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Basic and Experimental Research

The Intrinsic Renal Compartment Syndrome: New Perspectives in Kidney Transplantation

Herrler, Tanja1,4; Tischer, Anne1; Meyer, Andreas1; Feiler, Sergej2; Guba, Markus1; Nowak, Sebastian3; Rentsch, Markus1; Bartenstein, Peter3; Hacker, Marcus3; Jauch, Karl-Walter1

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doi: 10.1097/TP.0b013e3181c40aba
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Abstract

The extent of ischemia-reperfusion injury is considered as an important initial condition affecting long-term renal function and ultimately allograft survival after kidney transplantation (1). Ischemia-reperfusion injury is believed to reduce the reserve of functional nephrons and to contribute to tubular necrosis, which is the histologic correlate of delayed graft function (2). The structural damage after renal ischemia-reperfusion injury may also activate immunologic cascades that trigger allograft rejection. Despite enormous efforts to reduce ischemia-reperfusion injury by optimizing preservation solutions and antioxidative treatment, little progress has been achieved in improving long-term results in kidney transplantation (3).

So far, the role of increased interstitial tissue pressure in this pathophysiological pattern has not been systematically evaluated. The renal parenchyma is enclosed by the tight fibrous texture of the kidney capsule with little ability to accommodate volume shift because of parenchymal edema. Therefore, edema formation within this structural barrier may result in elevated tissue pressure within the renal compartment with further devastating effects on functional and structural integrity of the transplanted kidney. Here, we provide evidence that interstitial edema after an event of ischemia-reperfusion increases intrarenal pressure. Subsequent development of an intrinsic compartment syndrome results in impaired tubular excretion function, reduced blood flow, and structural damage of the kidney.

METHODS

Animals

Male Balb/C wildtype mice, aged 10 to 12 weeks and weighing 22 to 24 g were purchased from Charles River Laboratories (Sulzfeld, Germany). Animals were fed a standard diet and allowed free access to water. All animal experiments were conducted in accordance with institutional guidelines and were approved by the Administrative Panel on Laboratory Animal Care.

Renal Ischemia-Reperfusion Injury Model and Capsulotomy

Mice were anesthetized by intraperitoneal injection with a combination of ketamine (150 mg/kg) and xylazine (15 mg/kg) and placed on a heated surgical pad to keep constant body temperature. The right kidney was exposed through median abdominal incision, and mice were subjected to ischemia by clamping the right renal pedicle with a nontraumatic microaneurysm clamp (Braun, Melsungen, Germany) which was removed after 35 and 45 min, respectively. Mice exposed to unilateral renal ischemia were divided into nontherapy group (n=5) and therapy group (n=8). In the therapy group, subcapsular pressure of the right ischemic kidney was relieved by a standardized 0.3 mm (30 Gauge) needle puncture at the lower pole of the kidney capsule. To avoid injury of the kidney parenchyma, the kidney capsule was carefully lifted with a forceps at the incision site. The abdominal incision was closed with a 5-0 suture and surgical staples.

Subcapsular Pressure Measurement

Subcapsular pressure was continuously measured using a Codman ICP microsensor (Johnson & Johnson Medical Limited, Berkshire, UK). Pressure measurement was performed in a separate collective apart from the treatment study to avoid alteration of results. Mice (n=5 for each time point) were anesthetized by intraperitoneal injection and placed on a heated surgical pad to keep constant body temperature. The right kidney was exposed through median abdominal incision. After minimal incision of the kidney capsule, the ICP probe was advanced 2 mm under the capsule. Subcapsular pressure was continuously monitored for 10 min. The probe was then removed, and the ischemic kidneys were collected for histologic analysis.

