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NDT Advance Access originally published online on November 5, 2007
Nephrology Dialysis Transplantation 2008 23(3):842-852; doi:10.1093/ndt/gfm694
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Macrophages contribute to the development of renal fibrosis following ischaemia/reperfusion-induced acute kidney injury

Gang Jee Ko, Chang-Su Boo, Sang-Kyung Jo, Won Yong Cho and Hyoung Kyu Kim

Division of Nephrology, Department of Internal Medicine, College of Medicine, Korea University, Institute of Renal disease, Seoul, Korea

Won Yong Cho, Division of Nephrology, Department of Internal Medicine, 5Ka, Anam-Dong, Sungbuk-Ku, Korea University Hospital, Seoul, Korea. Tel: + 82-2-920-5599; Fax: + 82-2-927-5344; E-mail: wonyong{at}korea.ac.kr



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Statistical analysis
 Results
 Discussion
 References
 
Background. Ischaemia/reperfusion is a major cause of acute kidney injury and can result in poor long-term graft function. Although most of the patients with acute kidney injury recover their renal function, significant portion of patients suffer from progressive deterioration of renal function. A persistent inflammatory response might be associated with long-term changes following acute ischaemia/reperfusion. Macrophages are known to infiltrate into tubulointersitium in animal models of chronic kidney disease. However, the role of macrophages in long-term changes after ischaemia/reperfusion remains unknown. We aimed to investigate the role of macrophages on the development of tubulointerstitial fibrosis and functional impairment following acute ischaemia/reperfusion injury by depleting macrophages with liposome clodronate.

Methods. Male Sprague–Dawley rats underwent right nephrectomy and clamping of left renal vascular pedicle or sham operation. Liposome clodronate or phosphate buffered saline was administered for 8 weeks. Biochemical and histological renal damage and gene expression of various cytokines were assessed at 4 and 8 weeks after ischaemia/reperfusion.

Results. Ischaemic/reperfusion injury resulted in persistent inflammation and tubulointerstital fibrosis with decreased creatinine clearance and increased urinary albumin excretion at 4 and 8 weeks. Macrophage depletion attenuated those changes. This beneficial effect was accompanied with a decrease in gene expression of inflammatory and profibrotic cytokines.

Conclusions. These results suggest that macrophages play an important role in mediating persistent inflammation and fibrosis after ischaemia/reperfusion leading to a development of chronic kidney disease. Strategies targeting macrophage infiltration or activation can be useful in the prevention of development of chronic kidney disease following ischaemic injury.

Keywords: acute renal failure; fibrosis; inflammation; ischaemia/reperfusion; long-term effect; macrophage



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Statistical analysis
 Results
 Discussion
 References
 
Ischaemic/reperfusion injury is the primary cause of acute kidney injury [1] and is also associated with delayed graft function and increased risk of acute rejection in kidney transplantation. Despite the high mortality associated with acute kidney injury, most surviving patients are thought to recover renal function completely [2]. However, recent studies have suggested that post-inflammatory renal scarring caused by ischaemic/reperfusion injury might be an important contributor to the development of chronic kidney disease [3]. Moreover, clinical evidence has shown that delayed initial graft function with prolonged ischaemic time is associated with impairment in long-term graft function [4]. Much attention has been focused on the events that occur during ischaemia or in the early recovery phase [5]; however, the long-term effects and mechanisms of progressive renal functional deterioration after ischaemic/reperfusion injury are not completely understood.

The tubulointerstitial influx of inflammatory cells has been observed in many forms of chronic kidney disease, such as diabetes and hypertension [6] and persistent inflammation in kidney is thought to contribute to the development of tubulointerstitial fibrosis with functional impairment. However, the role of inflammation in the development of fibrosis after ischaemic/reperfusion injury has not been studied.

