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Nephrology 19 (2014) 410–419
Original Article
Acute kidney injury in rats with or without pre-existing chronic kidney disease: Cytokine/chemokine response MARTIN SKOTT,1,2 RIKKE NØRREGAARD,2,3 HANNE BIRKE-SØRENSEN,3 JOHAN PALMFELDT,4 TAE-HWAN KWON,5 JØRGEN FRØKIÆR2,3 and SØREN NIELSEN1,2 1 Department of Biomedicine, 2The Water & Salt Research Center and 3Institute of Clinical Medicine, Aarhus University, 4Research Unit for Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark; and 5Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea
KEY WORDS: acute kidney injury, chronic kidney disease, cytokines and chemokines, intestinal ischaemia and reperfusion. Correspondence: Professor Søren Nielsen, Department of Biomedicine, The Water and Salt Research Center, Aarhus University, Health, DK-8000 Aarhus, Denmark. Email: [email protected] Accepted for publication 9 April 2014. Accepted manuscript online 15 April 2014. doi:10.1111/nep.12263 Conflict of interest: The authors declare no competing interests.
SUMMARY AT A GLANCE Established a rat model of pre-existing chronic kidney injury and provided evidence that a second acute kidney injury challenge did not alter the immediate cytokine response to the acute injury compared with rats without chronic kidney injury.
ABSTRACT: Aim: Evidence suggests the possibility that pre-existing chronic kidney (CKD) disease may result in a more severe outcome of acute kidney injury (AKI). The aim of this study was to examine whether CKD enhances the inflammatory response in the kidney, as well as other organs, in response to AKI in rats. Methods: CKD was induced by 5/6 nephrectomy (Nx) and AKI by intestinal ischaemia and reperfusion (IIR). Results: For 6 weeks following Nx there was a progressive increase in serum creatinine with associated development of albuminuria. The increment in creatinine above baseline determination 90 min following IIR was comparable in 5/6 Nx and in the sham 5/6 Nx. Similarly, increased levels of serum alanine transaminase and histomorphological changes in the lungs were observed in the rats exposed to IIR compared with those exposed to sham IIR, with no additional significant impact of 5/6 Nx. In kidney tissue the levels of cytokines/chemokines were equally elevated regardless of exposure to sham IIR or IIR. In lung and liver tissue the levels of cytokines/ chemokines were equally elevated in the rats that were exposed to IIR, regardless of exposure to sham Nx or Nx. Conclusion: We conclude that the immediate severity of AKI induced by IIR in rats with CKD is similar to that induced in rats without CKD. However, the impact of Nx on the cytokine/chemokine response after AKI is not uniform in kidney, lung or liver tissue.
Chronic kidney disease (CKD) is a common risk factor in the development of acute kidney injury (AKI), and the risk of AKI increases proportionally with CKD stage.1,2 Additionally, the need for renal replacement treatment (RRT) is greater if AKI is superimposed in patients with pre-existing CKD than in those without CKD.3 This association between CKD and AKI has been supported widely epidemiologically but to a lesser extent biologically.4–7 Therefore, in one part of the present study, we hypothesized that rats with CKD were more vulnerable to acute episodes of renal injury induced by intestinal ischaemia and reperfusion (IIR). IIR induces a state of severe systemic inflammation7 that occurs in many forms of severe illness.8 Central to this process is the release of a 410
cascade of potent pro-inflammatory mediators (e.g. cytokines, chemokines and leukocytes) that are required for the initiation of an effective inflammatory response.8 Clinical studies have investigated the prognostic role of several cytokines in predicting the outcome of severe illness.9 In relation to AKI, higher levels of cytokines are found in non-survivors than in survivors.10 Other studies have shown that a state of low-grade chronic inflammation occurs in patients with CKD. Part of the mechanism behind this condition is an increased production and reduced clearance of cytokines and their soluble receptors.11,12 Thus, we hypothesized that a state of chronic inflammation caused by CKD enhances or attenuates the inflammatory response within © 2014 Asian Pacific Society of Nephrology
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the kidneys as well as other organs in response to IIRinduced AKI. To examine this, we used a recently developed 2-stage rodent model in which CKD was induced by 5/6 nephrectomy (Nx) and AKI was induced by IIR.7 Briefly, 5/6 nephrectomy is an experimental model that is widely used to gain insight into the mechanisms responsible for the development of CKD.13,14 Six weeks after the 5/6 nephrectomy, IIR was performed. Intestinal injury activates a variety of circulating inflammatory mediators such as leucocytes and induces the synthesis and release of pro-inflammatory cytokines.15,16 Together these mediators induce multiple organ failure (MOF), including acute pulmonary, hepatic and renal injury.17–20 In this model of IIR-induced AKI, decreased renal blood flow, microvascular injury and compromised tubular function are observed within the kidneys.19,20
METHODS Animals Fifty-one male Wistar rats (Taconic Eiby, Denmark) were included in the study. The animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996) and were approved by the Danish Ministry of Justice. All rats were housed in at least pairs at a room temperature of 21°C with alternating 12:12-h light-dark cycles and fed standard rat chow (Altromin, Lage, Germany) with free access to tap water.
Experimental design and surgery The rats were randomly divided into the following 5 groups: Nx + IIR group, nephrectomy and IIR (n = 11): S(Nx) + IIR group, sham nephrectomy and IIR (n = 12): Nx + S(IIR) group, nephrectomy and sham IIR (n = 7): S(Nx) + S(IIR) group, sham nephrectomy and sham IIR (n = 9): and Control group, untreated rats (n = 12). Blood was drawn from the tail vein at the following 3 time points: ‘At Right 1/1 Nx’ (total right nephrectomy), ‘Two weeks after 5/6 Nx’ and ‘Before IIR’. Prior to this, each rat was housed in a metabolic cage for 3 days for 24-h urine collection. On the day of surgery, animals were anesthetized with 2% isoflurane (Abbot, Illinois, USA) and atmospheric air at 2 L/min. The renal mass reduction (5/6 nephrectomy) was performed by excision of 2/3 of the left kidney. One week later, total nephrectomy of the right kidney was performed (Fig. 1). Sham operations (S(Nx)) were performed by manipulation of the kidneys without removal of any renal mass. Calculations of the amount of kidney tissue removed were based on the assumption that the kidneys weighed the same. The following equation was used: [(total nephrectomy weight + partial nephrectomy weight)/(2 × total nephrectomy weight)] × 100. Six weeks after the 5/6 nephrectomy, IIR was performed as previously described.7 Briefly, the small intestine was exteriorized through a midline laparotomy and the superior mesenteric artery (SMA) was occluded at its origin from the abdominal aorta for 45 min. Before the abdominal wall was closed, 1 mL of 37°C saline © 2014 Asian Pacific Society of Nephrology
was instilled into the peritoneal cavity and 1 mL was administered subcutaneously. After waking, the rats were monitored in cages until 90 min after the ischaemia, when euthanasia was performed. Sham operations (S(IIR)) were performed by laparotomy and manipulation of the SMA without occlusion. During each surgical procedure, the rats were placed on a heated table to maintain rectal temperatures of 37–38°C. Initially at each surgical procedure buprenophium 0.3 mg/kg (Temgesic; Reckitt and Colman) was injected subcutaneously to relieve the pain and thereafter administered in their drinking water at a similar dose.