Renal Scintigraphy Using 99mTechnetium-Mercapto-Acetyl-tri-Glycine (99mTc-MAG3)

Scintigraphy using 99mTc-MAG3 (Technescan MAG3, Covidien, Neustadt/Donau, Germany), a radioactive compound predominantly excreted by tubular secretion, was performed in analogy to a previously described protocol (4). Briefly, after hydration with sterile saline and induction of anesthesia with a combination of ketamine (150 mg/kg) and xylazine (15 mg/kg) mice underwent whole-body scintigraphy in a triple-headed gamma camera (Philips - former Picker - Prism 3000 XP, Cleveland, OH) using dynamic imaging protocols with 99mTc-MAG3. Each detector head was equipped with a LEHR collimator, but only one head was used. Intravenous injection of a standardized dose of approximately 3.7×107 Bq per mouse and acquisition in a dynamic planar technique were simultaneously started. One frame per 5 sec was collected with a total scan time amounting to 10 min. The image acquisition magnification was set to four times. To determine baseline renal function, 99mTc-MAG3 imaging was carried out 4 days before ischemia-reperfusion surgery. Postoperative renal scans were performed on day 2 and 18 for the assessment of early and long-term kidney function. To minimize experimental stress due to surgery, day 2 was chosen as the earliest postoperative time point for renal scintigraphy.

Image Analysis

Image files were analyzed using Hermes dynamic analysis software version 4.1 (Hermes Medical Solution, Stockholm, Sweden) by standard manual region of interest (ROI) analyses of the whole body, both kidneys including their background regions and the site of injection (Fig. 1A). Data were exported to Microsoft Excel to assess renal function represented as percentage of injected dose (%ID). Values for %ID were obtained by division of the background corrected kidney ROI by the injection site corrected whole body ROI. In addition to renal function curves (%ID), peak (%ID) and renal excretion capacity reflected by the delta of [peak (%ID) minus %ID at 10 min examination time] were determined (Fig. 1B). In analogy, background ROI of the ischemic kidneys were analyzed for the detection of potential leakage of radioactivity due to incision of the kidney capsule and depicted as %ID.

F1-5
FIGURE 1.:
Assessment of renal function by scintigraphy. (A) Image files were analyzed by standard manual region of interest (ROI) determination of the whole body, both kidneys including their background regions and the site of injection. (B) Renal function is represented as %ID. Renal function peak and the tubular excretion rate reflected by the delta of [peak %ID minus %ID at 10 min examination time] depict the extent of organ failure. %ID, percentage injected dose.

Renal Perfusion Measurements

After final 99mTc-MAG3 imaging on day 18, blood flow of the ischemic (right) and nonischemic (left) kidney was assessed using the O2C laser Doppler blood flow analyzer with the LFM-2 micro probe (2 mm tissue penetration; both Lea Medizintechnik, Giessen, Germany). Relative perfusion was calculated as the ratio of blood flow in the ischemic and the healthy kidney.

Weight Analysis

Before embedding for histologic analysis, both the right ischemic kidney and its healthy contralateral counterpart were weighed using a special accuracy weighing machine (Sartorius BP3105, Goettingen, Germany). Considering interindividual differences, kidney wet weight was expressed as the ratio of the ischemic and the healthy kidney, as similar wet weight was recorded for both healthy kidneys in an individual.

Histologic Analysis

Tissue specimens (ischemia-reperfusion injured right kidney and contralateral healthy kidney) were collected for histologic analysis. After paraffin embedding, histologic sections were stained with H&E.

Statistical Analysis

Statistical analysis was performed using paired or unpaired t test (two-sided). Data are expressed as mean±SEM. P values less than 0.05 were considered statistically significant.

RESULTS

Ischemia-reperfusion injury is associated with increased pressure within the renal compartment. After ischemia-reperfusion injury, a significant increase in pressure values within the renal compartment in an ischemia time-dependent manner was found (Fig. 2A). Overall, prolonged ischemia time of 45 min resulted in significantly higher pressure values compared with mild ischemia of 35 min, 6 hr and 12 hr after ischemia (P<0.05). Compared with baseline (0.9±0.3 mm Hg; n=5), 45 min ischemia led to a 7.6-fold increase 6 hr after reperfusion (7.0±1.0 mm Hg; P<0.001; n=5), whereas only a 3.6-fold increase was found for 35 min ischemia time (3.3±0.0 mm Hg; P<0.01; n=5). In the 35 min ischemia group, pressure already declined to baseline at 24 hr reperfusion time (2.2±0.5 mm Hg; n.s. vs. baseline; n=4). In contrast, pressure values in the 45 min ischemia group continued to be unphysiologically high for 24 hr. Notably, we found another moderate rise in pressure during long-term follow-up examination on day 18 (4.7±1.5 mm Hg; P<0.05; n=5). Histologically, the extent of interstitial edema after renal ischemia time of 45 min varied depending on the time of examination. Although histology was characterized by markedly dilated tubules at 6 hr reperfusion time and only slight interstitial edema, pronounced interstitial edema was seen 12 and 24 hr after ischemia. At 48 hr after ischemia, edema was considerably reduced (Fig. 2B).