Macrophages show heterogeneity depending on the nature of the injury, location or their activation status. The role of macrophages in inducing tissue injury by releasing reactive oxygen species, nitric oxide, complement factors and proinflammatory cytokines, or even an opposite role in resolution of inflammation or assisting regeneration has also been reported [7]. Previous studies reported the contribution of macrophages in mediating injury in ischaemia/reperfusion-induced acute kidney disease by showing that depletion of macrophages had beneficial effects on renal function following ischaemia/reperfusion [8]. However, the role of macrophages on the long-term changes in renal function after ischaemic/reperfusion injury remains unclear. In this study, we investigated the role of macrophages in the development of tubulointerstitial fibrosis and functional impairment following ischaemia/ reperfusion.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Statistical analysis
 Results
 Discussion
 References
 
Animals and experimental protocol
Male Sprague–Dawley rats weighing 150–200 g were purchased from Orient (Seoul, Korea) and were given free access to water and chow prior to manipulation. Rats were kept under a 12-h light/dark cycle at 25°C and fed standard rat chow and water at libitum, and were cared for the following criteria from the Animal Care Committee of Korea University. The rats (n = 6–9/each group) were anaesthetized via intraperitoneal (i.p.) injection of 100 mg/kg ketamine, 12.5 mg/kg xylazine and were subjected to unilateral (left) renal pedicle clamping for 50 min on the fifth day after unilateral (right) nephrectomy. Following the procedure, the rats were given 5 mL of warm normal saline and prophylactic antibiotics (cefazolin 500 mg/kg) (i.p.). Sham operations were performed in a similar manner, with the exception of renal pedicle clamping. At 4 and 8 weeks of reperfusion, the animals were killed. Blood was collected by intracardiac puncture and their kidneys were processed for molecular and histological examination (Figure 1).


Figure 1
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  		Fig. 1 Schematic schedule of the experiment. {downarrow}: liposome clodronate or phosphate buffered saline (PBS) injection.

 
Liposome preparation and in vivo depletion of monocyte macrophages
Clodronate was purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA) and liposome-encapsulated clodronate was prepared according to the methods previously described [9]. One milliliter of liposome clodronate or phosphate buffered saline was first injected at Day 3 after reperfusion via tail vein and every 5 days thereafter.

Histological examination
Paraformaldehyde (4%)-fixed and paraffin-embedded kidney tissues were stained with haematoxylin and eosin (H&E) or Masson's trichrome. The interstitial volume of the renal cortex was determined in the sections stained with Masson's trichrome. Ten randomly chosen, non-overlapping fields from the same section of renal cortex were captured (x200, Olympus BX51, Japan). The interstitial space was traced along the tubular basement membrane, with the exclusion of Bowman's capsule, peritubular capillaries and large vessels, on each photograph using Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA). The renal interstitial volume was expressed as the proportion of interstitial space over the entire field.

Interstitial collagen deposition was measured by Sirius red staining. After deparaffinization, the kidney sections were hydrated and incubated in picrosirius red solution (1% Sirius red in saturated picric acid) for 18 h, followed by 0.01N HCl treatment for 2 min and dehydration. Eight to ten high power fields (HPFs, x200) were captured and the collagen deposition was expressed as the proportion of Sirius red- stained area in the renal cortex. For immunohistochemical staining of ED-1 and TGF-β1, kidney sections were hydrated in graded ethanol solutions, treated with 0.3% H2O2 to quench the endogenous peroxidase activity, treated with 0.1% trypsin (Zymed, San Francisco, CA, USA) and incubated with blocking serum (Vector, Peterborough, UK) to suppress the nonspecific binding. Sections were incubated at 4°C overnight with anti-ED-1 (1 : 1000 Serotec, UK) and anti-TGF-β1 (1 : 50 Santa Cruz Biotechnology, Birmingham, AL, USA) and incubated with a biotin-conjugated secondary antibody followed by an avidin-biotin reagent (Vector Laboratories, Burlingame, CA, USA). Napthol AS-D chloroacetate (Sigma, St. Louis, MO, USA) was used to identify neutrophils. Eight to ten HPFs were captured and the mean number of positive cells in the field was calculated in order for quantification.

Quantitation of myeloperoxidase (MPO) activities
Tissue MPO activities were measured by homogenizing kidney tissue with 0.5% hexadecyltrimethylammonium bromide (HTAB) in 50 mM phosphate buffer (pH 6.0) and centrifuged at 17 000 g for 20 min in order to assess the degree of neutrophil and macrophage activation. The pellet was resuspended in HTAB, freeze-thawed (20 min at –80°C), homogenized for 60 s, sonicated three times for 30 s and centrifuged at 17 000 g for 20 min. Fifty microliter of supernatant was mixed with 200 µL of o-danisidine dihydrochloride (1.25 mg/mL in PBS) containing H2O2 (0.05%) and the absorbance was read at 450 nm. The results were adjusted according to the amount of protein and were expressed as fold differences compared to the levels of a sham-operated animal.

