Contribution of Autologous Omentum Transposition to the Regeneration of Renal Injuries in the Rat Model

Aim: After renal trauma, surgical treatment is vital, but sometimes there may be loss of function due to brosis. We aimed to evaluate the repair effect of transpositioned autologous omentum on injured renal tissue in a rat model. Methods: A total of 30 female Wistar Albino rats were included and they were randomly separated into a sham group and four study groups. Iatrogenic renal injuries were repaired using a surgical technique (primary repair 1 and 2 groups) or transpositioned autologous omentum (omentum repair 1 and 2 groups). In all groups, blood samples were taken preoperatively and on the 7 th postoperative day in all groups and also on the 18 th postoperative day in the control and two study groups. All rats were sacriced on the 7 th or 18 th day postoperatively and their right kidneys were taken for histopathological evaluation. Results: There was a trend toward decrease in urea and creatinine levels in all the groups. There was no signicant correlation between urea and creatinine levels and histological nding scores. The omentum repair group had signicantly lower inammation and granulation scores compared with the primary repair and sham groups. There was a signicant and positive correlation between inammation and granulation and brosis scores. There was a signicant and negative correlation between healing completion score and either inammation and granulation scores. There were also positive correlations between histological ndings in the kidney specimen and surrounding tissues. Conclusion: The use of the autologous omentum tissue for repair of kidney injury had attenuation effects on inammation and granulation compared with primary repair. These results imply that use of omentum tissue to facilitate healing of kidney injury may theoretically lead to a more effective healing process and reduced brosis and tissue and function loss. These potentially benecial effects of autologous omentum tissue should be investigated in further well-designed experimental and clinical studies.


Introduction
Renal trauma can occur associated with various mechanisms. Etiological factors are generally described as blunt kidney injuries (80-90%) and penetrating kidney injuries (10%). The main sign of renal trauma is hematuria which does not always happen (1). The main purpose of treatment in kidney trauma is to ensure that the kidney functions return to normal as soon as possible (2). Renal injuries are classi ed according to severity into 5 grades. Especially, grade 4-5 injuries in the renal tissue are candidate for surgical treatment. Major kidney injuries caused with blunt trauma are treated with conservative management (2,3). The other preferences of treatment are open or endoscopic surgical procedures such as laparoscopic/robot-assisted or open partial/total nephrectomy, nephrorraphy, autotransplantation, and embolization.
It is important to accelerate wound healing in renal traumas requiring surgical treatment in order to decrease morbidity and mortality rates. Wound healing includes three dynamic phases, namely: in ammation, proliferation, and remodeling (4). Angiogenesis, in amation, cellular proliferation, collagenization, granulation, and epithelialization are important processes in the remodeling of tissue (5).
There are many molecules which have a role in tissue regeneration such as vascular endothelial growth factor (VEGF) and nitric oxide (NO) (6). Synthetic or autologous materials such as fat tissue, omentum, meshes and fascias can be used for regeneration of injured kidney tissue (7).
The omentum is a quite vascular and fatty structure and includes many growth and angiogenic factors in the progenitor cells (8,9). Therefore, it can migrate to damaged tissues and aid in the regeneration process (10,11). It has been used for many surgical procedures in the treatment of bone fractures, spine injuries, ischemic heart diseases, and hepatic injuries (12). Progenitor stem cells have high proliferation and differentiation capabilities. However, they have a very short life span in the tissue if they are injected into a target. In many studies, the omentum has been used to wrap the injured tissue and has been shown to be useful (13). According to the results of a previous study, progression to chronic kidney failure has been shown to slow down after subtotal nephrectomy when omentum was used to cover the kidneys (11). This is a unique contribution in the context of nephron-sparing.
We aimed to evaluate the repair effect of transpositioned autologous omentum on the injured renal tissues in a rat model. We measured renal injury biomarkers (creatinine and urea) and made a histopathological evaluation to determine the degree of injury and repair and also assessed the correlation of biomarkers with histopathological ndings.