F2-5
FIGURE 2.:
Increase in subcapsular pressure following renal ischemia-reperfusion injury. (A) Renal ischemia-reperfusion injury led to a significant increase in pressure within the renal compartment in an ischemia time-dependent manner. Prolonged ischemia time of 45 min resulted in significantly higher pressure values compared with mild ischemia of 35 min, 6 hr, and 12 hr after ischemia. In the 35 min ischemia group, pressure already declined to baseline at 24 hr reperfusion time, whereas pressure values in the 45 min ischemia group continued to be unphysiologically high. Prolonged ischemia was characterized by a biphasic pressure increase with another significant rise in pressure 18 days after ischemia. (B) Following renal ischemia time of 45 min, the extent of interstitial edema varied depending on the time of examination. Although histology was characterized by markedly dilated tubules at 6 hr reperfusion time and only slight interstitial edema, pronounced interstitial edema was seen 12 and 24 hr after ischemia. At 48 hr after ischemia, edema was considerably reduced. Hematoxylin-eosin stain. Original magnification was ×200.

Prolonged ischemia-reperfusion injury leads to irreversible loss of tubular excretion function. Renal function curves obtained from 99mTc-MAG3 imaging were evaluated for peak (maximum uptake) and tubular excretion rate (peak [%ID] minus [%ID] at 10 min examination time). After 45 min ischemia, scintigraphy revealed early marked impairment of tubular excretion function in reference to the healthy kidney (pre/baseline=100%) with no evidence for spontaneous restoration in the long term (35.4% baseline ±2.6% on day 2; 33.0% baseline ±3.5% on day 18; P<0.001; n=5). Renal function peak was also considerably reduced on day 2 (79.2% baseline ±3.4%; P<0.001; n=5) and further declined during subsequent measurements on day 18 (41.6% baseline ±3.2%; P<0.001; n=5; Fig. 3A). In contrast, no significant decline in renal function peak (94.7±6.7% on day 2, 95.3±3.9% on day 18; n.s. vs. baseline; n=7) and excretion rate (59.0±7.0% on day 2, 99.9±5.2% on day 18; n.s. vs. baseline; n=7) was recorded after mild ischemia of 35 min. (Fig. 3B). Analysis of the background ROI of the ischemic kidneys showed significantly increased perirenal radioactivity in the therapy group (0.49±0.05%ID) compared with the nontreated group (0.34±0.05%ID) 2 days after ischemia.

F3-5
FIGURE 3.:
Prolonged ischemia-reperfusion injury leads to irreversible loss of tubular excretion function that is effectively prevented by incision of the kidney capsule. (A) After 45 min ischemia, scintigraphy revealed early marked impairment of tubular excretion function and renal function peak in reference to the healthy kidney with no evidence for spontaneous restoration in the long term. (B) Shorter ischemia time of 35 min was not associated with a significant decline in renal function peak and excretion rate. (C) Surgical pressure relief by surgical incision was able to significantly prevent loss of renal function after prolonged renal ischemia of 45 min. (D) Renal function peak was only reduced by 20% in the incision-treated kidneys vs. almost 60% reduction in the nontherapy group. (E) Tubular excretion rate in the therapy group was significantly higher than in nontreated animals. %ID, percentage injected dose. Pre, baseline renal function before surgery.

Incision of the kidney capsule preserves renal function after prolonged ischemia. Incision of the kidney capsule effectively preserved renal function as assessed by scintigraphy after prolonged renal ischemia of 45 min (Fig. 3C). Surgical pressure relief was able to significantly prevent loss of renal function. In contrast to the nontherapy group (peak: 41.6% baseline ±3.2%), renal function peak was only reduced by 20% (peak: 80.2% baseline ±10.7%; P<0.05 vs. nontherapy; Fig. 3D). Tubular excretion rate in the therapy group was as high as 62.5% baseline ±6.8% compared with 33.0% baseline ±3.5% for nontreated animals (P<0.01; Fig. 3E).