Quantitation of TNF-{alpha}, IL-1β, IL-6, MCP-1 and TGF-β1 mRNA by real-time RT-PCR
Total RNA was isolated using TRIzol reagent (Life Technology, Rockville, MD, USA) according to the manufacturer's protocol. After precipitation by isopropyl alcohol, total RNA was purified using an RNeasy minikit (Qiagen, Valencia, CA, USA). RNA quantification was done using spectrophotometer at A260/280 nm and 1 µg of total RNA was then reverse transcribed in a reaction volume of 50 µL containing 10x RT buffer, 5.5 mM MgCl2, 500 µM of each dNTP, 2.5 µM random hexamer, 0.4 U/µL RNase inhibitor and 3.125 U/µL MultiScribeTM Reverse Transcriptase (Taqman® Reverse Transcription Reagents; Applied Biosystem Inc., Foster City, CA, USA). The reaction was carried out at 25°C for 10 min, 48°C for 30 min and 95°C for 5 min. Real-time polymerase chain reaction (PCR) was then run in triplicate using the iCycler system (Bio-Rad, Hercules, CA, USA) under the amplification condition of 40 cycles at 95°C for 15 s and 60°C for 1 min. Gene-specific primer and probe sets were provided as pre-developed Taqman® Assay Reagents (Applied Biosystem Inc., Foster City, CA, USA) and were used according to the manufacture's protocol (rat TNF-{alpha}, IL-1β, IL-6). Primer sets for rat MCP-1 and TGF-β1 were designed using Beacon Designer software version 2 (Bio-Rad, Hercules, CA, USA) based on the sequences from GenBank and were as follows: MCP-1: sense, 5'-GATCTCTCTTCCTCCACCACTATG-3'; antisense, 5'-AATGAGTAGCAGCAGGTGAGT-3'; probe: 5'-AGGTCTCTGTCACGCTTCTG GCC-3'. TGF-β1: sense, 5'-CTCCAGCTCCACAGAGAAGAACTGC-3'; antisense, 5'-CACGATCATGTTGGACAACTGCTCC-3'; probe, 5'-TTTCGCCTCAGTGCCCA CTG-3'. Taqman® probe was labelled with 6-carboxy-flourescein as a reporter dye and 6-carboxy-tetramethyl-rhodamine as a quench dye. 18S ribosomal RNA (Taqman® ribosomal control reagent; Applied Biosystem Inc., Foster City, CA, USA) was used as an internal control to normalize all the data. The dynamic range of each primer/probe set was verified by a serial dilution of the cDNA template. The abundance of cytokine and chemokine genes was normalized to that of the 18S rRNA and was expressed as fold differences relative to the sham-operated control animals.



   Statistical analysis
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 Abstract
 Introduction
 Subjects and methods
 Statistical analysis
Results
 Discussion
 References
 
All data were presented as mean ± standard deviation and were analysed by the Mann–Whitney test using SPSS 10.0. P-value <0.05 was considered statistically significant.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Statistical analysis
 Results
Discussion
 References
 
Systemic macrophage depletion attenuated the long-term changes of renal functional impairment after ischaemia/reperfusion injury
In the preliminary study, liposome clodronate alone did not show any renal effect in rats. The effect of liposome clodronate on systemic monocyte-macrophage depletion was previously demonstrated in many laboratories including ours [8]. The percentage of monocytes in peripheral blood and the number of ED-1 positive cells in liver were markedly decreased 24 h after liposome clodronate injection and the depletion persisted until 5 days (data not shown). Ischaemic/reperfusion injury to kidney resulted in decreased creatinine clearance rate at 8 weeks (sham group: 2656.4 ± 324.5 versus ischaemia/reperfusion group: 1561.8 ± 208.1 mL/day, P < 0.05) and increased urinary protein/creatinine ratio at 4 and 8 weeks compared to the sham-operated animals (0.90 ± 0.03 versus 1.28 ± 0.23, 0.48 ± 0.07 versus 1.08 ± 0.23 at 4 and 8 weeks respectively P < 0.05), suggesting development of chronic kidney disease following ischaemia/reperfusion. However, systemic macrophage depletion resulted in attenuation of these functional parameters (creatinine clearance at 8 weeks: 2096.4 ± 144.8 mL/day, P < 0.05) (urinary protein excretion ratio: 0.99 ± 0.03 and 0.52 ± 0.07, P < 0.05 at 4 and 8 weeks respectively) (Figure 2).