Assessment of renal function Serum creatinine and urine albumin were determined colorimetrically (Vitros 5.1; Johnson & Johnson). Urine osmolality was measured with a vapour pressure osmometer (Osmomat 030: Genotec, Berlin, Germany). Blood was collected from the tail vein except at euthanasia, at which time it was drawn from the right ventricle of the heart. In this study, AKI was defined as a significant increase in serum creatinine during the IIR procedure. Enzyme-linked immunosorbent assays were performed to detect urinary neutrophil gelatinase-associated lipocalin (NGAL); briefly, the urine samples were centrifuged to remove debris and the supernatant was analysed in triplicate (Bioporto Diagnostics, Gentofte, Denmark).
Measurement of pro-inflammatory cytokines and chemokines in the lung, kidneys and liver The pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF-α) as well as the chemokines monocyte chemotactic protein-1 (MCP-1α) and RANTES were measured in the kidney, lung and liver homogenates using a Procarta Cytokine Assay kit (Invitrogen A/S, Taastrup, Denmark) according to the manufacturer’s instructions. The samples were analysed on a Luminex 100 IS flow system (Luminex Corporation, Austin, TX, USA). Briefly, the colour code of each bead identifies the target protein being assayed, while the fluorescent intensities on the beads measure target concentration. The tissue samples were diluted in the assay buffer and measured in duplicate. Total system raw median fluorescence intensity values were used to calculate the concentrations (pg/mL) from 4-parameter logistic fitted standard curves using StarStation version 2.3 software. The fits expressed by the R-squared value were all >0.995. To adjust measurements for differences in total protein content of the samples, values are expressed in picograms per total gram of protein. Total protein concentrations were determined using a Pierce BCA Protein Assay Kit.
Lung histomorphometry and immunohistochemistry After immersion fixation, lung specimens were embedded in paraffin. Sections were stained with eosin for light microscopic analysis. Only peripheral lung parenchyma was examined. Microscopic fields containing other structures, such as airways, large vessels and pleura, were excluded. High-contrast images at 10 × magnification were obtained and processed by ImageJ 1.43u software (National Institutes of Health, Bethesda, MD, USA) to measure the ratio between the airspace and the lung parenchyma. 411
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Fig. 1 Experimental design. 5/6 Nephrectomy (Nx) was performed in two steps, 2/3 left Nx followed by right total (1/1) Nx one week later. Six weeks after 5/6 Nx, intestinal ischaemia and reperfusion (IIR) were performed. Sham operations were performed for each surgical procedure as well. The control group was not subjected to any surgical procedures. The arrows indicate time of blood and urine sampling.
Liver enzymes Serum alanine aminotransferase (ALT) values were determined by using the Vitros 5.1 system (Ortho Clinical Diagnostics, New Brunswick, NJ, USA) with slide technology.
Statistical analysis Data are presented as mean ± standard error of the mean. Comparisons among groups were performed using one-way ANOVA. When the statistical model was significant, further post hoc analyses were performed using the Tukey–Kramer method for multiple comparisons using STATA v.10.1 (Stata Corporation, Texas, USA). Statistical analyses of the cytokine and chemokine measurements were performed on log-transformed data to yield normality. Analysis of normality was performed using Shapiro–Wilk test in STATA. A twosided P-value of < 0.05 was accepted as statistically significant.
RESULTS Physiological data Body weight was significantly lower in the 5/6 nephrectomized (Nx) rats compared with the sham 5/6 412
nephrectomized (S(Nx)) and control rats at time point ‘Before IIR’ (data not shown). During the 6 weeks of follow-up after the 5/6 nephrectomy, 9 (32%) of the 5/6 nephrectomized rats and 3 (13%) of the sham nephrectomized rats were excluded. Exclusion criteria included wound infections, extreme weight loss and spontaneous death. The excluded rats were not included in the data analysis at any time point. On average, 79% (data not shown) of the kidney mass was removed in the rats that underwent nephrectomy. As hypertrophy of the right kidney after 2/3 left nephrectomy may occur, the amount of the removed kidney mass may be underestimated.
Renal function after 5/6 nephrectomy Urine volume and water intake at the ‘Right 1/1 Nx’, ‘Two weeks after 5/6 Nx’ and ‘Before IIR’ time points were significantly higher in the 5/6 nephrectomized rats (Nx-rats) than in the sham 5/6 nephrectomized rats (S(Nx)-rats) (Table 1). Consistent with these changes, urine osmolality was significantly lower in the Nx-rats than in the S(Nx)-rats and control rats (Table 1). No changes or differences in serum © 2014 Asian Pacific Society of Nephrology
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Table 1 Urine analyses during the development of Chronic kidney disease (CKD) Group (n) UV (μL/min/day)
WI (μL/min/day)
UAlbumin (mg/day)
UOsm (mosmol kgH2O)
Period
Control (n = 12)
S(Nx) + S(IIR) (n = 9)
S(Nx) + IIR (n = 12)
Nx + S(IIR) (n = 7)
Nx + IIR (n = 11)
R1/3 Nx T-W a 5/6Nx Before IIR R1/3 Nx T-W a 5/6Nx Before IIR R1/3 Nx T-W a 5/6Nx Before IIR R1/3 Nx T-W a 5/6Nx Before IIR
31.8 ± 2.2 36.8 ± 1.5 31.2 ± 1.7 68.6 ± 4.1 69.1 ± 2.3 66.2 ± 1.9 ND ND ND 1722 ± 76 1627 ± 46 1617 ± 89
37.8 ± 6.2 28.4 ± 2.3* 30.1 ± 4.9 67.91 ± 9.13 75.16 ± 3.20 64.92 ± 6.56 ND ND 1.07 ± 1.07 1493 ± 16 2062 ± 17* 1601 ± 16
35.6 ± 5.2 29.1 ± 2.0* 29.8 ± 2.9 73.85 ± 7.76 74.08 ± 3.35 64.44 ± 3.17 ND ND 6.34 ± 6.26 1641 ± 12 1986 ± 91* 1531 ± 14
77.1 ± 16.8* 121.5 ± 18.3*#+ 100.7 ± 16.6*#+ 97.23 ± 14.2 172.20 ± 19.0*#+ 152.08 ± 16.8*#+ ND 6.32 ± 5.05 15.35 ± 5.99#+ 400 ± 63*#+ 461 ± 96*#+ 419 ± 56*#+
64.1 ± 5.2*#+ 82.3 ± 8.8*#+ 73.6 ± 11.6*#+ 97.8 ± 5.0*#+ 133.3 ± 11.0*#+ 114.6 ± 18.8*#+ ND 5.93 ± 3.25 12.75 ± 4.16#+ 619 ± 46*#+ 676 ± 81*#+ 758 ± 16*#+
#+
Urine was collected during housing in metabolic cages for 24 h. Values are shown as mean (±SE). *P < 0.05 versus Control; +P < 0.05 versus S(Nx) + S(IIR); #P < 0.05 versus S(Nx) + IIR. ND, non-detectable (lower detectable limit was 6 mg/L); UAlbumin, urinary albumin excretion; UOsm, urinary osmolality; UV, urine volume; WI, water intake.
sodium were seen in the groups exposed to Nx or sham Nx during the 6 weeks of follow-up (data not shown). Albuminuria was an additional indication of pre-existing renal impairment. At the ‘Two weeks after 5/6 Nx’ time point, urinary albumin excretion was detectable in the Nx-rats but not the S(Nx)-rats. At the ‘Before IIR’ time point, albumin was also detectable in the urine of the S(Nx)-rats; however, the urinary albumin levels were significantly lower than those of the Nx-rats. Urinary albumin was not detectable in the control rats (lower detection limit was 6 mg/L) throughout the study. At the ‘Right 1/1 Nx’, ‘Two weeks after 5/6 Nx’ and ‘Before IIR’ time points, serum creatinine was significantly higher in the Nx + IIR group than in the S(Nx) + IIR and Control group (Fig. 2A). A novel biomarker of CKD, NGAL was measured in the urine (Fig. 2B), and at the ‘Right 1/1 Nx’, ‘Two weeks after 5/6 Nx’ and ‘Before IIR’ time points, the level of urinary NGAL was significantly higher in the Nx + IIR group than in the S(Nx) + IIR and Control group (Fig. 2B).
