A PHASE 1 STUDY OF MESENCHYMAL STROMAL CELLS AND ISCHAEMIA REPERFUSION INJURY IN DECEASED DONOR RENAL TRANSPLANTATION

A IRISH1,2, S SWAMINATHAN1, S FIDLER4,6, LD’ORSOGNA2,4,6, R SINNIAH2,5,6, M STURM3

1Fiona Stanley Hospital, Murdoch, Australia; 2University of Western Australia,Nedlands, Australia; 3Cell and Tissue Therapies Western Australia, Perth, Australi; 4Department of Immunology, Murdoch, Australia; 5Department of Histopathology,Murdoch, Australia; 6Pathwest Laboratory, Nedlands, Australia

Background: Ischaemia Reperfusion Injury (IRI) is associated with delayed graft function (DGF) and reduced graft survival. Mesenchymal Stromal cells (MSCs) have anti-inflammatory and immune regulating properties that may mitigate IRI.

Aims: A Phase 1 study to assess safety and tolerability of bone-marrow derived allogeneic MSCs in deceased donor renal transplant recipients (RTR).

Methods: We administered MSCs from 2 donors to 12 RTRs in a dose of 2 x 106 cells/kg within 2 hours of reperfusion and on day 7. Patients received standard induction and immunosuppression. Patients were followed for 12 months with monitoring of graft function, HLA antibody, infectious complications and graft biopsy at 3 and 12 months. We compared them with 98 deceased donor controls (DC) performed between 2012-15.

Results: This is the first trial of allogeneic MSCs in deceased donor renal transplant recipients. 12 patients (5 male, mean age 45) were included in the study. There were no infusion reactions. Compared with DC, the MSC recipients had a higher proportion of regrafts (33% vs 13%) and shipped kidneys but no difference in ischaemic time (10±3 vs 10±4 hours) or DGF (17% vs 18%). Compared with DC, creatinine at 1 (134 vs 182 μmol/L) and 3 (123 vs 168 μmol/L) months was not significantly different. 2/12 MSCr had BPAR; one due to recurrent non-compliance and another receiving a steroid free protocol. There were no significant infectious complications (viral or bacterial).  Serial HLA monitoring detected de novo DSA against 1 kidney but no MSC donor at 12 months.

Conclusions: Immediate infusion of MSCs was well tolerated with no early safety signals of allogenicity, graft injury or over-immunosuppression. Larger controlled trials are warranted.

LOCALLY SECRETED ICOS-IG PROLONGS SURVIVAL OF XENOGRAFTS AND ALLOGRAFTS

L. IERINO1, D. CHRISTIANSEN2, E. MOUHTOURIS2, M. SANDRIN2

1Department of Nephrology and University of Melbourne, St Vincent’s Health, Fitzroy, Victoria;  2Department of Surgery and University of Melbourne, Austin Health, Heidelberg, Victoria.

Background: Our research has focused on the therapeutic strategy of locally secreted immunomodulatory molecules by cellular xenografts and previously demonstrated that survival of the porcine endothelial cell line (PIEC) engineered to secret soluble ICOS-Ig is specifically prolonged compared to wild-type PIEC.  Xenograft prolongation was associated with CD4+CD25+Foxp3+ graft infiltrating T cells consistent with regulatory T cells.  The effect of locally secreted ICOS-Ig on allograft survival was also examined.

Aim: (a) Study the mechanism of xenograft prolongation in more detail and (b) Determine whether allografts are prolonged using locally secreted ICOS-Ig.

Method and Results: CD4+25+ T cells from ICOS-Ig secreting xenografts were purified and the phenotype shown to express a range of markers consistent with regulatory T cells including Foxp3, Helios, CD127low, CD275 (ICOSL), CD278 (ICOS), CD39 and other regulatory T cell markers.  Gene analysis of CD4+25+ T cells demonstrated increased Foxp3, IL-10, ICOS, granzyme B (GzmB) and perforin (pfp).  The functional role of IL-10, pfp, GzmB in mediating xenograft prolongation was confirmed by transplanting ICOS-Ig PIEC and wild type PIEC into IL-10-/-, pfp-/- and GzmB-/- mice which demonstrated that ICOS-Ig PIEC and wild type PIEC were rejected in a similar time.  The outcome of allografts was examined using locally expressed ICOS-Ig.  A stable clone of NS-1 cell line (BALB/c origin) secreting ICOS-Ig was generated and effect on allograft survival examined by grafting onto C57BL/6 mice. Graft survival was significantly prolonged from 8 days to 15 days (p=0.008).