Irreversible impairment of vascular perfusion after prolonged ischemia is prevented by incision of the kidney capsule. No significant change in vascular perfusion was recorded 18 days after induction of ischemia-reperfusion injury for animals undergoing 35 min of renal ischemia as assessed by laser Doppler (99.5±1.5%; n.s. vs. healthy; n=7). In contrast, prolonged ischemia time of 45 min led to significant impairment of renal perfusion (64.5±6.8% healthy; P<0.005; n=5). Incision of the kidney capsule significantly preserved renal blood flow (96.2±4.8%; P<0.05 vs. nontherapy, n.s. vs. healthy; n=8; Fig. 4).

F4-5
FIGURE 4.:
Laser Doppler assessed perfusion measurement. No change in vascular perfusion was recorded 18 days after induction of ischemia-reperfusion injury for treated ischemic kidneys, whereas prolonged ischemia time (45 min) resulted in significant impairment of renal perfusion.

Excessive ischemia-reperfusion induces irreversible tissue damage and atrophy. In reference to their healthy counterpart, kidneys exposed to prolonged ischemia time of 45 min showed considerably more reduction in size than incision-treated kidneys (Fig. 5A). Nontreated kidneys exposed to ischemia exhibited marked loss in weight (66.6% healthy ±8.0%; P<0.05 vs. healthy; n=5) than kidneys with capsular incision (87.3% healthy ±7.9%; P<0.01 vs. nontherapy, n.s. vs. healthy; n=8; Fig. 5B).

F5-5
FIGURE 5.:
Kidney size and weight following ischemia-reperfusion injury. Excessive ischemia-reperfusion induces irreversible tissue damage and atrophy. (A) In reference to their healthy counterpart, kidneys exposed to prolonged ischemia time of 45 min showed considerably more reduction in size than incision-treated kidneys. (B) Consistently, nontreated ischemic kidneys exhibited marked loss in weight, whereas only moderate weight loss was seen in ischemic kidneys with capsular incision.

Surgical therapy by incision of the kidney capsule prevents tubular necrosis after prolonged ischemia. In reference to the healthy kidney, the extent of tissue damage correlated with increasing ischemia time. On day 18 after induction of ischemia-reperfusion injury, 35 min of ischemia resulted in only mild changes in histologic appearance including dilated tubules, granular degeneration within the cytoplasm, and without any signs of necrosis. Glomeruli were well preserved. In contrast, kidneys subjected to 45 min of ischemia exhibited extensive tissue damage including tubular atrophy and necrosis, excessive dilatation and cytoplasmic degeneration, loss of brush borders, protein cylinders, and multiple areas of calcification within the cortex as a consequence of previously occurred tubular necrosis. No distinct changes except for hyperemia were found within the glomeruli. Kidneys exposed to prolonged ischemia but treated by surgical incision presented completely preserved renal structures and excellent viability with no signs for necrosis (Fig. 6).

F6-5
FIGURE 6.:
Surgical therapy by incision of the kidney capsule prevents tubular necrosis after prolonged ischemia. In reference to the healthy kidney, kidneys subjected to 45 min of ischemia exhibited extensive tissue damage. No distinct changes except for hyperemia were found within the glomeruli. Kidneys exposed to prolonged ischemia but treated by surgical incision presented completely preserved renal structures and excellent viability with no signs for necrosis. Original magnification was ×200.

DISCUSSION

Generally, a compartment syndrome is an acute disease pattern characterized by increased pressure and, as a consequence, critically compromised microcirculation within a confined space. This pathophysiologic situation most commonly occurs in fascial compartments of the limbs and the abdominal compartment (5, 6). Depending on the extent and duration of pressure increase, this condition may lead to impaired blood supply, neurologic deficit, and organ/tissue damage. Compartment syndromes require prompt treatment to avoid chronic functional loss. This is achieved by management of the etiologic problem and compartmental pressure relief (7–10).

