Many institutions still continue to use standard pressure pneumoperitoneum at 12 – 14 mmHg because of its surgical space convenience, although several studies have demonstrated the negative effects of positive-pressure pneumoperitoneum on cardiovascular and organ perfusion. The unfavorable consequences are not expected during most elective laparoscopic operations in healthy or low-risk individuals. However, the increased intraabdominal pressure have a significant clinical impact for high-risk patients such as elderly, cardiac dysfunction or critically ill patients.4,10
The kidneys are very susceptible to the increased intraabdominal pressure, even a slight increase in pressure of 10 mmHg has begun to affect kidney function and at a pressure of as high as 20 mmHg kidney function begins to be disrupted.5,11 Animal study showed that CO2 pneumoperitoneum at 12 to 18 mmHg induced renal cell apoptosis in outer medulla and cortex.12 In humans, increased intra-abdominal pressure cause hypoperfusion in the abdominal or splanchnic region regardless with or without hypotension. Research on animals showed pneumoperitoneum with a pressure at 12 mmHg resulted in hypoperfusion that induced the release of inflammatory cytokines and neutrophil migration.5,12 The advanced venous congestion and decreased renal blood flow lead to tissue hypoperfusion or ischemia that trigger inflammatory responses. After desufflation, reperfusion occurs when renal blood is normalized then leads to oxidative stress that stimulates synthesis of inflammatory cytokines. Inflammatory cytokine has been postulated to mediate the association between blood flow changes and endothelial-epithelial injury.4,13
The increased intraabdominal pressure causes mechanical compression of the inferior vena cava and of the renal vasculature and parenchyma. It increases sympathetic activity, which is regulated through the mechanism of mediated baroreceptors together with the effects of CO2 that can lead to renal cortical vasoconstriction and its sequelae.12,13 The kidneys have autoregulation which is influenced by vascular (myogenic) and tubuloglomerular feedback (TGF) factors. Vascular factors affect autoregulation of renal perfusion through blood flow (pressure) and pressure on blood vessels (pressure), which depends on cardiac output and blood pressure as long as it is on the threshold of autoregulation.14 Under normal condition, blood flow is a laminar flow which gives constant pressure to the blood vessel walls. Changes in blood flow will cause shear stress due to turbulent or oscillatory flow. Shear stress will stimulate the synthesis of endothelial nitric oxide, which will activate the NF-?B signaling pathway through VEGFR-2 which is present on the surface of endothelial cells. Furthermore, stimulation of pro- or anti-inflammatory responses will play a role in the activation, injury, repair, apoptosis of endothelial and epithelial cells. Shear stress in the form of a constant or uniform laminar flow has a protective effect on the endothelium. Low or turbulent shear stress caused by impaired blood flow, not constant and uniform, will stimulate the inflammatory response, increase the expression of endothelial adhesion molecules and their interactions with neutrophils and monocytes in the endothelium.15
When pneumoperitoneum insufflation is given, an increase in RI values represents an increased intra-abdominal pressure will cause a decrease in interlobar arterial blood flow. It will stimulate a systemic inflammatory response that trigger the release of IL-6.15 Our study showed a higher release of IL-6 during the increasing intra-abdominal pressure in the standard pressure group compared to the low-pressure group. Pneumoperitoneum insufflation using CO2 gas has a disadvantage since it was high solubility gas that can be absorbed by the tissue, resulting in sympathetic stimulation such as tachycardia. Although CO2 and surgical techniques can contribute to the release of pro-inflammatory cytokines,16 our study showed a short-time and slight increasing intraabdominal pressure result in significantly increasing IL-6 levels, and using the low-pressure pneumoperitoneum can attenuate that response. The low-pressure group showed the trend of heart rate that significantly lower than the standard pressure group.
Studies on the impact of low-pressure versus standard pressure pneumoperitoneum have shown the various results. A laparoscopic cholecystectomy study performed with low-pressure or standard pressure showed no difference in the increase of IL-6, IL-8, and IL-10. Another laparoscopy study found significantly higher IL-1, IL-6, and CRP levels in the standard pneumoperitoneum pressure group compared with low-pressure pneumoperitoneum.13 Yap et al. found that laparoscopic donor nephrectomy resulted in nearly 50% of their subjects showed an increased tumor necrosis factor alpha (TNF-?) excretion at both 5 and 24 hours and increased urine neutrophil gelatinase-associated lipocalin (NGAL) after donor nephrectomy, that was suggesting that elevated cytokine levels may be due in part to increased endogenous production. The results of this study validate their previously published study which demonstrated that animal models of nephrectomy AKI resulted in increased TNF-?, IL-6, and monocyte chemoattractant protein-1 (MCP-1) expression. IL-6 has been shown to induce neutrophil infiltration with increased macrophage infiltration.17 An animal study showed the extrarenal IL-6 production from liver after unilateral nephrectomy.18 Our results suggested that the increased of plasma IL-6 was due to the increased endogenous production, not because of decreased renal excretion since the urine output and serum creatinine were within the normal limit before and after the procedure.