Conclusion: Prolonged survival of xenografts expressing ICOS-Ig was mediated by CD4+CD25+Foxp3+ Regulatory T cells.  Allograft survival was also prolonged using locally secreted ICOS-Ig.  The data indicates this therapeutic strategy may have broad clinical applications in transplantation.

FLT3 INHIBITOR ATTENUATES RENAL INJURY IN ADRIAMYCIN NEPHROPATHY BY SUPPRESSING CD103+ DC-MEDIATED T CELL ACTIVATION

Q CAO1, R WANG1, T CHEN1, P RAO1, Z ZHANG1, VENCENT LEE1, G ZHENG1, Y WANG1, DC HARRIS1

 1The Westmead Institute for Medical Research, The University of Sydney, NSW.

Aim and Background:

Chronic kidney disease (CKD) is a global public health problem, and lacks effective treatments. In our previous study, CD103+ DCs were shown to play a pathogenic role in Adriamycin nephropathy (AN), a model of focal sclerosing GN. FMS-like tyrosine kinase 3 (Flt3) is a receptor which is highly and specifically expressed on tissue resident CD103+ DCs. To test the effect on renal injury of inhibition of Flt3 on CD103+ DCs, we used a selective Flt3 inhibitor (AC220) to treat mice with AN.

Methods:

AN was induced in BALB/c mice, who were treated daily for 14 days with 10mg/kg AC220 or Vehicle (n=8/group) from day 7 after adriamycin, when AN was established. Renal functional and structural injury, as well as inflammatory cytokine expression and cell infiltration were assessed.

Results:

The number of kidney CD103+ DCs, but not CD103- DCs or plasmacytoid DCs, was significantly decreased in AN mice after AC220 administration. Treatment with AC220 significantly improved renal function and reduced the renal injury and fibrosis in AN mice. AC220-treated AN mice had decreased levels of inflammatory cytokines of IL-1beta, IL-6 and TNF-a. AC220 treatment decreased infiltration of CD4 T cells, CD8 T cells and macrophages in kidney, and reduced inflammatory cytokine and cytotoxic molecule expression of kidney CD8 T cells in AN mice.

Conclusions:

Flt3 inhibitor AC220 effectively reduced renal injury in AN mice, suggesting that this inhibitor might be a useful pharmaceutical agent to treat chronic kidney disease.

DEVELOPMENT OF MONOCLONAL ANTIBODIES FOR USE IN A NEW MOUSE MODEL OF VASCULITIS

H HUTTON1,2, J OOI1, S HOLDSWORTH1,2, A.R KITCHING1,2

1Centre for Inflammatory diseases, Monash University, Clayton, VIC; 2Dept Nephrology, Monash Health, Clayton, VIC

Aim: To develop and characterise anti-MPO monoclonal antibodies (mAbs) for use in a model of ANCA associated vasculitis (AAV).

Background: Currently available mouse models of anti-MPO vasculitis vary between centres, and are resource intensive, often necessitating the generation of polyclonal anti-MPO. Human studies suggest that epitope specificity is important in the pathogenicity of anti-MPO antibodies. MPO447-459 (human sequence numbering) is a B cell epitope, conserved in mice, that was exclusively associated with disease in humans.

Methods: Anti-MPO hybridoma cells were produced by Monash Antibody Technology Facility. 2000 single cells were cultured, and supernatants tested against mouse and human MPO by ELISA. Nine clones were selected for further assessment: ELISAs assessed binding to mouse and human MPO, and MPO447-459. Assessment of mAb induction of neutrophil reactive oxygen species (ROS) was performed using a flow cytometric dihydrorhodamine assay. Neutrophil recruitment at 4 hours and 7 days was assessed by injecting mice with 10ug LPS followed by 1mg mAb, polyclonal MPO-ANCA, or control IgG2a; immunohistochemistry on kidney sections was performed.

