed (humid weight), and submitted to histological analysis by light microscopy.
Statistical analysi\s of the creatinine, testosterone, serum corticosterone and proteinuria was obtained by means of an experimental plé}nwith two factors as sacrifice date with four leveIs (7th, 15th, 30th and 60th day) and surgery with two leveIs, ligation and control, and their values by variance analysis for an hierarchical model.119 The results of percentual variation between the weight of the animaIs.and their kidneys, testes and suprarenal gland as a routine after the section of the LRV.The blood flow to an organ detreases when the venous pressure nears the diastolic blood pressure.7 5 ,76The LLRV enables the inversion of the venous flow and venous hypertension in the kidney, testes and suprarenal gland.8,14,21,50,60,67, 71-89 Baptista-Silva (1994)1 in more than 200 surgeries of the aorta, never had the need to perform a ligature of • LRV in elective surgery such as aneurysm emergencies or trauma to the aorta.In other words, when the approach to the aorta was difficult, he used other surgi cal techniques recommended by other authors,39, 53,69,71,72,87,88,90-103 to facilitate the dissection of the aorta and avoid a LLRV.
Many researchers, worried about the complications of LLRV and renal vein thrombosis, and interested in investigating varicocele developed experimental models in animals with similar clinic':ll findings.Dogs and rats were used in these studies due to the similarity of their vascular anatomy, hemodynamic repercussion and immunology system to that of mano 11. was demonstrated that in dogs a LLRV increased 20 times the hilar renal venous pressure, reducing the renal arterial pressure, the thickening of the basal renal membrane, the high concentration of nephrotoxics antigen, proteinuria, hypoproteinemia, hypoalbuminemia, hypercholesterolemia, atrophy of the left kidney, arterial hypertension in the kidneys with renal congestion, reaction to immunocomplex with lesion of the right kidney and increase in mortality of animalS.47 ,75, 76,83, 104-117 Based on the current controversy in relation to renal lesion, and in the absence of literature on the incidence of lesion to the testes and suprarenal glands after the ligature of the left renal vein in man, we decided to research in rats the functional and histopathologic alterations caused by the LLRV in the kidneys, testes and suprarenal glands.

MATERIAL AND METHODS
Sixty four male adult EP.M1-WISTAR rats were used, weighing around 264 to 469 grams, and were maintained under controlled light conditions from 6 A.M. to 6 P.M. and darkness from 6 P.M. to 6 A.M., at room temperature of 24:t 1°C and free access to rat ration and water (1,118).The rats were randomized into 8 equal groups (8 animaIs each) -four groups for ligation of the left renal vein and four control groups.The groups were randomly chosen to be sacrificed on the 7th, 15th 30th and 60th day after initial surgery, always including one from the LIGATURE group (L7, L15, L30, L60) and one from the CONTROL group (C7, C15, C30, C60).

RESULTS
The examination of the abdominal wall of the rats showed good cicatrization and no infection was detected.AlI the rats of the ligature and control groups were submitted to an exam of the intraperitoneal cavity and no visceral abnormalies were found.The alterations observed in the retroperitoneum were related to the size and color of the kidneys aild suprarenal glands and main veins and tributaries.No thrombosis of the renal vessels and tributaries was observed either in the ligation or control groups.
The left kidneys of ligations.were swollen and had a violet coloring by day 7 and 15, but on day 30 they had reduced in size and were"more cyanotic.On the 60th day the reduction in size was more apparent and the coloring was darker with the left kidney showing an increase in size.The ligations of the LRV and their tributaries were always dilated.
The testes of ligation groups, to the left, the venous network was dilated; on the 60th day the left testis was always smaller than the"right one.
The suprarenal glands were toa small for the observation of macroscopic differences between them.The suprarenal venous network to the left of the ligation was enlarged compared to the controI.(Fig 1 , Table 2) The results of the proteinuria, serum creatinine, serum testosterone and serum corticosterone were statistically different in some periods of the controls but did not exceed the values of normality for rats.

Kidneys
In the ligature groups, we found severe lesions due to ischemia, classified as atrophy of the parenchyma, papilla necrosis, lesion of the distaI tubuli, severe ischemia, infarction and necrosis of the kidneys of the rats sacrificed

TESTES
observed on the 30th day and to the Ieft on the 60th day, in reIation to the controI group, and were more severe to the Ieft than to the right.p< 0,05 (TabIe 3, Fig. 3)

Suprarenal Glands
In the ligature groups, we found severe Iesions due to ischemia, cIassified as corticaI atrophy, medullar and corticaI necrosis of the suprarenaI.gIands of the rats sacrificed on the 7th, 15th, 30th and 60th days after the initiaI operation, but without statisticaI differences in relation to the controI group.(Table 3) p> 0,05.on the 7th, 15th, 30th and 60th days after the initial operation.They were more severe to the left than to the right.Statistical differences were only observed on the left side, in the ligation groups, compared with the control groups in the 7th, 15th, 30th and 60th day.p< 0,05 (TabIe. 3, Fig. 2)

Testes
In the ligature groups, we found severe lesions due to ischemia, classified as atrophy, necrosis, diffuse fibrosis with çalcification of the testes of tJ1erats sacrificed on the 7th, 15th, 30th and 60th days after the initial operation.Significant statistical bilateral differences were only

