Mesenchymal Stem Cells Infusion Prevents Acute Cellular Rejection in Rat Kidney Transplantation

Mesenchymal Stem Cells Infusion Prevents Acute Cellular Rejection in Rat Kidney Transplantation M. De Martino, S. Zonta, T. Rampino, M. Gregorini, F. Frassoni, G. Piotti, G. Bedino, L. Cobianchi, A. Dal Canton, P. Dionigi, and M. Alessiani ABSTRACT Mesenchymal stem cells (MSC) are multipotent cells that differentiate into various mature cell lineages. MSC show immunomodulatory effects by inhibiting T-cell proliferation. We evaluated the effect of the infusion of MSC in rats experimental kidney transplantation. Sprague-Dawley transgenic rats (SD) able to express the green fluorescent protein (EGFP) were used as MSC donors. Syngeneic (Lewis to Lewis, n ⫽ 10) and allogeneic (Fischer to Lewis, n ⫽ 10) kidney transplantations were performed after bilateral nephrectomy. Five transplanted rats who received syngeneic grafts, were treated with 3 ⫻ 106 MSC (Gr B), while the other 5 did not received MSC (Gr A). Five rats with allogenic grafts received 3 ⫻ 106 MSC (Gr C) and another 5 did not receive MSC (Gr D). The MSC were infused directly into the renal artery of the graft. No immunosuppressive therapy was provided. The animals were killed after 7 days. Biochemical analysis for renal function, histological (Banff criteria) and immunohistological analysis (ED1⫹ and CD8⫹) were performed on treated animals. MSC improved kidney function in Gr B and D vs Gr A and C. The tubular damage appeared to be less severe among Gr B and Gr D with respect to Gr A and C (P ⬍ .01). Vasculitis was more accentuated in Gr A and C (P ⬍ .01). MSCs reduced the inflammatory infiltrate; in Gr B and D, the number of ED1⫹ cells was lower than in Gr A and C (P ⬍ .005), which was also observed for CD8⫹ cells (P ⬍ .05). Our study demonstrated that the infusion of MSC attenuated histological damage from acute rejection by reducing the cellular infiltration. N INTERESTING stem cell population from a therapeutic perspective is multipotent mesenchymal stromal cells (MSC). Several studies have suggested that MSC may play a role to modulate immune responses.1,2 They are poor antigen-presenting cells that do not express major histocompatibility complex class II or costimulatory molecules. In accordance, expanded MSC do not stimulate T-cell proliferation in mixed lymphocyte reactions (MLR). They are able to downregulate alloreactive T-cell responses when added to mixed lymphocyte cultures (MLC).3 Recently, MSCs have been demonstrated to alter cytokine secretion profiles of naïve and effector T,2 dendritic (DC), and natural killer (NK) cells inducing a more anti-inflammatory or tolerant phenotype.1,4 In addition, when present in an inflammatory microenvironment, MSC inhibited interferon (IFN)-␥ secretion from Th1 and NK cells and increase interleukin (IL)-4 secretion from Th2 cells, thereby promoting a Th1-to-Th2 shift. Furthermore, MSC have been reported to induce T-cell

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division arrest,4 to inhibit differentiation and maturation of DCs,5 to inhibit B-cell proliferation,6 and to decrease the production of inflammatory cytokines by various immune cell populations.1 These immunomodulatory properties make MSC especially attractive for potential use to treat autoimmune and inflammatory conditions including rheumatoid arthritis, systemic lupus erythematosus (SLE), acute and chronic From the Chirurgia Epatopancreatica, IRCCS Pol. San Matteo e Dipartimento di Scienze Chirurgiche, Università di Pavia, Italy (M.D.M., S.Z., L.C., P.D., M.A.); Struttura Complessa di Nefrologia e Dialisi, IRCCS Pol. San Matteo e Università di Pavia, Italy (T.R., M.G., G.P., G.B., A.D.C.); Dipartimento di Ematologia, Ospedale San martino di Genova, Italy (F.F.). Address reprint requests to Zonta Sandro, MD, PhD, IRCCS Fondazione Pol. San Matteo, Istituto di Chirurgia Epatopancreatica, viale Golgi n° 21, 27100, Pavia, Italy. E-mail: s.zonta@ smatteo.pv.it

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0041-1345/–see front matter doi:10.1016/j.transproceed.2010.03.079

Transplantation Proceedings, 42, 1331–1335 (2010)