To the best of our knowledge, this is the first study to investigate the influence of increased pressure within the renal capsule on the functional outcome and the therapeutic potential of a surgical treatment option. So far, an internal compartment syndrome of the kidney after severe parenchymal trauma, ischemia-reperfusion injury, or transplantation has not been described. Our observation that ischemia-reperfusion injury increases subcapsular kidney pressure suggests that pathophysiologic changes similar to other compartment syndromes may occur within the tight fibrous kidney capsule. In fact, decompression of the renal compartment by a controlled capsulotomy relieved subcapsular pressure and significantly improved renal function. Importantly, linear incisions led to uncontrolled rupture of the kidney capsule, microcirculatory impairment due to traction/compression of the incisional margins, and protrusion of renal parenchyma. Only standardized minimal puncture was necessary to confer renoprotection and beneficial effects were evident even in long-term follow-up. The exact mechanisms of action are not entirely clear yet. In particular, it remains to be elucidated how a relatively small circular incision exerts considerable relief of subcapsular pressure to essentially normal values. Further studies using preclinical models closer to humans may provide valuable information in this regard. However, background ROI measurements of the kidneys revealed a significant perirenal accumulation of radioactivity in the therapy group 2 days after ischemia. This may be due to the drainage of interstitial fluid via the capsulotomy and suggests that this tiny incision works as a pressure compensating valve. The circular shape of the incision may also reduce surface tension of the kidney capsule, thus mediating intracapsular pressure relief. Again, it is unclear how this measurable subcapsular pressure changes translate into transparenchymal pressure distribution and how these effects in detail affect nephron function, but it is tempting to believe that this internal kidney compartment syndrome may initiate a vicious circle similar to other compartment syndromes (11–13).

In this study, prolonged ischemia resulted in a biphasic rise in pressure characterized by an early edema-related pressure elevation within the first 24 hr after ischemia and a late increase in pressure on day 18. The latter may be due to contraction due to atrophy and fibrosis. This is in line with the well-known consequences of compartment syndromes of the limb (14). Early subcapsular pressure increase is attributed to tubular dilatation 6 hr after ischemia, whereas interstitial edema seems to play a major role at 12 and 24 hr reperfusion time. As interstitial edema causes more pronounced restriction of the confined intracapsular space, this may also explain the higher pressure levels compared with the moderately increased pressure 18 days after ischemia. Mild ischemia only resulted in early moderate pressure increase but had no consequences on subcapsular pressure, renal function, and blood flow in the long term. In contrast, chronically elevated pressure involved persistent impairment of renal function and perfusion.

Using an optimized protocol for 99mTc-MAG3 scintigraphy-based determination of renal function, we were able to precisely assess long-term alterations of renal function in the same individual, thus overcoming the limitations of standard parameters such as serum creatinine and blood urea nitrogen that regularly decline to baseline levels within a few days in murine models of kidney injury (15–17). Importantly, this technique also bears the major advantage of collecting separate data sets for each kidney thus allowing the use of a less harmful protocol for unilateral renal ischemia. In this setting, compensation of renal function by the contralateral healthy kidney reduces the experimental burden for the animals and enables the use of injury strategies that go beyond the spontaneous regenerative capacity of the mice.

The role of an intrinsic compartment syndrome of the transplant kidney may have been underestimated. Although the relevance of our findings to renal transplantation remains speculative, the results of this study suggest that increased pressure within the graft may contribute to graft dysfunction. Moreover, standardized capsulotomy alone was efficient enough to preserve renal function and perfusion. Our data strongly support the existence of an intrinsic renal compartment syndrome after ischemia-reperfusion injury. Considering the complexity of the renal system, the development of novel therapeutic approaches to prevent, treat, or even reverse chronic allograft dysfunction represents a great challenge in the field of transplantation research. The major goal is to prolong function and survival of the renal allograft, and to improve quality of life for the allograft recipient. Beneficial effects of decompression seen in a murine model of renal ischemia-reperfusion injury appear promising, and it is tempting to speculate whether it will succeed in humans. Further studies will have to be performed to elucidate the precise pathomechanisms and consequences of elevated subcapsular pressure for the functional outcome of renal allografts, until novel therapeutic options can be translated into clinical practice.

ACKNOWLEDGMENTS

The authors thank Nikolaus Plesnila, M.D., Royal College of Surgeons in Ireland, Dublin, Ireland, for his support and Cornelia Arszol-Ihli, Department of Nuclear Medicine, University of Munich, Munich, Germany, for her excellent technical assistance. This report includes data that were generated during the doctoral thesis of Andreas Meyer at the Medical School of the University of Munich, Munich, Germany.

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Keywords:

Transplantation; Compartment; Kidney; Renal function; Scintigraphy

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