DISCUSSION.
In this study we found biochemical, functional and histological alterations as a result of the' ligation of the left renal vein.Confmning previous sriIdies on the subject, dilation of the left renal vein 'and its tributaries was found only in the ligation groups, in all periods.61,75,86,106,115,122,123 In the LLRV animaIs, we found a significant variation of body weight (less weight increase compared to the control group ) on the 7th, 15th and 60th day after the initial operation.However, this vatiation was' not present when only a stenosis of the LRV was performed.86 At the end of th~experiment hypertrophy was found in the right kidney, and atrophy in the left.These alterations showed that the right kidney incre~sed in size because of overload, and the left kidney lost weight due to severe histological injuries caused by ischemia due to an extendend venous stasis.These results are the same reported by other authors. 2 ,61,75,124-127 In dogs submitted to ligation of the left renal vein there was a decrease of blood flow on the left as a consequence of venous stasis 75and in ' man the variation of the pressure in the hilar renal venous stump was dependent on the flow through the tributaries of the LRV with the pressure above 60 em of water in the ' hilar renal venous stump possibly leading to a 'decrease of the glomerular filtration due to the decrease of the renal arterial flOW.17 ,54 We found an increase in creatinine serum and proteinuria in the ligature groups, although these values were practically normal.Light proteinuria was observed in rats submitted to LLRV.Massive proteinuria, such as that observed in nephrotic syndrome, was only possible in severe renal injury.124It is difficult to obtain massive proteinuria in an experimental study.128Failure to produce massive proteinuria from an affected kidney or nephrotic syndrome may be partially due to the insufficient increase of the renal venous pressure.Significant proteinuria occurred only in a dog with high renal venous pressure (30 em of saline).126Inacute experiments Wegria et aI 129 found that protein was not present in the urine when the pressure in the left renal vein of the dog was of less than 24,5 em of water.Apressure between 24,5 and 42 em of water in the left kidney excreted protein in 17 of the 27 animaIs under study.In pressures above 42 em of water, proteinuria was practically always present.129 In dogs the pressure of the left renal vein after ligature varied between 14 and 30 em of water, and was not associated with signifieant proteinuria from either the affected or the control kidney.126 In the histo~ogical analysis we observed that the kidneys had a severe lesion due to diffuse ischemia, atrophy of the parenchyma, necrosis of papilla, infarction and fibrosis in atl groups of ligation especially in the left kidney, with the biochemical alterations (serum creatinihe and proteinuria) remaining near the limits of normality for rats.Histological alterations were related to an increase in protein droplets in the tubular cells, leukocyte margination in the glomerular capillaries, and thickening of the glomerular membrane.126 This showed that in the case of LLRV biochemical analysis alone is not sufficient to uncover a renallesion as already reported in medicalliterature.61 ,75,114,124,125,127,13o Our results showed that renal lesions were predominantly on the left side in all periods.Nevertheless, lesions on the right side were present only in the ligature groups suggesting that there are other faetors eontributing to a lesion in the right kidney.This could possibly be due to an immunocomplex reaction as a result of an injury to the left kidney, similar to that described by other investigators.75,110 In the testes, atrophy was found on the left on the 60th day, a finding already described in experimental stenosis of LRV.86There was practically no variation of the serum testosterone leveI throughout the periods.Similar altération of the serum testosterone leveI was reported by other authors in relation to patients with varicocele and also in animaIs with stenosis of LRV.These authors have also found an increase of luteinizing hormones, follicle-stimulating, prolactin, oligosperrnia and zoosperrnia.I04, 107,108,117,131 The histological anal ysis of our study showed a severe bilateral testicular lesion in three of the four groups of ligation, more pronounced to the left on the 30th and 60th day.Other researchers found bilateral testicular lesions and they considered that these could be induced by immunocomplex reaction.86 ,132-137 Other studies must be conducted to further investigate testicular injuries caused by varicocele,138but it has been .. suggested that a defect in the testicular energetic metabolism caused by varicocele may be associated to the worsening of spermatogenesis in rats.139 In the suprarenal glands we found bilateral histologicallesions in most periods, but with no statistical difference, although we could find nothing in medicaI literature concerning suprarenal gland lesion provoked by varicocele or by LLRV.Other authors reported suprarenal gland lesions such as hemorrhage and necrosis, caused by stress, operation or post thrombosis of the inferior vena cava or of LRV in the newborn.64,77,78,84 We can report that the ligation of the left renal vein close to the inferior vena cava in male adult EPM 1-WISTAR rats leads to a severe bilateral histological lesion in the kidneys, testes especially on the left side.The results of the biochemical analysis were not sufficient to demonstrate functional alterations.Histopathological analysis was considered the best test for assessment of lesions caused by LLRV.

Figure 2 -Figure 1 -
Figure 2 -Left kidney of rat # 55 of the Iigature group, showing alterations of the ischemic type, difuse, with retracted, enlarged mesangial matrix and tubuli with atrophy signs, surrounded by interstitium with fibrosis and rare Iymphocyte infiltrates.(HE -126x)

Table 1
Distribution of the rats per group, type of procedure and sacrifice day ( * p < 0.05 by Fisher exact test )