Figure 2
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  		Fig. 2 Effect of monocyte-macrophage depletion on long-term follow-up of renal function after ischaemic/reperfusion (I/R) injury. BUN and creatinine levels were not changed with liposome clodronate (LC) treatment (A and B), but the increase of the urinary protein/creatinine ratio (C) and the decrease of the creatinine clearance rate (D) after ischaemic/reperfusion injury were attenuated with liposome clodronate treatment. *P < 0.05 compared with sham, #P < 0.05 compared with PBS.

 
Systemic macrophage depletion attenuated the long-term histological changes after ischaemia/reperfusion injury
Histologic examination at 4 and 8 weeks after ischaemic/reperfusion injury showed tubulointerstitial injury characterized by the dilatation of tubules, tubular cell atrophy, inflammatory cell infiltration and increased interstitial areas. These changes were attenuated by systemic macrophage depletion (Figure 3).


Figure 3
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  		Fig. 3 Effect of monocyte-macrophage depletion on renal histology (H&E, x40). Histologic examination of the renal cortex demonstrates inflammatory cells (Figure 3) infiltration, increased interstitial area (Figure 3), tubular atrophy and dilatation (Figure 3) at 4 and 8 weeks after I/R injury. These changes were diminished with LC treatment. (A) and (C) PBS, (B) and (D) LC. (A) and (B) 4 weeks, (C) and (D) 8 weeks.

 
Systemic macrophage depletion reduced proinflammatory cytokine/chemokine mRNA expression in the long-term follow-up after ischaemia/reperfusion injury
We examined various proinflammatory cytokines and chemokine expression. The mRNA expression levels of the proinflammatory cytokines TNF-{alpha} and IL-1β and chemokine MCP-1 were significantly increased at 4 and 8 weeks after ischaemic/reperfusion injury than in the sham-operated animals. However, systemic macrophage depletion led to a significant decrease in these cytokine and chemokine gene expression (TNF-{alpha}: 2.5/0.6, IL-1β: 9.7/2.4 at 4 weeks, MCP-1: 3.1/1.4 at 8 weeks, fold differences, P < 0.05, Figure 4). The level of IL-6 expression did not show any differences between groups.


Figure 4
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  		Fig. 4 Effect of monocyte-macrophage depletion on cytokine mRNA expression. IL-1β and MCP-1 expressions were decreased with LC treatment at both 4 and 8 weeks after I/R injury (A and B). TNF-{alpha} expressions were also significantly decreased at 4 weeks (C) *P < 0.05 compared with PBS.

 
Systemic macrophage depletion reduced inflammation in the long-term follow-up after ischaemia/reperfusion injury
ED-1-stained macrophages and esterase-positive neutrophils were increased at 4 and 8 weeks after ischaemic/ reperfusion injury compared to sham controls. Systemic macrophage depletion by liposome clodronate treatment decreased not only the number of kidney macrophages but also neutrophil infiltration (Figures 5 and 6). The MPO activities also increased significantly at 4 and 8 weeks after ischaemia/reperfusion injury compared to sham controls and macrophage depletion resulted in a significant decrease of MPO activities (Figure 7).


Figure 5
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  		Fig. 5 Effect of monocyte-macrophage depletion on ED-1 cell infiltration in kidney. Macrophage infiltration in renal cortex was increased at 4 and 8 weeks after I/R injury. LC treatment suppressed this increase. (A) and (D) sham, (B) and (E) PBS, (C) and (F) LC. (A), (B) and (C) 4 weeks, (D), (E) and (F) 8 weeks (ED-1 staining, x200). (G) Number of ED-1 positive cells per high power field (HPF). *P < 0.05 compared with sham, #P < 0.05 compared with PBS.

 

Figure 6
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  		Fig. 6 Effect of monocyte-macrophage depletion on leukocytes infiltration in kidney. Neutrophil infiltration was increased at 4 and 8 weeks after I/R injury, which were suppressed with LC treatment. (A) and (D) sham, (B) and (E) PBS, (C) and (F) LC. (A), (B) and (C) 4 weeks, (D), (E) and (F) 8 weeks (esterase staining, x200). (G) Number of esterase positive cells per HPF. *P < 0.05 compared with sham, #P < 0.05 compared with PBS.