Results: Of the nine selected, two mAbs (1D1 and 2H1) bound to all three of human, mouse, and MPO447-459. These two mAbs induced neutrophil ROS comparable to polyclonal ANCA (ANCA 11.2, 1D1 mAb 17.4, 2H1 mAb 8.3% neutrophils ROS+, control IgG2a mAb 1.7, all other anti-MPO mAbs<2). At 4 hours, 1D1 administration resulted in significantly increased glomerular neutrophil recruitment compared to control IgG2a (1.50± 0.08 vs 0.42±0.12 neutrophils per glomerular cross section, p=0.036) but, as with models using polyclonal anti-MPO antibodies, prominent neutrophil recruitment was not seen after 7 days.

Conclusion: mAbs 1D1 and 2H1 show promise for in vitro assays, and in a mouse model of AAV.

 

INTERLEUKIN (IL)-17 PRODUCTION BY TUBULOINTERSTITIAL HUMAN GAMMA-DELTA T CELLS IN RENAL FIBROSIS AND CHRONIC KIDNEY DISEASE

AJ KASSIANOS1,2,3,4, B LAW1,2,3, X WANG1,2, K KILDEY1,2, M LINDNER1,2,4, MJ RIST1,2, K BEAGLEY3, R WILKINSON1,2,3,4, H HEALY1,2

1Conjoint Kidney Laboratory, Pathology Queensland, Brisbane, Queensland; 2Kidney Health Service, Royal Brisbane and Women’s Hospital, Brisbane, Queensland; 3Queensland University of Technology, Brisbane, Queensland; 4Medical School, University of Queensland, Brisbane, Queensland

Aim: To characterise γ/δ T cells present in human fibrotic chronic kidney disease (CKD).

Background: γ/δ T cells are effector lymphocytes recognised as having important functional roles during chronic inflammatory processes. Mouse studies suggest a pathological role for γ/δ T cells in immune-mediated models of kidney disease. This study evaluates γ/δ T cells in human CKD.

Methods:  We extracted γ/δ T cells from healthy kidney tissue and diseased biopsies with and without fibrosis. γ/δ T cells were identified, enumerated and phenotyped by twelve-colour flow cytometry. Localisation of γ/δ T cells was examined by multi-colour immunofluorescent microscopy.

Results: We detected significantly elevated numbers of γ/δ T cells (CD45+CD3+γ/δ+) in diseased biopsies with interstitial fibrosis compared with diseased biopsies without fibrosis and healthy kidney tissue. The increased numbers of γ/δ T cells correlated significantly with loss of kidney function (eGFR). Immunofluorescent analysis of fibrotic kidney tissue localised the accumulation of γ/δ T cells within the tubulointerstitial compartment, adjacent to proximal tubular epithelial cells (PTEC), defined as tubular cells expressing aquaporin-1. Notably, we identified tubulointerstitial γ/δ T cells as a key source of pro-inflammatory cytokine IL-17.

Conclusion: The correlation of IL-17-producing γ/δ T cells with histologically and functionally more severe CKD suggests a pathological role. Further functional dissection of γ/δ T cells is now required for the development of therapeutics capable of blocking this previously untargeted immune cell population.

RENAL EXPRESSION OF TOLL-LIKE RECEPTORS IN PATIENTS WITH CRESCENTIC GLOMERULONEPHRITIS

O’SULLIVAN, K.M1, FORD, S.L1, KITCHING, A.R.1,2 HOLDSWORTH, S.R.1,2

1Centre for Inflammatory Diseases, Dept. Of Medicine, Monash University, Clayton; Australia, 2Dept. of Nephrology, Monash Health Clayton, Australia

Aim: We sought evidence of aberrant renal TLR expression in 46 patients with acute crescentic glomerulonephritis (38 ANCA associated vasculitis, 8 Lupus Nephritis)

Background: Infections can initiate and exacerbate disease in patients with crescentic glomerulonephritis (GN) and provoke disease relapse. TLRs may be the possible link between infection and autoimmunity.

