EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: In this study, we evaluated the effects of CXC chemokine receptor type 4 and stromal cell-derived factor 1 signaling in the progression of chronic allograft nephropathy in a rat model.

Materials and Methods: Experimental rats were divided into 3 groups: Lewis-to-Lewis isograft transplant (group A), Fisher 344 rat-to-Lewis allograft transplant with immunosuppressant cyclosporine (group B), and Fisher 344 rat-to-Lewis allograft transplant treated with cyclosporine and the CXC chemokine receptor type 4 antagonist AMD3100 (1 mg/kg/d) (group C). On day 90 after the operation, renal graft function, proteinuria, and histologic Banff score were measured. The expression levels of transforming growth factor β1 and collagen IV were determined by quantitative real-time polymerase chain reaction.

Results: Renal function and urinary protein were increased in allografts of groups B and C compared with isografts of group A. The Banff score was significantly decreased in the AMD3100-treated animals (group C), with renal fibrosis being reduced. In addition, overexpressed levels of transforming growth factor β1 and collagen IV in group B allografts were significantly reduced versus that shown with treatment with the CXC chemokine receptor type 4 antagonist in group C.

Conclusions: Together, these data strongly implicate that CXC chemokine receptor type 4 antagonism alleviated renal interstitial fibrosis in long-term surviving allografts by down-regulating expression of transforming growth factor β1.

Key words : Chronic allograft nephropathy, CXCR4 antagonist, Renal transplantation, Stromal cell-derived factor 1

Kidney transplant is the best treatment for patients with end-stage renal failure. The development of chronic allograft nephropathy (CAN) remains a major issue for kidney transplant recipients.¹ Therefore, it is necessary to develop interventional strategies to prevent long-term surviving allografts from CAN progression.

The CXC chemokine receptor type 4 (CXCR4) is an alpha-chemokine receptor specific for stromal cell-derived factor 1 (SDF-1); their interaction has a potent chemotactic activity in the trafficking of various bone marrow-derived cells, including lymphocytes, fibrocystoid cells, and CD34-positive stem cells.² CXCR4 has been shown to be up-regulated in fibrotic renal samples.³ Overexpression of CXCR4 in renal podocytes causes glomerular disease, and blocking CXCR4 improves glomerulonephritis.⁴ Recent research has shown that CXCR4 antagonism attenuates cardiorenal fibrosis.⁵ In a unilateral ureteral obstruction rat model, Yuan and associates⁶ demonstrated that CXCR4 contributes to renal fibrosis via its multiple effectors. Our previous study⁷ proved that prolonged cold ischemia induced the elevation of leukocyte recruitment associated with the up-regulated expression of the chemokine receptor. Thus, it is worthy to further evaluate the role of the CXCR4/SDF-1 signaling pathway in the progression of chronic allograft disease in rat renal grafts with long cold ischemia.

Fisher 344 rat (F344)-to-Lewis rat kidney transplant was performed, in which cyclosporine was used as the immunosuppressive regimen in this investigation. The CXCR4 antagonist AMD3100 is a drug that is widely used to block the CXCR4 receptor. Accordingly, we investigated the effects of AMD3100 on the development of CAN in a rat model.

Materials and Methods

Animals
Inbred male Lewis rats (RT1) and F344 rats (RT1lvr) weighing 200 to 250 g were purchased from an experimental animal center (Vital River Laboratory Animal Technology Co. Ltd., Beijing, China). All animals were maintained under standard conditions in a specific pathogen-free animal room and were fed standard rodent chow and water ad libitum. Rat studies were conducted in accordance with the Guidelines of the Committee on the Care and Use of Laboratory Animals and Good Laboratory Practice.

Experimental protocol
Orthotopic rat kidney transplant was performed as previously described.⁷ Donor kidneys were procured and stored in 4°C Belzer University of Wisconsin solution for 12 hours until implantation. The renal grafts were implanted into bilaterally nephrectomized recipients. Renal vessel and ureter anastomoses were performed end-to-end using 10/0 sutures. The duration of anastomosis was maintained within 20 minutes. The CXCR4 antagonist AMD3100 (Mozobil, Genzyme Corporation, Cambridge, MA, USA) and immunosuppressant cyclosporine (Sigma-Aldrich, San Quentin, CA, USA) were used in the recipient animals.