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kidney injury, and allograft rejection.1,3 The administration of MSC that produce a mix of anti-inflammatory, antifibrotic, and angiogenic cytokines may offer a novel multidrug delivery system. An attractive potential indication for MSC therapy in view of potent effects on DC and T-cell biology is kidney transplantation. In the current study we hypothesized that MSC downregulate the immune response controlling acute cellular rejection (ACR). We studied their effect in a rat kidney transplantation model in vivo. MATERIALS AND METHODS Animals Experiments were performed according to guidelines of our ethical committee for animal studies. Eleven-week-old Fisher F344 rats, (Charles River, Calco, Italy) were used as donors and 7-week-old Lewis RT1 rats (Charles River) as recipients. Transgenic SpragueDawley rats (SD) that express enhanced green fluorescent protein (Japan Slc, Inc, Hamamatsu, Japan) were used as MSC donors. Rats carrying the EGFP transgene were established using the same construct and technique as used to produce EGFP transgenic mice.7

Isolation and Culture of MSC Sprague-Dawley EGFP transgenic rats were killed by CO2 inhalation. Marrow cells, in both femurs and tibias were flushed from the bones using a syringe with a 26-gauge needle. Whole EGFPpositive bone marrow cells were counted, plated at a concentration of 106 cells/mL with Murine MesenCult medium (Stemcell Technologies, Vancouver, BC, Canada), and incubated at 37°C in a 5% humidified CO2 atmosphere. After 48 hours of culture, nonadherent cells were discarded, and fresh medium was added and replaced twice per week. When cultures reached subconfluence, adherent cells were detached with 0.05% trypsin (Sigma-Aldrich, St Louis, Mo). Collected cells were replated at a concentration ranging between 0.05 and 0.15 ⫻ 105/mL of MesenCult medium for several passages.

Immunocytochemical, Characterization, and Osteogenic and Adipogenic Differentiation of MSC Cultured EGFP MSC were evaluated by flow cytometry starting from the 3rd passage for CD90, CD73, CD105 and CD45, CD11b, CD34, CD79, HLA-DR (Becton Dickinson, San Josè, Calif) using a FacsCalibur flow cytometer (Becton Dickinson). Differentiation

into osteocytes and adipocytes was evaluated as previously described.8

Group of Study Lewis RT1 rat kidneys, were transplanted into Lewis RT1 rats for the syngeneic transplantation model. Fisher F344 rat kidneys were transplanted into Lewis RT1 rats for the allogeneic transplantation model. All recipients were bilaterally nephrectomized immediately before transplantation. We studied 4 groups of rats: Group A: 5 rats with Lewis RT1 to Lewis RT1 grafts received 200 ␮L of saline injected into renal artery soon after reperfusion. Group B included 5 rats with Lewis RT1 to Lewis RT1 transplants received 1 mL of PBS containing 3 ⫻ 106 MSC into renal artery soon after reperfusion. Group C were 10 Fisher F344 to Lewis RT1 allografts received 200 ␮l of saline into the renal artery soon after reperfusion. Group D was 10 rats with Fisher F344 to Lewis RT1 allografts that received 1 mL of PBS containing 3 ⫻ 106 MSC into the renal artery soon after reperfusion. No immunosuppressive therapy was administered. All rats were sacrificed at day 7. Thereafter the kidneys were removed with half fixed in 10% neutral-buffered formalin and half frozen in liquid nitrogen. All rats were males, weighing 200 to 250 g. They were housed in metabolic cages at constant room temperature (20 °C) and humidity (75%) under controlled light/dark cycle, with free access to water and standard chow diet. Rats were weighed daily using a Mettler PE-2000 balance. A 24-hour urine sample was collected on days ⫺1, 2, and 6, to measure volume before storage at ⫺20°C. Blood was drawn from the caudal vein on days 0, 3, and 7 rapidly centrifuged with the serum stored at ⫺20°C.

