A CRUCIAL ROLE FOR INTERLEUKIN-18 IN THE DEVELOPMENT OF RENAL INFLAMMATION AND ELEVATED BLOOD PRESSURE IN 1 KIDNEY/DOCA/SALT-INDUCED HYPERTENSION

J THOMAS1, Y LING1, S KRISHNAN2, D FERENS2, B KEMP-HARPER2, C SOBEY1, G DRUMMOND1, B HUUSKES1, A VINH1
1Department of Physiology, Anatomy and Microbiology, La Trobe University, Bundoora, Australia, 2Department of Pharmacology, Monash University, Clayton, Australia

Aim: To test the hypothesis that Interleukin-18 (IL-18) signalling contributes to the development of experimental hypertension and renal inflammation.
Background: Clinical studies have shown that levels of IL-18 are elevated in hypertensive patients. However, whether IL-18 plays a causal role in hypertension is unknown.
Methods: Hypertension was induced in male wild-type (WT) and IL-18-/- mice by uninephrectomy and treatment with deoxycorticosterone acetate (2.4 mg/d, s.c.) and saline (0.9%) drinking water (1K/DOCA/salt). Blood pressure (BP) was measured via tail cuff and radiotelemetry, and after 21 days kidneys were harvested for histopathological, mRNA and flow cytometric analyses.
Results: 1K/DOCA/salt-treated mice had higher BPs (140±3mmHg vs 118±2mmHg; n≥17; P≤0.0001) and renal weights (390±8mg vs. 291±7mg; n≥17; P<0.05) than 1K/placebo mice, and more than double the number of CD45+ leukocytes in their kidneys. 1K/DOCA/salt-treated mice also displayed >2-fold increases in renal mRNA expression of pro-IL-18 and its receptor, IL-18R (n≥6; P<0.01), with the latter due partly to an increased number of IL-18R-expressing T cells within the kidneys (22039±3246 vs 2958±1100; n≥7; P< 0.001). Phenotypic profiling of IL-18R+ T cells revealed that they were an important source of IL-17 in the kidneys of 1K/DOCA/salt mice (P<0.05 vs IL-18R-T cells). T cell-dependent production of IL-17 was abolished in IL-18-/- mice (n=3-8; P<0.05) and this was associated with protection from the development of renal fibrosis and a blunted hypertensive response to 1K/DOCA/salt (122±3 vs 143±4mmHg in WT mice; n≥9; P≤0.001).
Conclusions: Experimental hypertension is associated with upregulation of IL-18 signalling in the kidney. IL-18 deficiency protected against renal T cell activation and fibrosis and the development of high BP, highlighting the IL-18 system as a potential target for future anti-hypertensive therapies.


Biography:
Jordyn Thomas is a PhD student in the Vascular Biology and Immunopharmacology Group at La Trobe University. Her project aims to elucidate how inflammasome activation and subsequent production of IL-18 contributes to hypertension and chronic kidney disease. Although only 12 months into her candidature, Jordyn has already presented at a national conference, and she recently delivered an oral presentation at Experimental Biology 2018 (San Diego, USA). Jordyn has also written a comprehensive review on “The diagnostic and therapeutic potential of the IL-18 signalling pathway in chronic kidney disease” which is under consideration at the British Journal of Pharmacology.

PERSONALISED IMMUNOLOGY IN THE MANAGEMENT OF COMPLEX AUTOIMMUNITY

P PURI1, G WALTERS1,3, K GIBSON1, A COOK2, D FULCHER3, C VINUESA2,3, SJIANG1,2,3
1The Canberra Hospital , Canberra City., Australia, 2Department of Immunology and Infectious Disease, John Curtin School of Medical Research, Canberra City , Australia, 3Centre for Personalised Immunology, NHMRC Centre for Research Excellence, Canberra city,  Australia