Organ-specific biomarkers in the serum after IIR Significant increases of 50% (±12%) and 72% (±10%) in serum creatinine (Fig. 2A) were observed after IIR in the Nx + IIR (n = 8) and S(Nx) + IIR (n = 11) groups, respectively, indicating that AKI continued until euthanasia. However, these increases in serum creatinine were not significantly different between the groups (P = 0.25). No increases in serum creatinine were seen after IIR within the Nx + S(IIR) and S(Nx) + S(IIR) groups (data not shown). Significantly higher levels of the liver enzyme ALT (Fig. 2C) were observed in the rats exposed to IIR than in the sham IIR or control rats. We did not detect any differences in C-reactive protein (CRP) levels among the groups (data not shown). © 2014 Asian Pacific Society of Nephrology
Lung histopathology Light microscopic examination of the lung parenchyma (Fig. 3) showed a decreased ratio of airspace to tissue (as a marker of tissue swelling) in the rats that were exposed to IIR. The volume of airspace was significantly lower in the Nx + IIR group than in the other groups.
Multiplex analysis of cytokines and chemokines in the kidney, lung and liver tissues In the kidney tissues (Fig. 4), the levels of IL-6, RANTES and MCP-1 were equally elevated in the Nx + IIR and Nx + S(IIR) groups, and significantly different from those in the S(Nx) + IIR, S(Nx) + S(IIR) and control groups. In contrast IL-1β levels were significantly higher in the Nx + IIR group than in the other groups. In the lung tissues (Fig. 5), the levels of RANTES and MCP-1 were equally elevated in the Nx + IIR and S(Nx) + IIR groups, and significantly different from those in the S(Nx) + S(IIR) and control groups. In contrast, IL-1β and IL-6 were significantly higher in the Nx + IIR group than in the other groups. In the liver tissues (Fig. 6), the levels of IL-1β and MCP-1 were equally elevated in the Nx + IIR and S(Nx) + IIR groups and significantly different from those in the Nx + S(IIR), S(Nx) + S(IIR) and control groups. No differences in IL-6 and RANTES were detected between any of the groups. We did not detect any differences in TNF-α levels among the groups (data not shown).
DISCUSSION The main findings of the present study were that (i) Severity of AKI induced by a surgical procedure (IIR) in rats with 413
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Fig. 2 Organ injury during the development of chronic kidney disease (CKD) and after intestinal ischaemia and reperfusion. Renal function after 5/6 nephrectomy (5/6 Nx) and intestinal ischaemia and reperfusion (IIR) was determined by serum creatinine levels (A). Urinary neutrophil gelatinase-associated lipocalin (NGAL) (B) levels were determined after 5/6 Nx. Liver function after IIR was determined using alanine transaminase (ALT) (C). Values are presented as means (±SE). *P < 0.05 versus control; +P < 0.05 versus S(Nx) + S(IIR); #P < 0.05 versus S(Nx) + IIR; §P < 0.05 versus Nx + S(IIR); B−IIRP < 0.05 compared with the ‘Before IIR’ time point within ) S(Nx) + IIR (#), ( ) Nx + IIR, ( ) control (*). C: ( ) control (*), ( ) S(Nx) + S(IIR) (+), ( ) S(Nx) + IIR (#), ( ) Nx + S(IIR) the same group. A and B: ( ) Nx + IIR. (§), (
CKD is similar to that induced in normal rats without CKD. (ii) Upon exposure to IIR, rats with CKD developed a more pronounced pro-inflammatory cytokine and chemokine response in organs remote to the kidney. (iii) Majority of pro-inflammatory cytokine and chemokine responses within the kidneys were equally increased in the 5/6 nephrectomized rats regardless of IIR exposure. 414
CKD as a state of ‘chronic low-grade inflammation’ Chronically elevated common phenomenon undergoing dialysis. chemokines contribute
inflammatory biomarkers are a in patients with CKD who are not Pro-inflammatory cytokines and to a state of persistent low-grade © 2014 Asian Pacific Society of Nephrology
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Fig. 3 Chronic kidney disease (CKD) enhances lung injury after intestinal ischaemia and reperfusion (IIR). Rats were exposed to nephrectomy (Nx) (D,E), sham Nx (A,B) and or IIR(B,E) or sham IIR (A,D). Untreated rats served as controls (C). Lung injury was determined histologically in sections of peripheral lung parenchyma (n = 7–8/group). Light microscopic pictures (n = 4/animal) presented in high contrast were processed by ImageJ software to measure the ration of airspace (black) to lung parenchyma (white). Calculated area fractions with means from within each group are shown (F). Values are presented as means (±SE). *P < 0.05 versus é control; +P < 0.05 versus S(Nx) + S(IIR); #P < 0.05 versus S(Nx) + IIR; §P < 0.05 versus Nx + S(IIR). ( ) control (*), (○) S(Nx) + S(IIR) (+), (▼) S(Nx) + IIR (#), ( ) Nx + S(IIR) (§), ( ) Nx + IIR.
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inflammation.12 Of the pro-inflammatory cytokines, increased IL-6 and TNF-α levels have been demonstrated to be correlated with the degree of chronic renal impairment in patients with CKD.11,21 In the present study, we found elevated IL-6 levels in the kidneys of rats with surgically induced CKD regardless of IIR exposure. However, we did not find any significant correlation between sCr, a biomarker © 2014 Asian Pacific Society of Nephrology
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of CKD severity and kidney tissue IL-6 levels in the CKD group exposed to sham IIR at the end of the study (data not shown). Additionally, we did not detect any differences in the renal TNF-α level among the groups (data not shown). Similarly to pro-inflammatory cytokines, accumulating data from clinical studies and animal models of CKD support the perception that chemokines play a role during the devel415
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Fig. 4 Kidney tissue cytokine levels after intestinal ischemia & reperfusion (IIR). Pro-inflammatory cytokines interleukin (IL)-1β (A) and IL-6 (B) as well as the chemokines RANTES (C), MCP-1 (D) were measured in whole kidney tissue homogenates using multiplex assays. Individual tissue concentrations and means (horizontal line) within each group are shown. *P < 0.05 versus control; +P < 0.05 versus S(Nx) + S(IIR); #P < 0.05 versus S(Nx) + IIR; §P < 0.05 versus Nx + S(IIR). é ( ) control (*), (○) S(Nx) + S(IIR) (+), (▼) S(Nx) + IIR (#), ( ) Nx + S(IIR) (§), ( ) Nx + IIR.