As hypothesized, we found the increasing plasma syndecan-1 corresponded to the elevated plasma IL-6. Interleukin-6 is a proinflammatory cytokine that will cause Syndecan-1 activation and shedding from the endothelial surface of blood vessels into the bloodstream. In accordance with the degree of inflammation that occurs, the shedding of syndecan-1 increased in both level of pneumoperitoneum pressure compared to the baseline conditions. However, the increasing plasma Syndecan-1 level was less and Syndecan-1 expression in proximal tubular cells was higher in the low-pressure group compared to the standard pressure group. There was a significant increase in the degradation of syndecan-1 glycocalyx products after major surgery in human and animal studies.19 The duration of laparoscopic nephrectomy is relatively longer than open nephrectomy with the addition of using high-pressure pneumoperitoneum lead to exposing the kidney with longer time and more profound of warm ischemia that contributes to the syndecan-1 shedding.20
The renal tubular epithelium not only passively injured, but also produced an active response to inflammation. The release of proinflammatory and chemotactic cytokines which activates T cells and its co-stimulating molecules. Proximal tubular cells respond to T-cell ligands through cell surface receptor activation.13 The increasing syndecan-1 expression and its shedding into the blood are considered as an adaptive response to repair an early cell injury.21 Syndecan-1 plays a role in the process of re-epithelialization during the inflammation including to be involved in promoting the survival of renal tubular epithelial cell in animal models of ischemia/reperfusion and in kidney transplantation patients. In early renal injury, tubular epithelial cells increase the regulation of syndecan-1 to proliferate and repair the injured cells. In respond to a mild inflammatory condition, the increasing tubular syndecan-1 expression showed a better re-epithelialization process in allografts, that correlates with less proteinuria, lower serum creatinine, less tubular atrophy, and lower risk of delayed graft function. Syndecan-1 becomes a tubular marker that correlates with kidney graft function and survival.22 In further injury, the epithelial cells will increasingly lose syndecan-1, because of the decreasing of their ability to proliferate and regenerate it. The sustained elevating plasma syndecan-1 and low of syndecan-1 expression correlated with the degree of loss of tubular function in the kidney.23 Syndecan-1 expression in the proximal renal tubules is related to the degree of proteinuria in various kidney diseases so that the level of syndecan-1 in plasma could become a sign of renal tubular injury.24
Vascular endothelial growth factor-A (VEGF-A) is a strong angiogenic cytokine that has a role in maintaining the microvascular system and increasing vascular permeability. One of the VEGF-A regulators is VEGFR-2 which is expressed during ischemic or inflammatory conditions.25,26 When the inflammation occurs, IL-6 and activated Syndecan-1 in the endothelial cells stimulate the synthesis of VEGF-A molecules and its binding to VEGFR-2 on the endothelial surface, and then increase the VEGFR-2 phosphorylation process in order to repair the endothelial injury.23,27 Plasma syndecan-1 level was hypothesized to be correlated with plasma soluble VEGF-A as a marker of endothelial damage, and plasma creatinine and urea levels as a marker of kidney function.23 In a normal human kidney, VEGFR-2 is expressed on glomerular endothelial cells, peritubular capillaries, as well as in tubular epithelial cells in a low degree. Regulation of protein expression through the VEGFR-2 receptor is important for the survival of endothelial cell tissue in the kidneys after ischemic injury.25,28
The synthesis and activation of VEGFR-2 in baseline condition occurs but is very mild. Our study found there was an increase in the synthesis and higher activation of VEGFR-2 in the standard pressure than the low-pressure group. The level of plasma sVEGFR-2 was significantly higher when the standard pressure was used, in comparison to the low-pressure pneumoperitoneum that attenuated inflammation response and produced lower sVEGFR-2 level. Activation of VEGFR-2 as the marker of vascular endothelial permeability factor depended on the extent of inflammation results in an increase in endothelial permeability and increased levels of VEGFR-2 in soluble form (sVEGFR-2) in plasma. Plasma soluble VEGFR-2 is the result of an increase in alternative splicing of mRNA or as a proteolytic product of membrane-bound VEGFR-2 released into the bloodstream. During ischemic / reperfusion injury, VEGFR-2 mRNA expression and sVEGFR-2 increase as the response of VEGFR-2 receptors. The increased VEGFR-2 expression is a direct effect of VEGF released by ischemic tubular epithelial cells to the adjacent endothelial cells to maintain capillary blood supply and to promote tubular cell survival and recovery. As a comparison, previous studies have shown that at laparoscopy there is an increase in protective VGEF-mRNA expression as a response to repair of injured tissue.25,29,30
Increased VEGFR-2 expression in tubular epithelial cells in our result may describe that inflammatory responses that occur in the circulation reach the extracellular matrix and renal tubules. The VEGFR-2 expression in tubular epithelial cells was higher in the standard pressure compared to the low-pressure group. The low-pressure group produced less injury to the kidney due to less inflammation and less stimulation of VEGFR-2 in the renal endothelial and tubular epithelial cell. A previous study showed the overstimulation VEGFR-2 occurring before unilateral nephrectomy in experimental animals induced endothelial proliferation, abnormal angiogenesis, and extracellular matrix deposition causing acute tubulointerstitial injury.28 There is a hypothesis that syndecan-1 act as a VEGFR-2 co-receptor and has a role in modulating VEGF-VEGFR-2 signal for endothelial cell proliferation and survival. It has been proposed that syndecan-1 and VEGFR-2 can be a new marker for AKI and its treatment.23,31