Donor Procedure Donors were inbred male Fisher rats of 250 g body weight (BW; Charles River). Both for kidney harvesting and transplantation, anesthesia was induced and maintained by intramuscular injection of droperidol (neuroleptanalgesic, 0.5 mg/100 g BW) and fentanyl (analgesic, 0.01 mg/100 g BW). After a midline incision, the left kidney was gently exposed; the ureter was isolated and a polyethylene stent (PE 10, inner 0.28 mm, Becton Dickinson) was placed next to the renal pelvis. A cannula was introduced into the infrarenal aorta to the ostium of the renal artery. A single dose of sodium heparin (1000 UI) was administered and the kidney immediately washed with University of Winsconsin solution (UW; 4 mL, 4°C, 20 mL/hr flow rate). The renal artery was excised with an aortic patch, the renal vein was cut proximal to its junction with

Table 1. Graded Score for Definition of the Renal Histological Damages Associated With Tubulitis, Arteritis, and Glomerulitis Tubulitis Score

t0 t1 t2

t3

Description

Arteritis Score

No mononuclear cells in tubules Foci with 1 to 4 cells/tubular cross-section Foci with 5 to 10 cells/tubular cross-section

v0 v1

Foci with ⬎10 cells/tubular cross section, or the presence of at least 2 areas of tubular basement membrane destruction

v3

v2

Description

No arteritis Mild-to-moderate intimal arteritis in at least 1 arterial cross-section Severe intimal arteritis with at least 25% luminal area lost in at least 1 arterial cross-section Transmural arteritis and/or arterial fibrinoid change and medial smooth muscle necrosis with lymphocytic infiltrate in vessel

Glomerulitis Score

Description

g0 g1

No glomerulitis Glomerulitis in ⬍25% of glomeruli

g2

Segmental or global glomerulitis in 25% to 75% of glomeruli

g3

Glomerulitis (mostly global) in ⬎75% of glomeruli

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Table 2. Baseline Features of the Study Groups

Group Group Group Group

A B C D

n

MSC

Warm Ischemia Time (min ⫾ SD)

Total Transplantation Procedure Time (min ⫾ SD)

5 5 5 5

No Yes No Yes

25 ⫾ 8 23 ⫾ 5 25 ⫾ 4 26 ⫾ 3

90 ⫾ 4 100 ⫾ 3 93 ⫾ 5 102 ⫾ 4

the cava and the ureter next to the stent. The kidney was transplanted immediately.

Transplantation Procedure Recipients were inbred male Lewis rats of the same BW with respect to the donor. The kidney was heterotopically grafted. Intra-arterial graft infusion was performed with saline solution for Gr A and C or MSCs suspended in PBS for Gr B and D. For this purpose, the host left kidney was gently exposed and the ureter cut next to the kidney pelvis. The host renal artery was isolated and a perfluorocarbon microcatheter (inner Ф 200 ␮m) was placed into the arterial lumen. Then, left and right nephrectomies were sequentially completed. The recipient aorta and cava vein were occluded with vascular clamps; 2 end-to-side anastomoses were performed with 9/0 monofilament sutures. After the vascular clamps had been removed, allowing the graft to be reperfused, the

ureter was anastomosed end-to-end introducing the donor endoureteral stent into the lumen of the recipient ureter and fixing it in the same fashion as in the donor. After 5 minute reperfusion, the intra-arterial graft infusion was performed using a microcatheter placed in the host left renal artery. To optimize the infusion into the graft, a vascular clamp was placed distal to the arterial anastomosis, temporarily occluding the aorta. In this way the renal graft was the first passage organ for the infusion since no other collateral arteries originate from the aortic tract between the host left renal artery and the graft renal artery. Finally, the laparotomy was closed with 3-0 silk sutures. A single intramuscular dose of ciprofloxacin (5 mg) was administered. The vascular anastomosis took no more than 45 minutes, and the total surgical time did not exceed 90 minutes. The animals housed in a light/dark cycle chamber were allowed free access to tap water and rat chow.

Biochemical Measurements Plasma creatinine and urinary protein concentrations were measured at days 0, 3, and 7 by an automated method (Abbott Laboratories, Abbott Park, Ill), and the Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, Calif), respectively.

Renal Morphology Along the maximal sagittal plane of formalin-fixed kidneys 3-␮mthick sections were cut and stained with hematoxylin-eosin, periodic acid-Schiff, and Movat pentachrome for evaluation according

Fig 1. Posttransplantation functional result of the renal graft in the groups of study. (A) Serum creatinemia; (B) cretinemia clearance; (C) proteinuria; (D) urinary output.

DE MARTINO, ZONTA, RAMPINO ET AL

1334 to the updated Banff classification.9 The 2 investigators were blinded. They used an Olympus IX8 microscope connected to a CCD camera with software imaging analysis Cell-R (Olympus Optical Co, Tokyo, Japan). To assess tubulitis, arteritis, and glomerulitis, we examined 250 tubular cross-sections, 10 arterial cross-sections, and 10 nonconsecutive renal sections, respectively, in 5 nonconsecutive renal sections per animal. The histological damage was then graded using the score shown in Table 1.