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opment of CKD.1,2,22 Our data confirm increased MCP-1 levels in the kidney tissues of 5/6 nephrectomized rats compared with the levels in sham 5/6 nephrectomized rats.3,23 Various mechanisms might be responsible for the accompanying ‘uremic inflammation’ in response to decreased renal function. In addition to increased production and reduced renal excretion of pro-inflammatory cytokines and their soluble receptors,4–7 sympathetic overactivity and blunted vagal nerve activity, which inhibit local cytokine release, may also lead to an increased inflammatory response.7,24 416
Pre-existing CKD may predisposes rats to a more severe inflammatory response in organs other than the kidneys In the present study, we determined that rats with CKD compared with non-CKD develop a partially more pronounced pro-inflammatory cytokine (IL-1β, IL-6) response in the lungs upon IIR exposure. Similar findings were observed with plasma IL-6 levels by Leelahavenichkul et al.8,25 in an alternative AKI model and others have reported increased levels of IL-6 and MCP-1 in kidney tissues after AKI induced by renal ischaemia and reperfusion (I/R)8,26,27 in © 2014 Asian Pacific Society of Nephrology
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Fig. 5 Lung tissue cytokines levels after intestinal ischemia & reperfusion (IIR). Pro-inflammatory cytokines IL-1β (A), IL-6 (B) and chemokines RANTES (C), MCP-1 (D) were measured in lung tissue homogenates by multiplex assays. Individual tissue concentrations and mean (horizontal line) within each group are shown. *P < 0.05 versus Control; +P < 0.05 versus S(Nx) + S(IIR); #P < 0.05 versus S(Nx) + IIR; §P < 0.05 versus Nx + S(IIR). ( ) control (*), (○) S(Nx) + S(IIR) (+), (▼) S(Nx) + IIR é (#), ( ) Nx + S(IIR) (§), ( ) Nx + IIR.
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rodents with normal kidney function prior to AKI. That model was fundamentally different from the one used in the current study, in which cytokine and chemokine responses within the kidney were equally increased (beside IL-1β) in rats with CKD regardless of IIR exposure.
Severity of IIR-induced AKI in rats with CKD is similar to that induced in rats without CKD We observed an increase in serum creatinine after IIR in the rats that were 5/6 nephrectomized and those that were © 2014 Asian Pacific Society of Nephrology
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sham 5/6 nephrectomized. The fractional increase in serum creatinine between the groups was not significantly different, consistent with a similar magnitude of response to IIR in rats with CKD compared with those without CKD. A plausible explanation is that as glomerular filtration rate (GFR) falls, creatinine secretion is increased, and thus the rise in serum creatinine is less. Therefore a similar relative increase in serum creatinine detected in the CKD rats compared with those without CKD may not be an accurate reflection of the GFR changes in the non-steady-state condition of AKI.28 417
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Fig. 6 Liver tissue cytokines levels after intestinal ischemia & reperfusion (IIR). Pro-inflammatory cytokines IL-1β (A), IL-6 (B) and chemokines RANTES (C), MCP-1 (D) were measured in lung tissue homogenates by multiplex assays. Individual tissue concentrations and mean (horizontal line) within each group are shown. *P < 0.05 versus Control; +P < 0.05 versus S(Nx) + S(IIR); #P < 0.05 versus S(Nx) + IIR; §P < 0.05 versus Nx + S(IIR). ( ) control (*), (○) S(Nx) + S(IIR) (+), (▼) S(Nx) + IIR é (#), ( ) Nx + S(IIR) (§), ( ) Nx + IIR.
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CONCLUSION The present study is not without its limitations. First, we measured cytokines in whole tissue homogenates instead of plasma, which is used in clinical studies of MOF.29 The reason for this are that a significant proportion of the cytokines act via paracrine mechanisms and have a short half-life in plasma, making them very difficult to detect.30 Second, we were not able to answer the question of why persistent uremic inflammation, which occurs during CKD, increases the inflammatory response within the lung tissue. One explanation might be a reduction in renal clearance of cytokine/chemokines as a consequence of pre-existing CKD. 418
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Another may be, that increased capillary permeability and decreased bacterial clearance shown in animals with CKD, predispose them to the development of MOF.4 Our model of surgically induced AKI superimposed onto CKD in rodents is suitable for further investigation of the pathophysiology behind effects on the immediate early organ dysfunction in this clinically relevant condition.
ACKNOWLEDGEMENTS We thank Inge Marete Poulsen, Line V. Nielsen, Gitte B. Kall, Gitte Skou, Kirsten Strauss and Birgitte K. Sahl for their expert technical assistance and Professor Robert Fenton for © 2014 Asian Pacific Society of Nephrology
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his feedback during the manuscript preparation process. This work was supported by the Water and Salt research Centre (established and supported by the Danish National Research Foundation), Faculty of Health Science at the University of Aarhus, Novo Nordisk Foundation, Aase and Ejnar Danielsens Foundation, Augstinus Foundation and C.C. Klestrup and Hustru Henriette Klestrups Mindelegat.
REFERENCES 1. James MT, Hemmelgarn BR, Wiebe N et al. Glomerular filtration rate, proteinuria, and the incidence and consequences of acute kidney injury: A cohort study. Lancet 2010; 376: 2096–103. 2. Hsu C-Y, Chertow G, McCulloch C, Fan D, Ordonez J, Go A. Nonrecovery of kidney function and death after acute on chronic renal failure. Clin. J. Am. Soc. Nephrol. 2009; 4: 891–8. 3. Lee P-H, Wu V-C, Hu F-C et al. Outcomes following dialysis for acute kidney injury among different stages of chronic kidney disease. Am. J. Nephrol. 2011; 34: 95–103. 4. Doi K, Leelahavanichkul A, Hu X et al. Pre-existing renal disease promotes sepsis-induced acute kidney injury and worsens outcome. Kidney Int. 2008; 74: 1017–25. 5. Vercauteren SR, Ysebaert DK, De Greef KE, Eyskens EJ, De Broe ME. Chronic reduction in renal mass in the rat attenuates ischemia/reperfusion injury and does not impair tubular regeneration. J. Am. Soc. Nephrol. 1999; 10: 2551–61. 6. Goldfarb M, Rosenberger C, Abassi Z et al. Acute-on-chronic renal failure in the rat: Functional compensation and hypoxia tolerance. Am. J. Nephrol. 2006; 26: 22–33. 7. Skott M, Nørregaard R, Sorensen HB, Kwon T-H, Frokiaer J, Nielsen S. Pre-existing renal failure worsens the outcome after intestinal ischaemia and reperfusion in rats. Nephrol. Dial. Transplant. 2010; 25: 3509–17. 8. Bone R. Immunologic dissonance: A continuing evolution in our understanding of the systemic inflammatory response syndrome (SIRS) and the multiple organ dysfunction syndrome (MODS). Ann. Intern. Med. 1996; 125: 680–87. 9. Gogos CA, Drosou E, Bassaris HP, Skoutelis A. Pro- versus anti-inflammatory cytokine profile in patients with severe sepsis: A marker for prognosis and future therapeutic options. J. Infect. Dis. 2000; 181: 176–80. 10. Simmons E, Himmelfarb J, Sezer M et al. Plasma cytokine levels predict mortality in patients with acute renal failure. Kidney Int. 2004; 65: 1357–65. 11. Herbelin A, Ureña P, Nguyen AT, Zingraff J, Descamps-Latscha B. Elevated circulating levels of interleukin-6 in patients with chronic renal failure. Kidney Int. 1991; 39: 954–60. 12. Landray MJ, Wheeler DC, Lip GYH et al. Inflammation, endothelial dysfunction, and platelet activation in patients with chronic kidney disease: The chronic renal impairment in Birmingham (CRIB) study. Am. J. Kidney Dis. 2004; 43: 244–53. 13. Kwon T, FrokiAer J, Knepper M, Nielsen S. Reduced AQP1, -2, and -3 levels in kidneys of rats with CRF induced by surgical reduction in renal mass. Am. J. Physiol. 1998; 275 (5 Pt 2): F724–41.