Material And Methods
A total of 30 female Wistar Albino rats were included, which were 5 to 6 months old and weighed 250 to 300 grams. They were randomly separated into a control and four intervention groups (6 rats per group).
In all groups, blood samples were taken preoperatively and on the 7 th postoperative day for creatinine and urea analyses. Additional blood samples were obtained on the 18 th postoperative day for the same analyses in the control group and two intervention groups (group 2 and group 4). A sham operation was performed to the rats in the control group. All rats in the control group and groups 2 and 4 were sacri ced on the 18 th postoperative day and right kidneys were taken for histopathological evaluation. The rats in the groups 1 and 3 were sacri ced on the 7 th postoperative day and right kidneys were taken for histopathological evaluation. In all of the intervention groups (groups 1-4) an 8 mm diameter and 4 mm deep parenchymal damage was created with a Stiefel biopsy forceps on the front surface of the right kidneys according to the well described Stiefel biopsy technique in the literature. In the groups 1 and 2 (primary repair groups), kidney injuries were primary repaired with interrupted atraumatic matrix suture technique (Ethicon VICRYL Rapid 8-0, fastest absorbable, synthetic, braided, composed of a copolymer made from 90% glycolide and 10% L-lactide, absorption time 7-10 days). In groups 3 and 4 (kidney omentum repair groups), transpositioned autologous omentum was used without primary sutures on the injured renal tissue for repair. We preferred the time of sacri ce as the 18 th postoperative day, according to the study of Miguel et al. (14) which reported that posttraumatic necrosis in the tissue disappeared on the 18 th day. In that study, after this period, collagen maturation took place in the renal capsule and connective tissue at the edges of the wound were contracted (14). The summary of the procedures performed in the study groups are outlined in Table 1.
The subjects were kept in standardized laboratory conditions of 20-24 C 0 , 50-60% relative humidity, controlled light (day-night cycle of 12 h: 8/20 h), fed on standardized rodent food, and given ltered and chlorinated water. The animals were anesthetized with an intraperitoneal injection of ketamine (75 mg/kg) and xylazine (5 mg/kg). All rats were protected against the postoperative infections with an antibiotic (Cefazolin 15 mg/kg, SC).
The only exclusion criterion for this study was the death of the rats before the end of the study.
Tissue sampling and histopathological examination All kidney samples were xed in a 10% formaldehyde solution. Kidney tissues were embedded in para n and 5 μm tissue sections were obtained for hematoxylin-eosin (H&E) and Masson's Trichrome (MTC) staining protocol for collagen bers. In addition to macroscopic view, histopathological evaluation consisted of granulation, in ammation, brosis, foreign body reaction, and healing in the injured kidney and surrounding tissue (omentum). All components were scored between 0 and 5 according to density of the changes in the tissue (normal:0, rare:1, mild:2, modest:3, common:4, and excessive:5). Macroscopic evaluation consisted only of macroscopic view of the kidney to review the surface of the kidney in terms of presence of abnormal structures and it was performed with a quantitative/semi-quantitative analysis. The degree of granulation was evaluated in the glomeruli and parenchymal tissue and by reviewing these structures in terms of edema, in ammatory cells, angiogenesis, and broblasts. In ammation was evaluated by reviewing the tissues regarding acute in ammatory cells, macrophages, and lymphocytes. The degree of brosis (connective tissue evaluation) was evaluated by the presence of broblasts and their density. Foreign body reaction was evaluated with the following: necrosis, erythrocytes, and chronic in ammation ndings. Healing was determined by the ndings of regeneration and normalization in the tissues. The cut sections were examined for completeness and one representative section of each kidney was selected for tissue processing. The histological damage was examined under a light microscope by a pathologist who was blind to the study design (sham vs. renal regeneration). All pathological slides were scanned using a digital pathology system (3D Histech company, P250 -Flash III Dijital Scanner, 20X) and microscopic photos were taken using a software (3D Histech company, CaseViewer program, .tiff format and 300 dpi).

Biochemical analysis
The concentrations of creatinine and urea in the serum were determined by an enzymatic assay (Roche Diagnostics GmbH, Mannheim, Germany). Serum samples for the measurement were collected and stored at -80 0 C until the analysis was carried out. All laboratory investigators were blind to each rat's clinical information.