From electron microscopy examination, the low-pressure group showed proximal and distal tubule ultrastructure with intact tubular cell membranes with clear cell boundaries and intact brush borders. Those morphologies were better compared to the standard pressure group that had a greater injury seen from the tenuous tubular cell membrane, detached brush border from the cell body and showed more vacuolizations. The extracellular matrix peritubular endothelial cell was also more edematous in standard pressure pneumoperitoneum group. The results supported the use of low-pressure pneumoperitoneum results in a lower degree of ischemia and tissue inflammation, thus reducing injury to the endothelial and tubular epithelial cells. Perioperative ischemia and reperfusion process cause injury to the donor kidney epithelial cells that can continue to induce the response from the vascular endothelium.20,22 Experimental animal studies treated with various pressure gradients of CO2 pneumoperitoneum indicate that the increased intraabdominal pressure caused reperfusion ischemic injury leading to cell apoptosis.12 Damage or loss of tubular epithelial cells is the main histological finding of tissue damage that occurs in renal ischemic reperfusion injury. In humans, acute tubular necrosis was observed in 44% of patients in the open nephrectomy group and 45% in the laparoscopic group. In patients undergoing laparoscopy nephrectomy, 54% of renal biopsy specimens taken showed subcapsular cortical injury. These injuries indicate that pneumoperitoneum and mechanical injury during laparoscopic surgical manipulation causes acute tubular necrosis accompanied by peritubular capillaries congestion.30
The increasing urinary KIM-1 during pneumoperitoneum insufflation expressed the stress injury on the proximal tubules cell injury accompanied by the formation of debris cells and apoptotic cells. The process causes an increase of the KIM-1 molecule synthesis that will be released into the lumen of the tubule and can be detected in the urine.32 In our study urinary KIM-1 level was lower during low-pressure pneumoperitoneum compared to the standard pressure. The reversible tubular injury that was represented by decreasing KIM-1 level into the baseline level after surgery could be due to the short length of laparoscopy procedure.
We found syndecan-1 expression in proximal and distal tubular epithelial cells, with the negative expression of syndecan-1 within glomerular or peritubular vasculature. Our study result was similar to Adepu et al. who conducted the study and found syndecan-1 in the basolateral layer in proximal tubular epithelial cells in human kidney biopsy samples.22 She made a hypothesis that the increase in plasma syndecan-1 levels was partly derived from the extravascular source such as renal tubular epithelial cells. Our study on living donor patients showed the contradictory results to a previous animals study that showed the presence of syndecan-1 protein in the glomerulus and peritubular capillaries.23 Syndecan-1 expression was not detected in glomerular endothelium might be because of the dominant type of proteoglycan expression in glomerular endothelial cells was syndecan-4, perlecan, and glypican based on research on human glomerular endothelial culture cells in vitro. Other studies also showed the dominant proteoglycans layer in IgA nephropathy are perlecan and biglycan.24,26 Urinary syndecan-1 level can be used as another alternative to detect extravascular shedding of glycocalyx layer.
Our present study found that laparoscopic donor nephrectomy resulted in increased plasma IL-6, syndecan-1, and sVEGFR-2 during pneumoperitoneum insufflation and after gas desufflation. The IL-6 as a mediator in the extrarenal effects of AKI is clinically important, since it may lead to the use of cytokine-binding proteins and other anti-inflammatory agents to improve outcome beyond what current supportive renal measures can offer. Despite the absence of syndecan-1 from the glomerular and peritubular endothelial glycocalyx, it was found in the membrane of the proximal and distal tubules and was important for the survival of renal tubular cells during inflammation. The VEGFR-2 can be a sensitive marker to detect endothelial injury due to perfusion disturbance and inflammation. Both increasing plasma syndecan-1 and sVEGFR-2 level can be interpreted as an early warning of underlying renal injury, rather than plasma creatine, BUN or urine output.
Endothelial injury and renal tubular injury are the earliest signs of hypoperfusion and inflammation due to increased intra-abdominal pressure.1,5,12 From our study results, although the pneumoperitoneum time is relatively short, the increased inflammation reaction, endothelial and renal tubular injury markers were higher especially when the standard pressure was used in comparison to the lower pressure pneumoperitoneum. The low-pressure pneumoperitoneum could attenuate systemic inflammatory response and vascular response compared using the standard pressure. Inhibiting syndecan-1 shedding and the release of VEGFR-2 are believed has renal-protective roles.22,23,28,31 Further experimental and clinical studies on inhibiting syndecan-1 shedding and sVEGFR-2 response to endothelial injury in preventing and reducing kidney injury are warranted.