 

Figure 7
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  		Fig. 7 Effect of monocyte-macrophage depletion on MPO activities in kidney. MPO activities were significantly increased at 4 and 8 weeks after I/R injury, which were suppressed with LC treatment. *P < 0.05 compared with PBS.

 
Systemic macrophage depletion reduced TGF-β1 expression in the long-term follow-up after ischaemia/reperfusion injury
The mRNA expression of TGF-β1, the major profibrotic cytokine, increased at 4 and 8 weeks after ischaemic/ reperfusion injury significantly compared to sham controls. (3.4/1.0 and 13.4/3.5 at 4 and 8 weeks, respectively, P < 0.05) Immunohistochemical staining showed increased TGF-β1 expression was localized in basolateral membrane of tubular cells and interstitial area. Macrophage depletion significantly suppressed TGF-β1 expression after ischaemic/reperfusion injury (Figure 8).


Figure 8
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  		Fig. 8 Effect of monocyte-macrophage depletion on TGF-β1 expression. Immunostain of anti-TGF-β1 antibody demonstrates the increase of TGF-β1 expression at 4 and 8 weeks after I/R injury, which were suppressed with LC treatment. (A) and (D) sham, (B) and (E) PBS, (C) and (F) LC. (A), (B) and (C) 4 weeks; (D), (E) and (F) 8 weeks (TGF-β1 staining, x200) (G) TGF-β1 mRNA expression measured by RT-PCR shows similar results. *P < 0.05 compared with PBS.

 
Systemic macrophage depletion reduced renal fibrosis in the long-term follow-up after ischaemia/reperfusion injury
Renal fibrosis, demonstrated as an increase in interstitial volume in Masson's trichrome staining increased significantly at 4 and 8 weeks after ischaemic/reperfusion injury in saline-treated animals compared to sham controls. Macrophage depletion resulted in a significant reduction in fibrosis and the interstitial volume at 4 and 8 weeks after ischaemia/reperfusion injury (26.3 ± 8.7 versus 6.8 ± 1.3 and 33.3 ± 7.8 versus 9.2 ± 2.4% area of interstitium, P < 0.01 at 4 and 8 weeks, respectively) (Figure 9). Interstitial collagen deposition, demonstrated by Sirius red staining also increased in the saline group at 4 and 8 weeks after ischaemic/reperfusion injury, which was also decreased significantly in liposome clodronate treated animals (Figure 10).


Figure 9
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  		Fig. 9 Effect of monocyte-macrophage depletion on interstitial fibrosis in kidney. Interstitial fibrosis was increased at 4 and 8 weeks after I/R injury, which were suppressed with LC treatment. (A) and (D) sham, (B) and (E) PBS, (C) and (F) LC. (A), (B) and (C) 4 weeks, (D), (E) and (F) 8 weeks (Masson's trichrome staining, x200). (G) Morphological semi-quantitative analysis. *P < 0.05 compared with sham, #P < 0.05 compared with PBS.

 

Figure 10
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  		Fig. 10 Effect of monocyte-macrophage depletion on interstitial collagen deposition in kidney. Interstitial collagen deposition was increased at 4 and 8 weeks after I/R injury, which were suppressed with LC treatment. (A) and (D) sham, (B) and (E) PBS, (C) and (F) LC. (A), (B) and (C) 4 weeks, (D), (E) and (F) 8 weeks (Sirius red staining, x200). (G) Quantitative results of interstitial collagen fibre deposition. *P < 0.05 compared with sham, #P < 0.05 compared with PBS.

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Statistical analysis
 Results
 Discussion
References
 
In this study, we demonstrated that in long-term follow-up after ischaemic/reperfusion injury, (i) inflammation sustained and resulted in progressive renal tubular atrophy and interstitial fibrosis with renal functional deterioration and (ii) systemic macrophage depletion not only attenuated both inflammation and fibrosis but provided functional protection as well.