Statistical analysis
It was estimated that 5 groups comprising 6 rats per group would be required to detect 3 units of improvement (with 1.5 units as standard deviation) as a signi cant effect in a wound healing model, assuming a power of 80% and a con dence level of 95%. SPSS 21.0 (IBM Corp., Armonk, NY) was used for statistical analysis. The descriptive statistics for categorical variables were given as the numbers and percentages. Ordinal variables were presented as medians and continuous variables were presented as means + standard deviations (SD). Kolmogorov-Smirnov test was used to assess normality of the variables. Ordinal variables were compared using the Kruskal Wallis and the Mann-Whitney U tests.
Nonparametric dependent variables were compared using the Wilcoxon test. Spearman's test was used for correlation analyses. Statistical signi cance was de ned as p < 0.05. Table 2 Table 2 outlines the urea and creatinine levels of the study groups. The mean creatinine level decreased signi cantly from day 1 to day 7 in the sham group (p=0.038). The mean creatinine and urea levels signi cantly decreased from day 1 to day 7 in the primary repair group (p=0.005 and p<0.001, respectively). In the primary repair 2 group, mean creatinine level decreased signi cantly from day 1 to day 7 (p=0.011). There was no signi cant change in urea or creatinine levels in the omentum repair group 1. On the other hand, mean urea level signi cantly decreased from day to day 7 in the omentum repair 2 group (p=0.005). There was no other signi cant change in urea or creatinine levels within the study groups.

Results of biochemical analysis are shown in
There was no signi cant difference in kidney macroscopic evaluation or kidney connective tissue scores between the study groups (p=0.56 and p=0.19, respectively). On the other hand, there were signi cant differences in granulation degree (p<0.001), in ammation degree (p<0.001), foreign body reaction (p=0.001), and completion score for healing process in the kidney specimen (p=0.004). There were also signi cant differences between granulation degree (p=0.009), in ammation degree (p=0.013), brosis degree (p=0.010), and foreign body reaction (p<0.001) in the surrounding tissue.
The primary repair 1 and 2 groups had signi cantly higher median granulation and in ammation scores in the kidney specimen compared with the sham and omentum repair groups. The omentum repair groups had similar granulation and in ammation scores with the sham group. The foreign body reaction score in the kidney specimen was signi cantly higher in the primary repair groups compared with the sham group. The completion score for healing process in the kidney specimen was signi cantly higher in the omentum repair groups compared with the primary repair groups. Figure 6 outlines granulation, in ammation, and completion of healing scores in kidney specimens in the study groups.
The sham group had a signi cantly lower median foreign body score in the surrounding tissue compared with all other study groups and signi cantly lower connective tissue reaction scores in the surrounding tissues compared with the primary repair groups and omentum repair 2 group. The primary repair groups had signi cantly lower median granulation and in ammation scores in the surrounding tissues compared with the omentum repair 2 group. The median histological scores in the surrounding tissues were similar in the remaining comparisons between the study groups.
There were no signi cant correlations between urea or creatinine levels and histological nding scores.
The granulation degree in kidney specimen had positive moderate or strong correlations with granulation degree (r=0.478, p=0.008), in ammation degree (r=0.591, p=0.001), brosis degree (r=0.394, p=0.031), and foreign body reaction in the surrounding tissue (r=0.635, p<0.001). In ammation degree in the kidney specimen had positive moderate or strong correlations with granulation degree (r=0.512, p=0.004), in ammation degree (r=0.507, p=0.004), brosis degree (r=0.434, p=0.017), and foreign body reaction in the surrounding tissue (r=0.660, p<0.001). Fibrosis degree in the kidney specimen had a moderate and positive correlation with brosis degree in the surrounding tissue (r=0.429, p=0.018). Foreign body reaction in the kidney specimen was strongly and positively correlated with brosis degree in (r=0.829, p<0.001) and foreign body reaction in the surrounding tissue (r=0.813, p<0.001). Healing process completion score in the kidney had negative moderate or strong correlations with granulation degree (r=-0.625, p=0.001) and foreign body reaction in the surrounding tissue (r=-0.425, p=0.039).
In ammation degree in the surrounding tissue had positive moderate or strong correlations with granulation degree (r=0.490, p=0.006), brosis degree (r=0.397, p=0.030), and foreign body reaction in the surrounding tissue (r=0.431, p=0.017). Fibrosis degree in the surrounding tissue had positive moderate or strong correlations with in ammation degree (r=0.397, p=0.030) and foreign body reaction in the surrounding tissue (r=0.708, p<0.001).
Granulation degree in the kidney specimen was strongly and positively correlated with in ammation degree (r=0.824, p<0.001) and foreign body reaction in the kidney specimen (r=0.872, p<0.001); and a strong and negative correlation with healing process completion score in the kidney (r=-0.627, p=0.001). In ammation degree in the kidney specimen was strongly and positively correlated with foreign body reaction in the kidney specimen (r=0.731, p=0.001) and strongly and negatively correlated with healing process completion score in the kidney specimen (r=-0.608, p=0.002).