A 72-year-old male presented with severe hypophosphatemia, hypokalaemia and hypomagnesaemia associated with recurrent pathological fractures. His background included a steroid-dependant eosinophilia-associated autoimmune disease (EAAD), associated with hypogammaglobulinaemia. Investigations demonstrated hyperphosphaturia (85mmol/24 hours) with normal renal function, parathyroid hormone (PTH) and 25- and 1,25-dihydroxycholecalciferol levels. Serum fibroblast growth factor (FGF23) levels ranged between 56 – 900ng/L suggestive of tumour-induced osteomalacia (TIO). Venous sampling and DOTATATE CT-PET were non-diagnostic. Aside from persistent eosinophilia, autoimmune serology was negative.
Treatment: Phosphate was corrected with parenteral phosphate (dose 80mmol/12hr) and calcitriol (3ug/daily). The patient was trialled on methotrexate, hydroxyurea, and secukinumab with limited clinical efficacy.  Despite normalization of eosinophilia by mepolizumab he remained prednisolone dependant requiring 15mg/day to maintain EAAD remission. Due to the severity of his autoimmune disease and steroid dependency, the patient underwent immunophenotyping and exome sequencing through the Canberra Hospital Glomerulonephritis clinic and ANU Centre for Personalised Immunology. Immunophenotyping demonstrated an increase in CD8+ T cells (61.1%vs 36.8% (58.5-63.8) of CD3+ in healthy controls (HC) p=0.005) and T regulatory cells (Tregs) (CD3+CD4+CD25+CD127-) (17.05 vs 5.54 (4.65-5.54) HC, p=<0.0001). CD8+ subsets demonstrated reduced naïve CD8+ (CD3+CD8+CCR7+CD45RA+) (8.5% vs 46.7 (37.8-58.8) p=0.0009) and expansion of CD8+ TEMRA (CD3+CD8+CCR7-CD45RA+) (66.4% vs 13.9 (11.1 – 19.6), p<0.0001) suggesting a state of chronic activation. We commenced tacrolimus aiming levels between 5-7ng/ml to control CD8+ activity. 2 months after initiation the patient was tapering on 9mg prednisolone with no inflammatory arthritis. Repeat immunophenotyping demonstrated a decline in Tregs (17.05 to 8.5% post-treatment) and CD8+ TEMRA (66.4 – 57.2% post-treatment).
Discussion: Immunophenotyping and genome sequencing may assist in individualising aetiology, pathophysiology and therefore therapeutic targets in difficult to treat or undifferentiated autoimmune disease.


Biography:
I am a first year Renal trainee at the Canberra hospital. I have an interest in renal immunology and transplantation. I hope to undertake more research in these fields over the course of my training both in a lab and clinical setting.

RARE VARIANTS IN BLK CONTRIBUTE TO THE PATHOGENESIS OF SYSTEMIC LUPUS ERYTHEMATOSUS

S JIANG1,2,3, V ATHANASOPOULOS1,2, J ELLYARD1,2, A CHUAH4, J CAPPELLO1,2, S PRABHU1, J CARDENAS5, J GU5, J ROCO1,2, I PAPA1,2, M YABAS1, G WALTERS1,2,3, G BURGIO1, K  MCKEON1,2, A ENDERS1,2, P CANETE1,2, S  ALEXANDER2,6, A KITCHING2,7, N SHEN8, J BABON9, M FIELD2, T ANDREWS2, V PASCUAL5, M COOK1,2, C VINUESA1,2
1Department of Immunology and Infectious Disease, ANU, Australia, 2Centre for Personalised Immunology, ANU, Australia, 3Department of Renal Medicine, The Canberra Hospital, Canberra, Australia, 4Genome Informatics Laboratory, ANU, Australia, 5Baylor Medical Institute, , Baylor, USA, 6Westmead Children’s Hospital, Sydney, Australia, 7Centre for Inflammatory Diseases, Department of Medicine, Monash University , Melbourne, Australia, 8China Australia Centre for Personalised Immunology, Renji Hospital, JiaoTong University, Shanghai , China, 9Walter and Eliza Hall Institute,, Melbourne, Australia