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14. Kaufman J, Siegel N, Hayslett J. Functional and hemodynamic adaptation to progressive renal ablation. Circ. Res. 1975; 36: 286–93. 15. Moore F. The role of the gastrointestinal tract in postinjury multiple organ failure. Am. J. Surg. 1999; 178: 449–53. 16. Grotz MR, Deitch EA, Ding J, Xu D, Huang Q, Regel G. Intestinal cytokine response after gut ischemia: Role of gut barrier failure. Ann. Surg. 1999; 229: 478–86. 17. Caty M, Guice K, Oldham K, Remick D, Kunkel S. Evidence for tumor necrosis factor-induced pulmonary microvascular injury after intestinal ischemia-reperfusion injury. Ann. Surg. 1990; 212: 694–700. 18. Yao J-H, Zhang X-S, Zheng S-S et al. Prophylaxis with carnosol attenuates liver injury induced by intestinal ischemia/reperfusion. World J. Gastroenterol. 2009; 15: 3240–45. 19. LaNoue J, Turnage R, Kadesky K, Guice K, Oldham K, Myers S. The effect of intestinal reperfusion on renal function and perfusion. J. Surg. Res. 1996; 64: 19–25. 20. Rothenbach P, Turnage R, Iglesias J, Riva A, Bartula L, Myers S. Downstream effects of splanchnic ischemia-reperfusion injury on renal function and eicosanoid release. J. Appl. Physiol. 1997; 83: 530–36. 21. Pecoits-Filho R, Heimbürger O, Bárány P et al. Associations between circulating inflammatory markers and residual renal function in CRF patients. Am. J. Kidney Dis. 2003; 41: 1212–18. 22. Chung ACK, Lan HY. Chemokines in renal injury. J. Am. Soc. Nephrol. 2011; 22: 802–9. 23. Cho K-H, Kim H-J, Rodriguez-Iturbe B, Vaziri ND. Niacin ameliorates oxidative stress, inflammation, proteinuria, and hypertension in rats with chronic renal failure. Ren. Physiol. 2009; 297: F106–13. 24. Tracey KJ. Fat meets the cholinergic antiinflammatory pathway. J. Exp. Med. 2005; 202: 1017–21. 25. Leelahavanichkul A, Huang Y, Hu X et al. Chronic kidney disease worsens sepsis and sepsis-induced acute kidney injury by releasing high mobility group box protein-1. Kidney Int. 2011; 80: 1198–211. 26. Efrati S, Berman S, Hamad RA et al. Effect of captopril treatment on recuperation from ischemia/reperfusion-induced acute renal injury. Nephrol. Dial. Transplant. 2012; 27: 136–45. 27. Gao J, Zhang D, Yang X, Zhang Y, Li P, Su X. Lysophosphatidic acid and lovastatin might protect kidney in renal I/R injury by downregulating MCP-1 in rat. Ren. Fail. 2011; 33: 805–10. 28. Bellomo R, Ronco C, Kellum JA, Mehta RL, Palevsky P, Acute Dialysis Quality Initiative Workgroup. Acute renal failure – definition, outcome measures, animal models, fluid therapy and information technology needs: The Second International Consensus Conference of the Acute Dialysis Quality Initiative (ADQI) Group. 2004. pp. R204–12. 29. Cuschieri J, Bulger E, Schaeffer V et al. Early elevation in random plasma IL-6 after severe injury is associated with development of organ failure. Shock 2010; 34: 346–51. 30. Kenis G, Teunissen C, De Jongh R, Bosmans E, Steinbusch H, Maes M. Stability of interleukin 6, soluble interleukin 6 receptor, interleukin 10 and CC16 in human serum. Cytokine 2002; 19: 228–35.
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Nephrology 19 (2014) 410–419
Original Article
Acute kidney injury in rats with or without pre-existing chronic kidney disease: Cytokine/chemokine response MARTIN SKOTT,1,2 RIKKE NØRREGAARD,2,3 HANNE BIRKE-SØRENSEN,3 JOHAN PALMFELDT,4 TAE-HWAN KWON,5 JØRGEN FRØKIÆR2,3 and SØREN NIELSEN1,2 1 Department of Biomedicine, 2The Water & Salt Research Center and 3Institute of Clinical Medicine, Aarhus University, 4Research Unit for Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark; and 5Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea
KEY WORDS: acute kidney injury, chronic kidney disease, cytokines and chemokines, intestinal ischaemia and reperfusion. Correspondence: Professor Søren Nielsen, Department of Biomedicine, The Water and Salt Research Center, Aarhus University, Health, DK-8000 Aarhus, Denmark. Email: [email protected] Accepted for publication 9 April 2014. Accepted manuscript online 15 April 2014. doi:10.1111/nep.12263 Conflict of interest: The authors declare no competing interests.
SUMMARY AT A GLANCE Established a rat model of pre-existing chronic kidney injury and provided evidence that a second acute kidney injury challenge did not alter the immediate cytokine response to the acute injury compared with rats without chronic kidney injury.
ABSTRACT: Aim: Evidence suggests the possibility that pre-existing chronic kidney (CKD) disease may result in a more severe outcome of acute kidney injury (AKI). The aim of this study was to examine whether CKD enhances the inflammatory response in the kidney, as well as other organs, in response to AKI in rats. Methods: CKD was induced by 5/6 nephrectomy (Nx) and AKI by intestinal ischaemia and reperfusion (IIR). Results: For 6 weeks following Nx there was a progressive increase in serum creatinine with associated development of albuminuria. The increment in creatinine above baseline determination 90 min following IIR was comparable in 5/6 Nx and in the sham 5/6 Nx. Similarly, increased levels of serum alanine transaminase and histomorphological changes in the lungs were observed in the rats exposed to IIR compared with those exposed to sham IIR, with no additional significant impact of 5/6 Nx. In kidney tissue the levels of cytokines/chemokines were equally elevated regardless of exposure to sham IIR or IIR. In lung and liver tissue the levels of cytokines/ chemokines were equally elevated in the rats that were exposed to IIR, regardless of exposure to sham Nx or Nx. Conclusion: We conclude that the immediate severity of AKI induced by IIR in rats with CKD is similar to that induced in rats without CKD. However, the impact of Nx on the cytokine/chemokine response after AKI is not uniform in kidney, lung or liver tissue.
Chronic kidney disease (CKD) is a common risk factor in the development of acute kidney injury (AKI), and the risk of AKI increases proportionally with CKD stage.1,2 Additionally, the need for renal replacement treatment (RRT) is greater if AKI is superimposed in patients with pre-existing CKD than in those without CKD.3 This association between CKD and AKI has been supported widely epidemiologically but to a lesser extent biologically.4–7 Therefore, in one part of the present study, we hypothesized that rats with CKD were more vulnerable to acute episodes of renal injury induced by intestinal ischaemia and reperfusion (IIR). IIR induces a state of severe systemic inflammation7 that occurs in many forms of severe illness.8 Central to this process is the release of a 410
cascade of potent pro-inflammatory mediators (e.g. cytokines, chemokines and leukocytes) that are required for the initiation of an effective inflammatory response.8 Clinical studies have investigated the prognostic role of several cytokines in predicting the outcome of severe illness.9 In relation to AKI, higher levels of cytokines are found in non-survivors than in survivors.10 Other studies have shown that a state of low-grade chronic inflammation occurs in patients with CKD. Part of the mechanism behind this condition is an increased production and reduced clearance of cytokines and their soluble receptors.11,12 Thus, we hypothesized that a state of chronic inflammation caused by CKD enhances or attenuates the inflammatory response within © 2014 Asian Pacific Society of Nephrology
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the kidneys as well as other organs in response to IIRinduced AKI. To examine this, we used a recently developed 2-stage rodent model in which CKD was induced by 5/6 nephrectomy (Nx) and AKI was induced by IIR.7 Briefly, 5/6 nephrectomy is an experimental model that is widely used to gain insight into the mechanisms responsible for the development of CKD.13,14 Six weeks after the 5/6 nephrectomy, IIR was performed. Intestinal injury activates a variety of circulating inflammatory mediators such as leucocytes and induces the synthesis and release of pro-inflammatory cytokines.15,16 Together these mediators induce multiple organ failure (MOF), including acute pulmonary, hepatic and renal injury.17–20 In this model of IIR-induced AKI, decreased renal blood flow, microvascular injury and compromised tubular function are observed within the kidneys.19,20
METHODS Animals Fifty-one male Wistar rats (Taconic Eiby, Denmark) were included in the study. The animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996) and were approved by the Danish Ministry of Justice. All rats were housed in at least pairs at a room temperature of 21°C with alternating 12:12-h light-dark cycles and fed standard rat chow (Altromin, Lage, Germany) with free access to tap water.