Discussion
The prevalence of renal trauma ranges between 0.3%-3.25% in the literature and the most common causes are blunt trauma followed by penetrating trauma. The most commonly used renal trauma classi cation is that of the American Association for the Surgery of Trauma (AAST) which ranges between grades 1-5 (15). Currently, except for the hemodynamically unstable grade 4-5 renal trauma, renal injuries are followed up with a conservative approach. Surgical intervention is also considered in case of signi cant vital changes related with renal injury. Partial/total nephrectomy or nephrorrhaphy can be preferred according to the type or degree of injury.
Usually transperitoneal surgical approach is more preferable because this route provides some advantages such as the early control of large veins and arteries. Surgery for a renal trauma comprises control of the bleeding by sutures, watertight closure of the collecting system, and closure of parenchymal injuries. Even preserving thirty percent of kidney capacity can provide adequate kidney functions. The renal capsule should be preserved at all possible cases for a successful repair (16).
Sometimes, if renal capsule is not available, a pedicle ap of omentum, free peritoneal graft, free fat graft, or polyglycolic acid mesh can be used for coverage of a large defect. In the technique, omentum is placed on the injured tissue and super cially sutured with mono lament absorbable sutures (17)(18)(19).
The omentum has long been known to have the capacity to migrate to injured organs such as bones, spinal cords, heart, liver, and pancreas and facilitate their healing. Many studies have shown that a reduction in total nephron capacity may cause kidney failure in the future, thus maximum protection of kidney tissue should be the main purpose. Some suture material and surgical techniques can be harmful to the kidney tissue. For this reason, alternative techniques have been developed to better protect the kidney tissue especially in case of large tissue lose. One of them is to use the omentum or fatty tissue for repairing of renal injury.
The mesenchymal stem cells (MSCs) can be obtained from adipose tissue, peripheral blood or bone marrow. Another alternative source for repairing of injured tissue is the omentum. It is a very vascular structure and suitable to use to facilitate repair in case of injury as it contains a large number of growth and angiogenic factors and progenitor cells for regeneration (20). MSCs were rst isolated from adipose tissue in 2001 by Zuk et al. (19). It is well-known that MSCs have the abilities of multipotency, selfrenewing, proliferation, regeneration, and differentiation (20). Of note, MSCs can accelerate tissue repair by direct migration to the injured sites (21,22). Alternatively, MSCs may be administered locally or systemically for treatment. It is widely agreed that transplanted MSCs can directly reconstruct impaired organs. They have some speci c features as endocrine (growth factors, chemokines, and cytokines with paracrine and autocrine activities), immunomodulatory (T-cells, dendritic cells, and natural killer cells), and anti-in ammatory effects (23). These factors suppress the local immune system, inhibit brosis and apoptosis, enhance angiogenesis, and stimulate proliferation and differentiation. Firstly, Iwai et al. discovered that local injection of adipose tissue derived MSCs facilitated attenuation of brosis (24).
Normal wound healing process includes endothelial injury, myo broblast activation, macrophage migration, in ammatory signal stimulation, immune activation, matrix deposition, and remodeling. Especially in the rst 24-28 hours, many molecular reactions occur in the tissue. Fibroblasts are very crucial members at the in ammation process. Moreover, functional microcirculatory bed has been shown to be of critical importance in the prevention of epithelial loss and brosis (25). Fibrosis is one of the most common and refractory pathological processes. Fibrosis is a redundant accumulation of extracellular matrix (ECM) in tissues by collagen reaction and at the end of the recovery process a thick brotic neocapsule can occur. On the other hand, MSCs can directly release HGF and BMP-7, which are important inhibitors of brosis. MSCs have been shown to exert anti-brotic effects in animal models by matrix metalloproteinases (26). Unlike synthetic meshes, autologous MSCs are immune compatible and this is an advantage in the remodeling process.