Analysis of the Cellular Infiltrate Analysis of the cellular infiltrate was performed blinded by assessing 40 consecutive high-power fields (⫻400 magnification). Tubular interstitial expression of ED-1 Ag, and CD8 were studied in formalin-fixed tissues, CD4 expression, in frozen tissues by immunohistochemistry. Briefly, 3-␮m-thick sections were exposed overnight at 4°C to the following antibodies: anti-ED-1 (AbD Serotec); or anti CD8 (AbD Serotec; or anti CD4 (AbD Serotec). The immune complex was visualized with the biotin-streptavidinperoxidase complex and 3,3-diaminobenzidine (DakoCytomation). Sections were faintly counterstained with Harris hematoxylin. Negative controls included omission of the primary Ab.

Statistical Analysis Analysis of variance followed by the Newman-Keuls test or Student’s t-test were used to compare the mean values. Differences in renal injury scores were evaluated with Mann-Whitney and Kruskal-Wallis tests.

RESULTS

The baseline features of the study groups are shown in Table 2 The mean duration of the surgical procedure was respectively 90 ⫾ 11 minutes for Gr A, 98 ⫾ 5 minutes for Gr B, 93 ⫾ 5 minutes for Gr C, and 102 ⫾ 7 minutes for Gr D. We did not observe any surgical complication associated with the procedures. In particular, we did not note any arterial infarction in Gr B and D after MSC infusion. There was no delayed graft function, which was achieved by maintaining the warm ischemia time below 30 minutes. The postoperative functional renal parameters are plotted in Fig 1. A significant difference (P ⬍ .05) was noted between Gr A and B and D versus Gr C for serum creatinine, creatinine clearance, proteinuria, and diuresis. No other significant differences were registered between Gr A, B, and D regarding functional parameters of the renal grafts (P ⫽ .87). The histological examination of graft specimens according to the Banff criteria regarding tubulitis, arteritis, and glomerulitis are shown in Fig 2. Gr C tubulitis score was different from Gr D for tubular damage (t0, t1, t2, and t3). (P ⬍ .001). The differences in t0 scores between Gr D and Gr A and Gr B were significant; data that were confirmed for the v0 score and g0 score: v0-Gr A vs Gr D and Gr B vs Gr D (P ⫽ .02) g0-Gr A vs Gr D and Gr B vs Gr D (P ⫽ .01). Regarding the arteritis score, Gr D show significant

Fig 2. Tubulitis (A), vasculitis (B), and glomerulitis (C) scores reported in the 3 groups of study according to the Banff criteria after the observation of 250 cross-sections in 5 nonconsecutive renal sections for animal. D; Mean number of CD4, CD8, and ED1-positive cells observed in 20 high-resolution fields for each tissue specimen in treated rats.

MESENCHYMAL STEM CELLS INFUSION

differences for v0 and v3 versus Gr C (P ⬍ .001); also for the glomerulitis, Gr D showed histological damage with respect to Gr C: g0-Gr D vs Gr C (P ⫽ .001); G3-Gr D vs Gr C (P ⫽ .001). Fig 2D shows the number of CD4/CD8 positive cells in the renal graft. The difference between the mean values of the numbers of CD4, CD8, and ED-1–positive cells for high power field reported in Gr D versus Gr C were highly significant (P ⫽ .000). DISCUSSION

It has been suggested that MSC may have beneficial roles toward immunomodulation and prevention of acute rejection after transplantation of solid organs in rats. After the establishment of a reliable, well-reproducible surgical model, we collected results that supported this hypothesis. First, we ensured that the MSC reached the renal graft at high concentrations using superselective arterial infusion. So we avoided the barrier of the pulmonary capillaries when the peripheral venous infusion is performed; in this site MSCs remain largely trapped because of their large size.10,11 Analyses of histological and immunohistochemical data confirmed the protective effects of MSC in kidney transplantation to reduce the inflammatory response and conserve organ function. Comparing our data with those of Zhang et al,12 the difference in outcomes and control of acute cellular rejection was mainly due to the route of MSC infusion. This observation implies that immunomodulating effects of MSCs strictly depend on a high concentration at the site of immunogenic injury.13 To transfer the use of MSC to a clinical setting to prevent acute rejection in solid organ transplantation, there are issues to be resolved concerning the residence time of MSC in the host and the maintenance of immunosuppressive capacity as well as frequency and optimal doses and the

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long-term effects. The transfer of the rodent animal model to a large size animal may provide answers to these questions.

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