Experimental design and surgery The rats were randomly divided into the following 5 groups: Nx + IIR group, nephrectomy and IIR (n = 11): S(Nx) + IIR group, sham nephrectomy and IIR (n = 12): Nx + S(IIR) group, nephrectomy and sham IIR (n = 7): S(Nx) + S(IIR) group, sham nephrectomy and sham IIR (n = 9): and Control group, untreated rats (n = 12). Blood was drawn from the tail vein at the following 3 time points: ‘At Right 1/1 Nx’ (total right nephrectomy), ‘Two weeks after 5/6 Nx’ and ‘Before IIR’. Prior to this, each rat was housed in a metabolic cage for 3 days for 24-h urine collection. On the day of surgery, animals were anesthetized with 2% isoflurane (Abbot, Illinois, USA) and atmospheric air at 2 L/min. The renal mass reduction (5/6 nephrectomy) was performed by excision of 2/3 of the left kidney. One week later, total nephrectomy of the right kidney was performed (Fig. 1). Sham operations (S(Nx)) were performed by manipulation of the kidneys without removal of any renal mass. Calculations of the amount of kidney tissue removed were based on the assumption that the kidneys weighed the same. The following equation was used: [(total nephrectomy weight + partial nephrectomy weight)/(2 × total nephrectomy weight)] × 100. Six weeks after the 5/6 nephrectomy, IIR was performed as previously described.7 Briefly, the small intestine was exteriorized through a midline laparotomy and the superior mesenteric artery (SMA) was occluded at its origin from the abdominal aorta for 45 min. Before the abdominal wall was closed, 1 mL of 37°C saline © 2014 Asian Pacific Society of Nephrology
was instilled into the peritoneal cavity and 1 mL was administered subcutaneously. After waking, the rats were monitored in cages until 90 min after the ischaemia, when euthanasia was performed. Sham operations (S(IIR)) were performed by laparotomy and manipulation of the SMA without occlusion. During each surgical procedure, the rats were placed on a heated table to maintain rectal temperatures of 37–38°C. Initially at each surgical procedure buprenophium 0.3 mg/kg (Temgesic; Reckitt and Colman) was injected subcutaneously to relieve the pain and thereafter administered in their drinking water at a similar dose.
Assessment of renal function Serum creatinine and urine albumin were determined colorimetrically (Vitros 5.1; Johnson & Johnson). Urine osmolality was measured with a vapour pressure osmometer (Osmomat 030: Genotec, Berlin, Germany). Blood was collected from the tail vein except at euthanasia, at which time it was drawn from the right ventricle of the heart. In this study, AKI was defined as a significant increase in serum creatinine during the IIR procedure. Enzyme-linked immunosorbent assays were performed to detect urinary neutrophil gelatinase-associated lipocalin (NGAL); briefly, the urine samples were centrifuged to remove debris and the supernatant was analysed in triplicate (Bioporto Diagnostics, Gentofte, Denmark).
Measurement of pro-inflammatory cytokines and chemokines in the lung, kidneys and liver The pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF-α) as well as the chemokines monocyte chemotactic protein-1 (MCP-1α) and RANTES were measured in the kidney, lung and liver homogenates using a Procarta Cytokine Assay kit (Invitrogen A/S, Taastrup, Denmark) according to the manufacturer’s instructions. The samples were analysed on a Luminex 100 IS flow system (Luminex Corporation, Austin, TX, USA). Briefly, the colour code of each bead identifies the target protein being assayed, while the fluorescent intensities on the beads measure target concentration. The tissue samples were diluted in the assay buffer and measured in duplicate. Total system raw median fluorescence intensity values were used to calculate the concentrations (pg/mL) from 4-parameter logistic fitted standard curves using StarStation version 2.3 software. The fits expressed by the R-squared value were all >0.995. To adjust measurements for differences in total protein content of the samples, values are expressed in picograms per total gram of protein. Total protein concentrations were determined using a Pierce BCA Protein Assay Kit.
Lung histomorphometry and immunohistochemistry After immersion fixation, lung specimens were embedded in paraffin. Sections were stained with eosin for light microscopic analysis. Only peripheral lung parenchyma was examined. Microscopic fields containing other structures, such as airways, large vessels and pleura, were excluded. High-contrast images at 10 × magnification were obtained and processed by ImageJ 1.43u software (National Institutes of Health, Bethesda, MD, USA) to measure the ratio between the airspace and the lung parenchyma. 411
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Fig. 1 Experimental design. 5/6 Nephrectomy (Nx) was performed in two steps, 2/3 left Nx followed by right total (1/1) Nx one week later. Six weeks after 5/6 Nx, intestinal ischaemia and reperfusion (IIR) were performed. Sham operations were performed for each surgical procedure as well. The control group was not subjected to any surgical procedures. The arrows indicate time of blood and urine sampling.
Liver enzymes Serum alanine aminotransferase (ALT) values were determined by using the Vitros 5.1 system (Ortho Clinical Diagnostics, New Brunswick, NJ, USA) with slide technology.
Statistical analysis Data are presented as mean ± standard error of the mean. Comparisons among groups were performed using one-way ANOVA. When the statistical model was significant, further post hoc analyses were performed using the Tukey–Kramer method for multiple comparisons using STATA v.10.1 (Stata Corporation, Texas, USA). Statistical analyses of the cytokine and chemokine measurements were performed on log-transformed data to yield normality. Analysis of normality was performed using Shapiro–Wilk test in STATA. A twosided P-value of < 0.05 was accepted as statistically significant.
RESULTS Physiological data Body weight was significantly lower in the 5/6 nephrectomized (Nx) rats compared with the sham 5/6 412
nephrectomized (S(Nx)) and control rats at time point ‘Before IIR’ (data not shown). During the 6 weeks of follow-up after the 5/6 nephrectomy, 9 (32%) of the 5/6 nephrectomized rats and 3 (13%) of the sham nephrectomized rats were excluded. Exclusion criteria included wound infections, extreme weight loss and spontaneous death. The excluded rats were not included in the data analysis at any time point. On average, 79% (data not shown) of the kidney mass was removed in the rats that underwent nephrectomy. As hypertrophy of the right kidney after 2/3 left nephrectomy may occur, the amount of the removed kidney mass may be underestimated.