In the present study, granulation and in ammation scores in the kidney specimen were signi cantly lower in the omentum repair groups compared with the sham and primary repair groups. This nding suggests that omentum attenuated granulation and in ammation related with kidney injury. Transpositioned autologous omentum may be effective by reducing macrophage in ltration as well as reducing brosis.
In many studies, the histological damage of the kidneys has been evaluated in tissues with the EGTI scoring system (27). This scoring system consists of histological damage in 4 individual components: endothelial, glomerular, tubular, and interstitial (EGTI Scoring system) and is scored from 0 to 4. This scoring is performed in the renal cortex, especially for glomerular units. Therefore, we preferred to use a new scoring system for histopathological evaluation, so that it was possible to evaluate different components of the regeneration on the whole kidney tissue.
There was a trend towards decrease in urea and creatinine levels in the study groups. Also there was no correlation between urea and creatinine levels and histological nding scores. These ndings can be explained by the fact that we could not produce su cient nephron damage with our trauma model. In the future, this model may be planned to be repeated with major kidney tissue damage. Contrary to our results, Garcia-Gomez et al. reported that the omentum was effective in treatment of kidney injuries. In the context of the use of omentum, progression to chronic kidney disease could be reduced in that rat model (12). But in that study, kidney injuries were larger (5/6 subtotal nephrectomy).
According to the results of the present study, granulation and in ammation in kidney specimens were positively correlated with granulation, in ammation, brosis, and foreign body reaction in the surrounding tissue. Healing process completion in the kidney specimen was inversely correlated with granulation and foreign body reaction in the surrounding tissue. As expected, in ammation in the surrounding tissue was positively correlated with granulation, brosis, and foreign body reaction in the surrounding tissue. Moreover, brosis in the surrounding tissue was positively correlated with in ammation and foreign body reaction. Therefore, one can consider that in ammation and granulation may lead to brosis and interventions to reduce in ammation and granulation after injury may aid in prevention of brosis and permanent tissue damage.
Granulation in the kidney specimen was strongly and positively correlated with in ammation and foreign body reaction in the kidney specimen and strongly and negatively correlated with healing process completion score in the kidney specimen. Moreover, in ammation in the kidney specimen were positively correlated with granulation and foreign body reaction in the kidney specimen, and negatively correlated with healing process completion score in the kidney specimen. Therefore, we can speculate that in ammation and granulation after injury are also related with a reduced healing capacity and measures to reduce may also aid in acceleration of healing.
Among the limitations of this study are the fact that only blood creatinine and urea levels were used for biochemical evaluation of renal injury and we did not measure the urine concentrations due to the technical inadequacy of urine collection in the rats. Moreover, we were able to evaluate histopathological analysis only qualitatively. We also lacked a kidney injury group without primary repair or omentum repair. The use of such a group might improve the quality of evaluation of the effect of primary repair and omentum repair in comparison. Lastly, the injury model used in this study did not cause an increase in urea or creatinine levels. Therefore, performing a similar study to see the effect of primary repair and omentum repair after a larger kidney injury model would provide a better of the effect of these interventions.

Conclusion
We aimed to determine the repair capacity of omentum tissue on renal injury in a rat model. According to our results, the use of the autologous omentum tissue for repair of kidney injury had attenuation effects on in ammation and granulation compared with primary repair. These results imply that use of omentum tissue to facilitate healing of kidney injury may theoretically lead to a more effective healing process and reduced brosis and tissue and function loss. These potentially bene cial effects of autologous omentum tissue should be investigated in further well-designed experimental and clinical studies.

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Competing interests
The authors declare that there are no con icts of interest.    Table   3: Score of histopathological evaluation in kidney tissue for control and study groups.