Aim: To assess the contribution of rare genetic variants in BLK to SLE
Background: SLE is a relapsing autoimmune disease of which excessive Type 1 Interferon (T1IFN) production is central to SLE pathogenesis. Genetics represent one of the most potent risk factors for SLE, yet no GWAS-associated variant has a proven contribution to pathophysiology. Although BLK is reproducibly associated with SLE by GWAS, its mechanism remains unelucidated.
Methods: SLE patients and controls underwent whole exome sequencing. BLK, BLK variants and interacting partners were purchased or generated with site directed mutagenesis. IRF5 interaction and phosphorylation was tested by western blot. IFNb activity was measured using a dual luciferase assay in HEK293T and CRISPR-Cas9 engineered Ramos cells. Patient lymphocyte RNA profiling was performed by microarray with QuSAGE algorithm analysis.
Results: Seven unique rare BLK SNPs were found in 22.9% (n=20/87) of SLE patients compared with 6.2% (n=6/97, p<0.0001) of healthy controls or 2.8% (n=3/102, p<0.002) of an immunodeficiency cohort. Computational modelling predicted these SNPs disrupted residues critical to BLK function. Overexpression confirmed significant impairment of kinase ability of BLK SNPs. Similar to the related Src-kinase LYN, wildtype BLK was capable of interacting with and repressing IRF5-mediated IFNb production, although BLK SNPs impaired repression of IFNb expression. CRISPR-Cas9 engineered BLK mutation into Ramos cells resulted in enhanced IFNb expression in response to R848 stimulation. Microarray confirmed increased T1 IFN responsive gene expression in SLE patients with BLK SNPs.
Conclusions: Rare variants in BLK are observed at high frequency in SLE patients. These variants are deleterious, altering protein function enhancing T1IFN expression directly linking genetic variation in BLK to SLE pathogenesis.

DEFICIENCY OF MINERALOCORTICOID SIGNALING IN MACROPHAGES PROVIDES PROTECTION AGAINST KIDNEY AND CARDIAC INJURY IN HYPERTENSIVE DIABETIC MICE.

G TESCH1,2, E OZOLS1, M YOUNG3, D NIKOLIC-PATERSON1,2
1Monash Health, Clayton, Australia, 2Monash University Centre for Inflammatory Diseases, Clayton, Australia, 3Hudson Institute of Medical Research, Clayton, Australia

Aim: To evaluate whether macrophage mineralocorticoid receptor signaling contributes to the development of kidney and cardiac disease in hypertensive diabetic mice.
Background: Mineralocorticoid receptor antagonists (MRAs) reduce hypertension, inflammation and tissue injury in human and experimental diabetes. Furthermore, inflammation and injury in diabetic kidneys and hearts is dependent on infiltrating macrophages. Therefore, we hypothesized that the tissue protective effects of MRAs in diabetes is partially due to inhibition of MR signaling in macrophages.
Methods: Transgenic mice with intact myeloid MR (MRflox/flox = WT) or myeloid MR deficiency (MRflox/flox LysMCre = MyMRKO) were developed on the hypertensive endothelial nitric oxide synthase deficient (Nos3-/-) mouse strain. Groups of these mice (n=13) were made diabetic with low dose streptozotocin (STZ) and were assessed after 15 weeks for the development of hypertension (systolic blood pressure), nephropathy (renal function impairment, albuminuria, renal injury) and cardiomyopathy (cardiac dysfunction–echocardiography).
Results: MR intact Nos3-/- mice with diabetes developed hypertension, albuminuria, renal function impairment (elevated cystatin-C), tubular injury (increased KIM-1 gene expression), and reduced cardiac function  (LV fractional shortening: diabetic 21% versus non-diabetic 31%), but no neutrophil infiltrate. In comparison, myeloid MR deficient Nos3-/- mice with similar diabetes had equivalent levels of hypertension, albuminuria and tubular injury, but showed improved renal function (plasma cystatin-C 48%↓, p<0.01) and improved cardiac function (LV fractional shortening  28%, p<0.01).
Conclusion: MR signaling in macrophages promotes injury in the diabetic kidneys and hearts of Nos3-/- mice without affecting hypertension. Therefore, MRAs can suppress diabetic nephropathy and diabetic cardiomyopathy by reducing macrophage-mediated injury independent of their ability to lower blood pressure.