Ischaemic/reperfusion injury is the leading cause of acute kidney injury [1]. Despite a high mortality, it appears that long-term renal impairment is unlikely [2]. However, recent studies suggested that an incomplete recovery of renal function might be responsible for the development of chronic kidney disease in patients with acute kidney injury [3]. In addition, ischaemic injury to allograft is the most common cause of delayed graft function and might result in the development of chronic allograft nephropathy with poor allograft survival [4]. Nonetheless, the long-term sequel of acute kidney injury still remains unclear due to paucity and limitation of a long-term follow-up study in a severely ill patient population.

Our long-term follow-up experiments after ischaemic/ reperfusion injury in rats demonstrated that an increase of urinary protein excretion with renal dysfunction developed at 4 and 8 weeks after initial injury and also this functional deterioration was accompanied by histological evidence of tubular atrophy and interstitial fibrosis. These findings might suggest the development of chronic kidney disease following ischaemia/reperfusion injury to kidney. Pathogenesis of progression of chronic kidney disease is multifactorial and complex and has been known to undergo a ‘common pathway’ irrespective of initiating insults [10]. Of a variety of mechanisms, inflammation seems to play an important role in mediating the progression of kidney diseases not only in traditional inflammatory diseases such as glomerulonephritis but also in those not usually thought to be inflammatory diseases such as diabetic nephropathy or obstructive nephropathy [5,6]. It is evident that inflammation has a pathogenetic role in acute kidney injury after ischaemic/reperfusion injury [11]; however, it is still unclear whether the inflammatory responses persist after the initial recovery and are responsible for further development of chronic kidney disease in long-term follow-up after ischaemic/reperfusion injury. First we investigated various proinflammatory cytokine and chemokine gene expressions and found that these gene expression levels were significantly elevated compared to the sham-operated control animals. Inflammatory cytokines produced by acute ischaemic insult might be sustained possibly due to chronic hypoxia caused by peritubular capillary loss after acute ischaemic/reperfusion injury [12]. Increased cytokine and chemokine gene expression were accompanied by an increase in kidney macrophage and neutrophil infiltration, showing inflammation persists after injury. More importantly, the inflammatory response was associated with higher urinary protein excretion and decreased creatinine clearance and this might suggest that persistent inflammation plays an important role in the pathogenesis of chronic kidney disease after ischaemic/reperfusion injury.

Macrophages have been known to mediate tissue injury in a variety of kidney diseases by producing cytokines, reactive oxygen species or nitric oxides [6,7] and recently, the role of macrophages in mediating acute kidney injury has also been demonstrated in two different species [8,13]. However, macrophages show heterogeneity according to their activation status or location and certain subsets of macrophages have been known to be essential in tissue regeneration or repair [7]. Although several recent studies suggested that macrophage accumulation mediated organ dysfunction in nonimmune renal diseases such as diabetic nephropathy [6,14], the precise role of macrophages in long-term kidney injury after ischaemic/reperfusion injury has not been examined.

To examine the role of macrophages in long-term changes of renal function and histology after ischaemic/ reperfusion injury, we used liposome clodronate. Intravenously injected liposome clodronate is rapidly taken up by macrophages and following liposome digestion by lysosomal phospholipases, free clodronate induces rapid apoptosis in macrophages. The selectivity of liposome clodronate on the depletion of macrophages, but not on neutrophils or lymphocytes, has been demonstrated previously [9]. In our experiment, liposome clodronate administration began on the third day after renal pedicle clamping when plasma creatinine levels returned to normal to avoid any interference by the effects of the acute stage after ischaemic/reperfusion injury. For prolonged macrophage depletion, we administered liposome clodronate every 5 days thereafter based on the preliminary study demonstrating that liver ED-1 positive cell depletion persisted at least for 5 days after injection.