Renal function after 5/6 nephrectomy Urine volume and water intake at the ‘Right 1/1 Nx’, ‘Two weeks after 5/6 Nx’ and ‘Before IIR’ time points were significantly higher in the 5/6 nephrectomized rats (Nx-rats) than in the sham 5/6 nephrectomized rats (S(Nx)-rats) (Table 1). Consistent with these changes, urine osmolality was significantly lower in the Nx-rats than in the S(Nx)-rats and control rats (Table 1). No changes or differences in serum © 2014 Asian Pacific Society of Nephrology
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Table 1 Urine analyses during the development of Chronic kidney disease (CKD) Group (n) UV (μL/min/day)
WI (μL/min/day)
UAlbumin (mg/day)
UOsm (mosmol kgH2O)
Period
Control (n = 12)
S(Nx) + S(IIR) (n = 9)
S(Nx) + IIR (n = 12)
Nx + S(IIR) (n = 7)
Nx + IIR (n = 11)
R1/3 Nx T-W a 5/6Nx Before IIR R1/3 Nx T-W a 5/6Nx Before IIR R1/3 Nx T-W a 5/6Nx Before IIR R1/3 Nx T-W a 5/6Nx Before IIR
31.8 ± 2.2 36.8 ± 1.5 31.2 ± 1.7 68.6 ± 4.1 69.1 ± 2.3 66.2 ± 1.9 ND ND ND 1722 ± 76 1627 ± 46 1617 ± 89
37.8 ± 6.2 28.4 ± 2.3* 30.1 ± 4.9 67.91 ± 9.13 75.16 ± 3.20 64.92 ± 6.56 ND ND 1.07 ± 1.07 1493 ± 16 2062 ± 17* 1601 ± 16
35.6 ± 5.2 29.1 ± 2.0* 29.8 ± 2.9 73.85 ± 7.76 74.08 ± 3.35 64.44 ± 3.17 ND ND 6.34 ± 6.26 1641 ± 12 1986 ± 91* 1531 ± 14
77.1 ± 16.8* 121.5 ± 18.3*#+ 100.7 ± 16.6*#+ 97.23 ± 14.2 172.20 ± 19.0*#+ 152.08 ± 16.8*#+ ND 6.32 ± 5.05 15.35 ± 5.99#+ 400 ± 63*#+ 461 ± 96*#+ 419 ± 56*#+
64.1 ± 5.2*#+ 82.3 ± 8.8*#+ 73.6 ± 11.6*#+ 97.8 ± 5.0*#+ 133.3 ± 11.0*#+ 114.6 ± 18.8*#+ ND 5.93 ± 3.25 12.75 ± 4.16#+ 619 ± 46*#+ 676 ± 81*#+ 758 ± 16*#+
#+
Urine was collected during housing in metabolic cages for 24 h. Values are shown as mean (±SE). *P < 0.05 versus Control; +P < 0.05 versus S(Nx) + S(IIR); #P < 0.05 versus S(Nx) + IIR. ND, non-detectable (lower detectable limit was 6 mg/L); UAlbumin, urinary albumin excretion; UOsm, urinary osmolality; UV, urine volume; WI, water intake.
sodium were seen in the groups exposed to Nx or sham Nx during the 6 weeks of follow-up (data not shown). Albuminuria was an additional indication of pre-existing renal impairment. At the ‘Two weeks after 5/6 Nx’ time point, urinary albumin excretion was detectable in the Nx-rats but not the S(Nx)-rats. At the ‘Before IIR’ time point, albumin was also detectable in the urine of the S(Nx)-rats; however, the urinary albumin levels were significantly lower than those of the Nx-rats. Urinary albumin was not detectable in the control rats (lower detection limit was 6 mg/L) throughout the study. At the ‘Right 1/1 Nx’, ‘Two weeks after 5/6 Nx’ and ‘Before IIR’ time points, serum creatinine was significantly higher in the Nx + IIR group than in the S(Nx) + IIR and Control group (Fig. 2A). A novel biomarker of CKD, NGAL was measured in the urine (Fig. 2B), and at the ‘Right 1/1 Nx’, ‘Two weeks after 5/6 Nx’ and ‘Before IIR’ time points, the level of urinary NGAL was significantly higher in the Nx + IIR group than in the S(Nx) + IIR and Control group (Fig. 2B).
Organ-specific biomarkers in the serum after IIR Significant increases of 50% (±12%) and 72% (±10%) in serum creatinine (Fig. 2A) were observed after IIR in the Nx + IIR (n = 8) and S(Nx) + IIR (n = 11) groups, respectively, indicating that AKI continued until euthanasia. However, these increases in serum creatinine were not significantly different between the groups (P = 0.25). No increases in serum creatinine were seen after IIR within the Nx + S(IIR) and S(Nx) + S(IIR) groups (data not shown). Significantly higher levels of the liver enzyme ALT (Fig. 2C) were observed in the rats exposed to IIR than in the sham IIR or control rats. We did not detect any differences in C-reactive protein (CRP) levels among the groups (data not shown). © 2014 Asian Pacific Society of Nephrology
Lung histopathology Light microscopic examination of the lung parenchyma (Fig. 3) showed a decreased ratio of airspace to tissue (as a marker of tissue swelling) in the rats that were exposed to IIR. The volume of airspace was significantly lower in the Nx + IIR group than in the other groups.
Multiplex analysis of cytokines and chemokines in the kidney, lung and liver tissues In the kidney tissues (Fig. 4), the levels of IL-6, RANTES and MCP-1 were equally elevated in the Nx + IIR and Nx + S(IIR) groups, and significantly different from those in the S(Nx) + IIR, S(Nx) + S(IIR) and control groups. In contrast IL-1β levels were significantly higher in the Nx + IIR group than in the other groups. In the lung tissues (Fig. 5), the levels of RANTES and MCP-1 were equally elevated in the Nx + IIR and S(Nx) + IIR groups, and significantly different from those in the S(Nx) + S(IIR) and control groups. In contrast, IL-1β and IL-6 were significantly higher in the Nx + IIR group than in the other groups. In the liver tissues (Fig. 6), the levels of IL-1β and MCP-1 were equally elevated in the Nx + IIR and S(Nx) + IIR groups and significantly different from those in the Nx + S(IIR), S(Nx) + S(IIR) and control groups. No differences in IL-6 and RANTES were detected between any of the groups. We did not detect any differences in TNF-α levels among the groups (data not shown).
DISCUSSION The main findings of the present study were that (i) Severity of AKI induced by a surgical procedure (IIR) in rats with 413
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Fig. 2 Organ injury during the development of chronic kidney disease (CKD) and after intestinal ischaemia and reperfusion. Renal function after 5/6 nephrectomy (5/6 Nx) and intestinal ischaemia and reperfusion (IIR) was determined by serum creatinine levels (A). Urinary neutrophil gelatinase-associated lipocalin (NGAL) (B) levels were determined after 5/6 Nx. Liver function after IIR was determined using alanine transaminase (ALT) (C). Values are presented as means (±SE). *P < 0.05 versus control; +P < 0.05 versus S(Nx) + S(IIR); #P < 0.05 versus S(Nx) + IIR; §P < 0.05 versus Nx + S(IIR); B−IIRP < 0.05 compared with the ‘Before IIR’ time point within ) S(Nx) + IIR (#), ( ) Nx + IIR, ( ) control (*). C: ( ) control (*), ( ) S(Nx) + S(IIR) (+), ( ) S(Nx) + IIR (#), ( ) Nx + S(IIR) the same group. A and B: ( ) Nx + IIR. (§), (
CKD is similar to that induced in normal rats without CKD. (ii) Upon exposure to IIR, rats with CKD developed a more pronounced pro-inflammatory cytokine and chemokine response in organs remote to the kidney. (iii) Majority of pro-inflammatory cytokine and chemokine responses within the kidneys were equally increased in the 5/6 nephrectomized rats regardless of IIR exposure. 414
CKD as a state of ‘chronic low-grade inflammation’ Chronically elevated common phenomenon undergoing dialysis. chemokines contribute
inflammatory biomarkers are a in patients with CKD who are not Pro-inflammatory cytokines and to a state of persistent low-grade © 2014 Asian Pacific Society of Nephrology
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Fig. 3 Chronic kidney disease (CKD) enhances lung injury after intestinal ischaemia and reperfusion (IIR). Rats were exposed to nephrectomy (Nx) (D,E), sham Nx (A,B) and or IIR(B,E) or sham IIR (A,D). Untreated rats served as controls (C). Lung injury was determined histologically in sections of peripheral lung parenchyma (n = 7–8/group). Light microscopic pictures (n = 4/animal) presented in high contrast were processed by ImageJ software to measure the ration of airspace (black) to lung parenchyma (white). Calculated area fractions with means from within each group are shown (F). Values are presented as means (±SE). *P < 0.05 versus é control; +P < 0.05 versus S(Nx) + S(IIR); #P < 0.05 versus S(Nx) + IIR; §P < 0.05 versus Nx + S(IIR). ( ) control (*), (○) S(Nx) + S(IIR) (+), (▼) S(Nx) + IIR (#), ( ) Nx + S(IIR) (§), ( ) Nx + IIR.