Biography:
The research career of Greg Tesch has focused on the role of inflammation in diabetic nephropathy and other chronic kidney diseases.

TISSUE-RESIDENT MUCOSAL-ASSOCIATED INVARIANT T (MAIT) CELLS IN HUMAN RENAL FIBROSIS AND CHRONIC KIDNEY DISEASE (CKD)

HG HEALY1,2,3, B LAW1,2,4,  X WANG1,2, K KILDEY1,2,  K GIULIANI1,2,  K BEAGLEY4, R WILKINSON1,2,3,4, A KASSIANOS1,2,3,4
1Pathology Queensland, Brisbane, Australia, 2Royal Brisbane and Women’s Hospital, Brisbane, Australia, 3University of Queensland, Brisbane, Australia, 4Queensland University of Technology, Brisbane, Australia

Aim: To functionally characterise mucosal-associated invariant T (MAIT) cells present in human chronic kidney disease (CKD).
Background: MAIT cells are a specialised lymphocyte population associated with chronic inflammatory disorders in peripheral tissues. To date, MAIT cell research has focused primarily on mucosal tissue, with limited studies on non-mucosal organs such as kidneys. In this study, we evaluated MAIT cells in human native kidneys with tubulointerstitial fibrosis, the pathological hallmark of CKD.
Methods: MAIT cells were identified, enumerated and phenotyped from human kidney tissue by multi-colour flow cytometry. Localisation of MAIT cells was performed by immunofluorescence microscopy. MAIT cells and human primary proximal tubular epithelial cells (PTEC) were co-cultured under hypoxic (1% O₂) conditions to examine mechanistic tubulointerstitial interactions.
Results: We detected significantly elevated numbers of MAIT cells (TCR-Vα7.2+ CD161++) in diseased biopsies with interstitial fibrosis compared with diseased biopsies without fibrosis and healthy kidney tissue. The increased numbers of MAIT cells also correlated significantly with loss of kidney function (eGFR). MAIT cells in fibrotic biopsies expressed cytokine receptors (IL-7Rα, IL-18Rα), activation receptor (NKG2D), extravasation marker (CD44) and tissue-resident markers (CD69, CD103 and CD49a). Immunofluorescent staining of fibrotic kidney tissue localised the accumulation of MAIT cells within the tubulointerstitial compartment, adjacent to PTEC. Notably, under in vitro pro-fibrotic/hypoxic conditions, PTEC induced the up-regulated expression of tissue-resident markers on MAIT cells.
Conclusion: We provide the first characterisation of MAIT cells in human kidney tissue. Collectively, our data suggest that human MAIT cells are retained as tissue-resident lymphocytes and are positioned to contribute to the fibrotic process via complex interactions with PTEC. Further dissection of kidney MAIT cells is now required for the development of novel CKD therapeutics.


Biography:
A/Prof Helen Healy is the Head of the Conjoint Kidney Research Laboratory and Director of Kidney Health Service, RBWH. A/Prof Healy has pioneered research into PTEC-immune cell interactions in the human system. She has a particular research focus on using our understanding of the mechanisms of CKD to develop effective treatments for this common chronic disease.

ROLE OF JNK SIGNALLING IN PODOCYTE DAMAGE AND PROTEINURIA IN EXPERIMENTAL GLOMERLULAR DISEASE.