Rats were randomly divided into the following 3 groups (n = 10). Group A received Lewis-to-Lewis isograft transplant without any treatment, group B received allograft transplant (F344-to-Lewis) with only cyclosporine treatment (10 mg/kg/d), and group C received allograft transplant with cyclosporine and AMD3100 (1 mg/kg/day) until sampling. The animals were killed by exsanguination under general anesthesia on day 90 after transplant. The samples were collected, and graft survival rates were monitored. The renal transplant model was stable, and 50% of rats survived until sampling.

Biochemical analyses
Blood samples were collected, sera were obtained using centrifugation of blood (3000g for 10 min), and samples were stored at -80°C until assaying. The levels of serum creatinine, serum urea nitrogen, and urinary protein were measured using an automatic biochemistry analyzer.

Renal histologic analyses
Kidney grafts were fixed in a 10% formalin solution and embedded with paraffin. Sections (4 μm thickness) were stained with hematoxylin and eosin or Masson stain. Pathologists performed the manipulations, and sections were photographed using a Leica camera (Leica Camera AG, Solms, Germany). The Banff score was calculated using the quantitative criteria of Banff 09, including the change in the degree (score 0-3, ranging from mild to severe) of renal glomerular sclerosis, tubular atrophy, inflammatory infiltration, arteriolar intimal thickness, and interstitial fibrosis. The total score ranged from 0 to 15. Values are shown as means ± standard deviation from 5 independent experiments.

Immunohistochemistry assay
Rabbit anti-SDF-1 and CXCR4 (1:200; BD Pharmingen, San Jose, CA, USA) antibodies were used to detect their expression. The secondary antibody used for immunostaining was biotin-conjugated antimouse immunoglobulin (1:200; Boster Biological technology, Wuhan, China). It was used for 30 minutes followed by horseradish peroxidase-conjugated streptavidin with 3,3′-diaminobenzidine as the chromogen. The entire process was performed with the use of standard immunohistochemistry protocols. We analyzed 3,3′-diaminobenzidine staining using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Ten representative fields per section were randomly selected by an investigator who was blinded to the treatment groups. Values were calculated as percentages of positive cells.

Real-time polymerase chain reaction
Quantitative real-time polymerase chain reaction (PCR) was performed for transforming growth factor β1 (TGF-β1) and collagen IV mRNA analyses. Kidneys were surgically removed and homogenized, and RNA was isolated using an RNeasy mini kit (Qiagen, Shanghai, China). RNA (1 μg) was subsequently transcribed into cDNA using Iscript (Bio-Rad, Hercules, CA, USA). A 1:10 dilution of this cDNA was used for quantitative real-time PCR analysis using the iTaq universal SYBR Green Supermix (Bio-Rad). A Bio-Rad CFX-96 real-time PCR machine was used to perform the reactions and analyze the cycle time values. The respective cycle time values were normalized to the Hprt1 coding gene, and values from all experimental groups were expressed relative to the uninjured kidney or vehicle control for in vitro studies. Primers for rat TGF-β1 and collagen IV were purchased from SA Biosciences (Qiagen). The primer sequences for the remaining genes are as follows: forward 5’-CCTGAGTGGCT GTCTTTTGAC-3’ and reverse 5’-CCTGTATTCCG TCTCCTTGGT-3’ for TGF-β1 (Tgfb1); forward 5’-CGGGTACCCAGGACTCA TAG-3’ and reverse 5’-GGACCTGCTTCACCCTTTTC-3’ for collagen IV; and forward 5’-CTGGTGCTGAGTATGTCGTG-3’ and reverse 5’-CAG TCTTCTGA GTGGCAGTG-3’ for GAPDH (Gapdh). The ratios of the TGF-β1 and collagen IV band intensities to the GAPDH band intensity were used to evaluate relative mRNA expression.

Statistical analyses
Data are expressed as means ± standard deviation. Comparisons between treatment groups were analyzed using one-way analysis of variance followed by a post hoc Student Newman-Keuls test using GraphPad Prism 5.0 software (GraphPad, La Jolla, CA, USA). Significance was analyzed using the log-rank test with SPSS software (SPSS: an IBM Company, version 17.0, IBM Corporation, Armonk, NY, USA). P < .05 was considered to be statistically significant.