Methods: Multi colour confocal microscopy was used to examine kidney biopsies from patients with a first presentation of AAV (38), Lupus Nephritis (8) and controls (10) and stained for TLR2, TLR4 and TLR9, CD34, nephrin, CD68 (macrophages), and neutrophil elastase. The extent of staining was measured using Image J, and correlated with both clinical and histological parameters.

Results: TLR2, TLR4 and TLR9 expression was significantly increased in the AAV patient biopsies in both the glomeruli and tubulointerstitium, compared to controls (all, P<0.05).TLR2 expression was significantly increased within the glomeruli and tubulointerstitium of AAV patients compared to the LN patients (P<0.05), whereas there was no significant difference in TLR4 and TLR9 expression between the two disease groups. There was significant col-localisation of each TLR and its known ligand (High Mobility Group Box 1, and Fibrinogen), indicative of a functional relevance.TLR2 and TLR4 were predominantly expressed on endothelial cells, podocytes and leukocytes, whereas TLR9 was expressed by podocytes and infiltrating glomerular macrophages. TLR2 and TLR4 expression correlated inversely with presenting eGFR (P=<0.02).

Conclusions: TLR2, TLR4 and TLR9 expression in biopsies from AAV and LN patients is dysregulated. LN patients had enhanced renal TLR expression but not to the same extent as AAV patients. These results suggest that TLR expression is significantly increased in AAV compared to LN, indicating these receptors may be a potential target for therapeutics.

THE IMPACT OF INTRAUTERINE INFLAMMATION ON FETAL RENAL DEVELOPMENT

N TRAN1, MR SUTHERLAND1, TJ FLORES1, D RYAN1, L MOORE2, AL KENT3, JE DAHLSTROM3,4, JF BERTRAM1, VG PUELLES5, MJ BLACK1

1Monash University, Clayton, VIC; 2SA Pathology, Women’s and Children’s Hospital, and the University of Adelaide, Adelaide, SA; 3Canberra Hospital, Australian National University Medical School, Canberra, ACT; 4ACT Pathology, Canberra, ACT; 5RWTH Aachen University, Aachen, Germany

Aim: To determine the effects of chorioamnionitis on nephrogenesis and podocyte number in the developing human kidney.

Background: Chorioamnionitis, a common cause of preterm birth, is the inflammation of fetal membranes due to a bacterial infection during pregnancy. The fetal inflammatory response associated with chorioamnionitis has the potential to disrupt ongoing organ development, and is known to lead to organ injury. The specific impact of chorioamnionitis on nephron formation (nephrogenesis) in the human kidney, however, is unknown.

Methods: Archived kidney tissue was collected at autopsy from infants aged 21-28 weeks gestation (term = 38 weeks) that had been exposed (n=12), or unexposed (control, n=12), to chorioamnionitis in utero. Any intrauterine growth restricted infants were excluded. Nephrogenic zone width, glomerular generation number and glomerular maturity were assessed histologically. Immunofluorescent staining and stereology were used to assess glomerular volume, podocyte number, and podocyte density of inner-cortical glomeruli.

Results: There was no significant difference in kidney weight, nephrogenic zone width, glomerular generation number or glomerular maturity between groups. Average podocyte number (control: 697±16, chorioamnionitis: 657±15, p=0.08) and density (control: 2,163±75, chorioamnionitis: 1,978±64 per 105 µm3, p=0.07) per glomerulus tended to be lower in chorioamnionitis-exposed infants compared to controls, but this did not quite reach statistical significance.

Conclusions: Exposure to chorioamnionitis during gestation does not appear to significantly affect nephrogenesis or podocyte endowment in the kidneys of non-growth restricted infants.

About ANZSN

The ASM is hosted by Australian and New Zealand Society of Nephrology.

The aims of the Society are to promote and support the study of the kidney and urinary tract in health and disease, and to ensure the highest professional standards for the practice of nephrology in Australia and New Zealand.

Conference Managers

Please contact the team at Conference Design with any questions regarding the Annual Scientific Meeting

© 2015 - 2016 Conference Design Pty Ltd