F MA1,2, J LIU1,2, E OZOLS1,2,  PCHEW1,2, J HO1,2, Y HAN1,2, K BLEASE3, B BENNETT3, G TESCH1,2, D NIKOLIC-PATERSON1,2
1Dept of Nephrology, Monash Medical Centre, Clayton, Australia, 2Monash University Centre for Inflammatory Diseases, Clayton, Australia, 3Celgene, San Diego, USA

Aim: To determine whether JNK signalling in podocytes contributes to podocyte damage and proteinuria in glomerular disease.
Background: Activation of the c-Jun amino terminal kinase (JNK) is evident in podocytes and infiltrating macrophages in human and experimental glomerulonephritis. In vitro studies indicate that JNK signalling facilitates podocyte damage in response to inflammatory cytokines and toxins (e.g. puromycin aminonucleoside, PAN): however, the role of JNK in podocyte damage in glomerular disease remains unclear.
Methods: Podocyte damage (area of synaptopodin staining), proteinuria and macrophage infiltration was assessed in two disease models. WKY rats received a JNK inhibitor (CC-930), or vehicle, on days 7-28 of anti-GBM disease. Mice with Jnk gene deletion in podocytes (Jnk1f/fJnk2-/-PodCre known as JnkPod) were examined in the anti-GBM disease model with wild type and Jnk2-/- control groups. Finally, SD rats received a JNK inhibitor (CC-401), or vehicle, on days 0-8 of PAN-induced podocyte damage.
Results: Systemic JNK inhibition prevented podocyte damage, proteinuria and glomerular lesions in rat anti-GBM disease (all P<0.001 vs vehicle). This was associated with reduced macrophage infiltration and activation. JnkPod mice develop normally and exhibit normal kidney structure and function. However, JnkPod mice were not protected from podocyte damage, albuminuria, glomerular damage, renal function impairment or macrophage infiltration on day 7 of anti-GBM disease. PAN induced JNK activation in podocytes on day 4 which preceded the peak of proteinuria on day 8. However, systemic JNK inhibition failed to protect rats against PAN-induced podocyte damage or proteinuria.
Conclusions: Contrary to in vitro studies, JNK signalling in podocytes does not induce podocyte damage in vivo. Systemic JNK blockade reduces podocyte damage in anti-GBM disease via indirect effects, most probably on macrophages.


Biography:
Dr Nikolic-Paterson is from the Department of Nephrology at Monash Medical Centre and his research interests focus on the mechanisms of renal inflammation and fibrosis.

LOW DOSE INTERLEUKIN-2 THERAPY MODULATES AUTOIMMUNITY AND RENAL INJURY IN EXPERIMENTAL ANTI-MYELOPEROXIDASE GLOMERULONEPHRITIS

J DICK1, P GAN1, M ALIKHAN1, R KITCHING1, S HOLDSWORTH1
1Monash University , Clayton , Australia