Relatively smaller numbers of ED-1 positive resident macrophages were found in the kidneys of the sham-operated animals like other studies. However, the number significantly increased in the long-term follow-up at 4 and 8 weeks after ischaemic/reperfusion injury and prolonged liposome clodronate administration led to a decrease in kidney ED-1 positive cell accumulation. A decrease in kidney macrophage infiltration was accompanied by functional and histological renal protection. These findings might suggest that infiltrating macrophages play an important role in mediating renal injury. There are several other studies that investigate the relationship between the sustained inflammatory response and progressive renal injury following acute ischaemic/reperfusion injury [15–18]. They also demonstrated the renoprotective effect of immunosuppressive drug in reducing the inflammation [19]. However, the mechanism of the drug was thought to be suppressing primarily T-cell activation by an indirect pathway, and the effects on the change of renal function varied between the drugs. We showed here for the first time the possible association between persistent macrophage infiltration and the development of chronic kidney disease after ischaemic/reperfusion injury to kidney. In addition to macrophages, we also observed esterase positive neutrophil infiltration increased in long-term follow-up at 4 and 8 weeks after ischaemia/reperfusion and macrophage depletion decreased neutrophil infiltration and MPO activity significantly. The role of neutrophils in acute kidney injury has been well known and is thought to be associated with liberation of various protease, reactive oxygen species or cytokines [20], but the role of neutrophils in the progression of chronic kidney disease has not been demonstrated previously. Macrophages, within microenvironment of damaged tissue, could be activated and also could produce a variety of signals that enhance neutrophil–endothelial interaction, leading to enhanced neutrophil infiltration into tissue. Although the mechanism how macrophage depletion led to a decrease in neutrophil infiltration is unclear in this study, this finding could suggest that neutrophil also plays an important role in progression of chronic kidney disease.

Tubulointerstitial fibrosis and collagen deposition were observed in the long-term follow-up after acute ischaemic/ reperfusion injury in our experiments. It was accompanied by an increase in TGF-β1, the major profibrotic cytokine expression. Renal fibrosis and TGF-β1 expression were reduced by macrophage depletion. This finding may suggest that the macrophages induce tubulointerstitial fibrosis after ischaemic/reperfusion injury via TGF-β1 signalling pathway. TGF-β1 might be originated from activated macrophages directly or adjacent tubular cells or renal fibroblast produced through epithelial mesenchymal transition, and it was compatible with our experiment that showed TGF-β1 positivity in dilated tubule and interstitium in immunostain. TGF-β1 may be produced with the stimulation of chemokines, such as MCP-1 and proinflammatory cytokines such as TNF-{alpha} and IL-1β [7]. The association of renal inflammation and the development of renal fibrosis have also been demonstrated in another chronic kidney disease model such as diabetic nephropathy [6]. A recent paper by Chow et al. also demonstrated the importance of MCP-1, a potent macrophage chemokine in renal inflammation and fibrosis in db/db mice by demonstrating that Ccl2–/– mice were protected from the development of kidney macrophage accumulation and tubulointerstitial fibrosis [14]. The significance of these effects was also noted by a decrease in urinary albumin excretion and plasma creatinine levels. Therefore it is likely that increased MCP-1 and other proinflammatory cytokines evoke sustained kidney inflammation and this could lead to the development of tubulointerstitial fibrosis with functional impairment after severe ischaemic/reperfusion injury. Although a direct causal relationship between macrophage accumulation and renal damage cannot be provided due to an absence of adoptive transfer experiments, this study could suggest that macrophage accumulation is an important player in the development and progression of chronic kidney disease after severe ischaemic/reperfusion injury. There are several limitations of this study: first, limited time points that we examined; longer time follow-up will provide more accurate information about progression of chronic kidney disease. Second, studies showing characteristic macrophage phenotype have not been done, and third, an adoptive transfer study may provide direct evidence of the pathogenetic role of macrophages.

In conclusion, our study provided evidence that progressive decline of renal function and structural changes are found in long-term follow-up after acute ischaemic/reperfusion injury alone, and might suggest that the macrophage plays an important role in those changes. Macrophages-mediated renal damage with the induction of the inflammatory and fibrotic pathways is thought to be presumably by releasing proinflammatory and profibrotic cytokines/chemokines. Therefore, strategies that limit this sustained macrophage activation may be novel and useful approaches in the prevention of chronic kidney disease following acute kidney injury or to improve the long-term graft survival rate in kidney transplantation.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Statistical analysis
 Results
 Discussion
 References
 

  1. Star RA. Treatment of acute renal failure. Kidney Int (1998) 54:1817–1831.[CrossRef][Web of Science][Medline]
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Received for publication: 23. 5.07
Accepted in revised form: 7. 9.07


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J. Y. Ghee, D. H. Han, H. K. Song, W. Y. Kim, S. H. Kim, H. E. Yoon, B. S. Choi, Y. S. Kim, J. Kim, and C. W. Yang
The role of macrophage in the pathogenesis of chronic cyclosporine-induced nephropathy
Nephrol. Dial. Transplant., December 1, 2008; 23(12): 4061 - 4069.
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