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inflammation.12 Of the pro-inflammatory cytokines, increased IL-6 and TNF-α levels have been demonstrated to be correlated with the degree of chronic renal impairment in patients with CKD.11,21 In the present study, we found elevated IL-6 levels in the kidneys of rats with surgically induced CKD regardless of IIR exposure. However, we did not find any significant correlation between sCr, a biomarker © 2014 Asian Pacific Society of Nephrology
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of CKD severity and kidney tissue IL-6 levels in the CKD group exposed to sham IIR at the end of the study (data not shown). Additionally, we did not detect any differences in the renal TNF-α level among the groups (data not shown). Similarly to pro-inflammatory cytokines, accumulating data from clinical studies and animal models of CKD support the perception that chemokines play a role during the devel415
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Fig. 4 Kidney tissue cytokine levels after intestinal ischemia & reperfusion (IIR). Pro-inflammatory cytokines interleukin (IL)-1β (A) and IL-6 (B) as well as the chemokines RANTES (C), MCP-1 (D) were measured in whole kidney tissue homogenates using multiplex assays. Individual tissue concentrations and means (horizontal line) within each group are shown. *P < 0.05 versus control; +P < 0.05 versus S(Nx) + S(IIR); #P < 0.05 versus S(Nx) + IIR; §P < 0.05 versus Nx + S(IIR). é ( ) control (*), (○) S(Nx) + S(IIR) (+), (▼) S(Nx) + IIR (#), ( ) Nx + S(IIR) (§), ( ) Nx + IIR.
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opment of CKD.1,2,22 Our data confirm increased MCP-1 levels in the kidney tissues of 5/6 nephrectomized rats compared with the levels in sham 5/6 nephrectomized rats.3,23 Various mechanisms might be responsible for the accompanying ‘uremic inflammation’ in response to decreased renal function. In addition to increased production and reduced renal excretion of pro-inflammatory cytokines and their soluble receptors,4–7 sympathetic overactivity and blunted vagal nerve activity, which inhibit local cytokine release, may also lead to an increased inflammatory response.7,24 416
Pre-existing CKD may predisposes rats to a more severe inflammatory response in organs other than the kidneys In the present study, we determined that rats with CKD compared with non-CKD develop a partially more pronounced pro-inflammatory cytokine (IL-1β, IL-6) response in the lungs upon IIR exposure. Similar findings were observed with plasma IL-6 levels by Leelahavenichkul et al.8,25 in an alternative AKI model and others have reported increased levels of IL-6 and MCP-1 in kidney tissues after AKI induced by renal ischaemia and reperfusion (I/R)8,26,27 in © 2014 Asian Pacific Society of Nephrology
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Fig. 5 Lung tissue cytokines levels after intestinal ischemia & reperfusion (IIR). Pro-inflammatory cytokines IL-1β (A), IL-6 (B) and chemokines RANTES (C), MCP-1 (D) were measured in lung tissue homogenates by multiplex assays. Individual tissue concentrations and mean (horizontal line) within each group are shown. *P < 0.05 versus Control; +P < 0.05 versus S(Nx) + S(IIR); #P < 0.05 versus S(Nx) + IIR; §P < 0.05 versus Nx + S(IIR). ( ) control (*), (○) S(Nx) + S(IIR) (+), (▼) S(Nx) + IIR é (#), ( ) Nx + S(IIR) (§), ( ) Nx + IIR.
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rodents with normal kidney function prior to AKI. That model was fundamentally different from the one used in the current study, in which cytokine and chemokine responses within the kidney were equally increased (beside IL-1β) in rats with CKD regardless of IIR exposure.
Severity of IIR-induced AKI in rats with CKD is similar to that induced in rats without CKD We observed an increase in serum creatinine after IIR in the rats that were 5/6 nephrectomized and those that were © 2014 Asian Pacific Society of Nephrology
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sham 5/6 nephrectomized. The fractional increase in serum creatinine between the groups was not significantly different, consistent with a similar magnitude of response to IIR in rats with CKD compared with those without CKD. A plausible explanation is that as glomerular filtration rate (GFR) falls, creatinine secretion is increased, and thus the rise in serum creatinine is less. Therefore a similar relative increase in serum creatinine detected in the CKD rats compared with those without CKD may not be an accurate reflection of the GFR changes in the non-steady-state condition of AKI.28 417
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Fig. 6 Liver tissue cytokines levels after intestinal ischemia & reperfusion (IIR). Pro-inflammatory cytokines IL-1β (A), IL-6 (B) and chemokines RANTES (C), MCP-1 (D) were measured in lung tissue homogenates by multiplex assays. Individual tissue concentrations and mean (horizontal line) within each group are shown. *P < 0.05 versus Control; +P < 0.05 versus S(Nx) + S(IIR); #P < 0.05 versus S(Nx) + IIR; §P < 0.05 versus Nx + S(IIR). ( ) control (*), (○) S(Nx) + S(IIR) (+), (▼) S(Nx) + IIR é (#), ( ) Nx + S(IIR) (§), ( ) Nx + IIR.
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CONCLUSION The present study is not without its limitations. First, we measured cytokines in whole tissue homogenates instead of plasma, which is used in clinical studies of MOF.29 The reason for this are that a significant proportion of the cytokines act via paracrine mechanisms and have a short half-life in plasma, making them very difficult to detect.30 Second, we were not able to answer the question of why persistent uremic inflammation, which occurs during CKD, increases the inflammatory response within the lung tissue. One explanation might be a reduction in renal clearance of cytokine/chemokines as a consequence of pre-existing CKD. 418
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Another may be, that increased capillary permeability and decreased bacterial clearance shown in animals with CKD, predispose them to the development of MOF.4 Our model of surgically induced AKI superimposed onto CKD in rodents is suitable for further investigation of the pathophysiology behind effects on the immediate early organ dysfunction in this clinically relevant condition.
ACKNOWLEDGEMENTS We thank Inge Marete Poulsen, Line V. Nielsen, Gitte B. Kall, Gitte Skou, Kirsten Strauss and Birgitte K. Sahl for their expert technical assistance and Professor Robert Fenton for © 2014 Asian Pacific Society of Nephrology
AKI on CKD
his feedback during the manuscript preparation process. This work was supported by the Water and Salt research Centre (established and supported by the Danish National Research Foundation), Faculty of Health Science at the University of Aarhus, Novo Nordisk Foundation, Aase and Ejnar Danielsens Foundation, Augstinus Foundation and C.C. Klestrup and Hustru Henriette Klestrups Mindelegat.
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