Aim: To determine the potential of low dose IL-2 therapy in experimental ANCA-associated vasculitis (AAV).
Background: Low dose IL-2 expands regulatory T cells (Tregs) and is a potential therapy for autoimmune disease.
Methods: Anti-MPO autoimmunity was induced in C57BL/6 mice by immunisation with MPO. T cell mediated anti-MPO glomerulonephritis was triggered on day 16 in immunised mice by anti-GBM globulin, and MPO-ANCA induced glomerulonephritis by passive transfer of MPO-ANCA/LPS. Mice were treated with IL-2, 25,000units/day for 7 days. Immune responses were measured by flow cytometry, ELISPOT and ELISA.
Results: IL-2 increased splenic Tregs (26.2±0.8vs17±0.3 %CD4+, P<0.0001) and up-regulated expression of the suppressive markers CTLA4 and PD-1. Mice treated with IL-2 before MPO immunisation developed attenuated Th1 and Th17 autoimmunity, measured by splenocyte ELISPOT (IFN-γ 68±12vs123±16 spots, P=0.009; IL-17A 31±7vs70±12 spots P=0.003). This was associated with reduced segmental glomerular necrosis (24±4vs48±6%, P=0.0054) and reduced albuminuria. IL-2 pre-treatment did not alter humoral immunity.Treatment with IL-2 before passive transfer of MPO-ANCA/LPS increased numbers of intra-renal Tregs (2316±402vs1246±170 CD4+Foxp3+cells/kidney, P=0.02) and reduced intra-renal F4/80hiCCR2hi inflammatory macrophages (P<0.01).To evaluate therapy in established autoimmunity, mice were treated with IL-2 12 days after MPO immunisation. Although splenic Tregs were expanded, T effector response was also enhanced with more IFN-γ and IL17A producing cells (both P<0.01). Conversely, IL-2 therapy attenuated ANCA production (0.49±0.04 vs 0.29±0.03 OD480, P=0.0019), this was associated with suppressed T follicular helper cell generation. The net effect of IL-2 therapy was exacerbation of glomerular necrosis (87±3vs26±4%, P<0.0001) and increased albuminuria.
Conclusions: IL-2 has diverse and context specific effects on cellular and humoral autoimmunity. The potentiation of T effector responses in established autoimmunity may limit its utility in AAV.


Biography:
Dr Jonathan Dick studied medicine at the University of Oxford and University College London before training in internal medicine and nephrology in London. He recently completed a PhD at Monash University supervised by Professors Steve Holdsworth and Richard Kitching on novel roles for complement in pre-clinical models of ANCA-vasculitis.

COMBINED INHIBITION OF CCR2 AND ACE PROVIDES ADDED PROTECTION AGAINST PROGRESSION OF DIABETIC NEPHROPATHY IN NOS3 DEFICIENT MICE.

G TESCH1,2,  N PULLEN3, D NIKOLIC-PATERSON1,2
1Monash Health, Australia, 2Monash University Centre for Inflammatory Diseases, Clayton, Australia, 3Pfizer Worldwide Research & Development, Cambridge, USA

Aim: To evaluate intervention with a CCR2 antagonist (CCR2A) in a model of progressive diabetic glomerulosclerosis and determine whether a CCR2A provides added benefit over conventional treatment with an angiotensin converting enzyme inhibitor (ACEi).
Background: Blockade of chemokine CC motif receptor 2 (CCR2) inhibits kidney macrophage accumulation and suppresses early glomerular damage in diabetic animals. CCR2As are in clinical trials for diabetic nephropathy prompting investigation into whether they provide added benefit over ACEi.
Methods: Diabetes was induced in hypertensive endothelial nitric oxide synthase (Nos3) deficient mice by streptozotocin (STZ). Groups of diabetic Nos3-/- mice received a CCR2A (PF-04634817) as an early intervention (2-15 weeks after STZ). The late intervention (weeks 8-15 after STZ) involved PF-04634817 alone, ACEi (captopril) alone, or combined ACEi + CCR2A.
Results: Early intervention with CCR2A inhibited kidney inflammation and glomerulosclerosis, albuminuria, podocyte loss and renal function impairment, but not hypertension. Late intervention with CCR2A also inhibited kidney inflammation, glomerulosclerosis, renal dysfunction but did not affect albuminuria. ACEi alone suppressed hypertension and albuminuria, partially reduced podocyte loss and glomerulosclerosis, but did not affect renal dysfunction. Compared to ACEi alone, combined intervention with ACEi + CCR2A provided better protection against kidney damage (inflammation, glomerulosclerosis, renal function impairment), but not albuminuria.
Conclusion: Combining CCR2A and ACEi provides broader and superior renal protection than ACEi alone in a model of established diabetic glomerulosclerosis with hypertension.


Biography:
Greg Tesch is head of diabetes research in the Nephrology Department at Monash Health. His work is primarily focused on understanding the inflammatory mechanisms which promote diabetic nephropathy and developing novel anti-inflammatory treatments for diabetic nephropathy and other chronic kidney diseases.

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