Characteristics and graft function in study animals
Characteristics of the transplant groups are shown in Table 1. There were no differences in body weight, graft weight, and warm ischemia time between all groups. Renal function and urinary protein were increased in groups B and C compared with group A, but there were no significant differences between groups B and C (Table 1).

Survival rates and histologic assessment
The 90-day survival rate was somewhat higher in group C; however, differences were not significant among the 3 groups. Pathologic changes in grafts stained with hematoxylin and eosin and Masson were scored according to Banff 09 standard. The mean score of group B was higher than in group A, which was significantly reduced by treatment with the CXCR4 antagonist in group B (Figure 1).

Expression of profibrotic genes and CXCR4/SDF-1 axis
Transforming growth factor β1 was overexpressed in group B compared with group A, and treatment with the CXCR4 antagonist in group C attenuated the mRNA expression of TGF-β1 in the allografts. The mRNA expression of collagen IV was also significantly increased in group B compared with group A, an effect that was suppressed by treatment with the CXCR4 antagonist in group C (Figure 2). Renal allografts showed a significant increase in SDF-1 and CXCR4 staining in groups B and C compared with isografts in group A. We also observed that CXCR4 was significantly decreased in group C versus group B (Figure 3), although no apparent differences in SDF-1 expression were seen between group B and group C.

Over the long term, kidney transplant patients gradually develop CAN, which leads to graft loss. Chronic allograft nephropathy is pathologically characterized by glomerulosclerosis, tubular atrophy, and interstitial fibrosis (IF/TA). The mechanism of CAN is complex and involves multiple factors such as ischemia/reperfusion injury and acute and chronic inflammatory responses. Episodes of chronic interstitial lymphocyte infiltration contribute to the progression of IF/TA.

The chemoattracting axis of CXCR4/SDF-1 plays an important role in kidney disease, such as nephritis5 and glomerulonephritis4 via lymphocyte recruitment and subsequent podocyte proliferation. The chemokine receptor CXCR4 has been shown to be overexpressed in IF/TA samples compared with normal kidneys.3 The development of IF/TA was the cumulative result of time-dependent chronic injuries, in which mononuclear cells infiltrate into ischemic tissue via this chemokine axis,^8,9 resulting in permanent damage and loss of nephrons.¹⁰ The persistent expression of the CXCR4/SDF-1 axis¹⁰ in long-term surviving grafts induced the profibrotic cytokine TGF-β1 and fibrotic protein collagen IV, promoting the development and progression of IF/TA. Inhibition of TGF-β in rats resulted in marked improvement of renal fibrosis.¹¹ Consistent with that finding, our results showed that continued treatment with the CXCR4 antagonist inhibited the expression of profibrotic and fibrotic genes and attenuated renal fibrosis.

There is a different effect of SDF-1 expression on renal grafts, based on the degree of kidney damage and the expression level of the CXCR4/SDF-1 axis. In a slightly damaged kidney, the CXCR4/SDF-1 signal facilitates the repair effect of CD34-positive stem cells and preserves microvascular integrity.¹² In allografts with severe ischemia/reperfusion-induced injury, the high expression of CXCR4/SDF-1 axis can overcome the repair effect and accelerate the chronic inflammatory process by chemoattracting mononuclear cells.¹³ This finding also explains the discrepancy in the role of the CXCR4/SDF-1 pathway in the ischemic kidney. Chen and associates showed that augmentation of this pathway attenuated the progression of chronic renal disease in a rat nephrectomy model,¹² whereas Gao and associates proved that down-regulated expression of SDF-1 provided a protective effect of allografts in a renal transplant model.¹⁰ As we have demonstrated, the CXCR4/SDF-1 axis is up-regulated in allografts, leading to renal fibrosis. This may be because the CXCR4/SDF-1 axis is expressed in multiple immune cells, which could trigger several signaling pathways. To further resolve this discordance, more detailed insights into the molecular mechanisms are essential.

In summary, these data show that CXCR4 antagonism is partially effective in delaying renal fibrosis in long-term surviving allografts. The effect is associated with dampened expression of the profibrotic gene.

Volume: 15 Issue: 4 August 2017

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