EARLY POSTNATAL IMPAIRMENT OF RENAL FUNCTION IN INDIGENOUS INFANTS BORN PRETERM

MR SUTHERLAND1, D RYAN1, B DAVISON2, M CHATFIELD2, S DIWAKARLA3, WE HOY4, G SINGH2,5, MJ BLACK1

1Monash University, Clayton, VIC; 2Menzies School of Health Research, Charles Darwin University, Darwin, NT;  3Florey Institute of Neuroscience and Mental Health, Parkville, VIC; 4University of Queensland, Brisbane, QLD; 5Royal Darwin Hospital, Darwin, NT.

Aim: To examine the impact of preterm birth (delivery prior to 37 completed weeks of gestation) on early postnatal renal function in Indigenous and non-Indigenous neonates.

Background: The incidence of renal disease in Australia is disproportionately high amongst Indigenous populations. The cause of renal disease is multifactorial, but impaired renal development in early life is likely an important antecedent. Preterm birth (affecting ~8% of all Australian infants, and ~14% of Indigenous infants) can occur when nephrogenesis is still ongoing; renal function in preterm neonates may be adversely affected by renal immaturity and/or injury.

Methods: Indigenous (n=60) and non-Indigenous (n=42) infants were grouped by gestational age at birth: < 28, 29–32, and 33-36 weeks. Twenty-four hour pooled urine samples were obtained on days 3-7, 14, 21 and 28 to assess creatinine clearance (CrCl), and the fractional excretion of sodium (FeNa). Spot urine samples taken weekly were assessed for urine total protein, albumin, and beta-2 microglobulin levels.

Results: Compared to non-Indigenous neonates, Indigenous neonates exhibited significantly increased beta-2 microglobulin excretion on days 8 and 15, and Indigenous neonates born extremely preterm (≤ 28 weeks gestation) also had significantly increased FeNa levels over the first month of life; these findings are indicative of impaired tubular function. Pathological proteinuria (urine total protein > 500 mg/L) was evident in 13% of Indigenous preterm infants compared to 4% of non-Indigenous preterm infants.

Conclusions: These findings demonstrate that early neonatal renal dysfunction following preterm birth is exacerbated in Indigenous infants, and this may be reflective of impaired intrauterine renal development and/or a greater vulnerability to postnatal renal injury.

INTRAUTERINE GROWTH RESTRICTION: ADVERSE EFFECTS ON RENAL GROWTH AND FUNCTION IN PRETERM INFANTS

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.

SOCIOECONOMIC STATUS AND HEALTH IN CHILDREN AND ADOLESCENTS WITH CHRONIC KIDNEY DISEASE – THE KIDS WITH CKD STUDY (KCAD)

M DIDSBURY1,2, A VAN ZWIETEN1,2, K CHEN1,2, L JAMES1,2, A FRANCIS2,3, S KIM1,2, G WILLIAMS1,2, K HOWARD2, S MCTAGGART3, A WALKER4, F MACKIE5, T KARA6, N NASSAR2, A TEIXEIRA-PINTO1,2, A TONG1,2, JC CRAIG1,2, G WONG1,2,7

1Centre for Kidney Research, The Children’s Hospital at Westmead, Sydney, New South Wales; 2Sydney School of Public Health, The University of Sydney, New South Wales; 3Child and Adolescent Renal Service, Children’s Health Queensland; 4Department of Renal Medicine, The Royal Children’s Hospital, Melbourne, Victoria 5Department of Nephrology, Sydney Children’s Hospital at Randwick, Sydney, New South Wales; 6Department of Nephrology, Starship Children’s Hospital, Auckland, New Zealand; 7Centre for Transplant and Renal Research, Westmead Hospital, Sydney, New South Wales

Aim: In children with chronic kidney disease (CKD), we aimed to assess the impact of low socioeconomic status (SES) on overall health, and to identify the mediators of any association observed.

Background: Striking disparities in health by SES exist in children without CKD. The relationship between SES and health, and the factors that contribute to the health disparities by SES in children with CKD are unknown.

Methods: Between 2012 and 2017, 377 children aged 6-18 years with CKD [stage 1-5 (n=200), dialysis (n=42), transplant (n=135)] were recruited from five paediatric units across Australia and New Zealand. We used adjusted logistic regression to assess the association between SES and self-rated health status, and mediation analyses to determine variables that mediated the SES and health relationship.

Results: The median ages of caregivers and children were 42.3 (interquartile range (IQR) 9.7) and 12.1 (6.6) years. Approximately 50% of households earned less than the population median income, with 41% of primary caregivers being unemployed. Lower SES, defined as lower income group [adjusted OR (95%CI): 3.62 (1.81-7.68)], financial hardship [3.26 (1.58-6.70)] and unemployment [3.40 (1.51-6.12)], was associated with poorer self-rated health in children with CKD (I-V) but not in those receiving dialysis or after transplant (p interaction = 0.001). Over 50% of the relationship between SES and poor health was mediated by co-morbidities including chronic infections and growth impairment, while 30% of the overall effect was mediated by poor parental health (Sobel p = 0.04).

Conclusions: Lower SES is associated with poor health in children with CKD stages I-V.  Modifiable factors including comorbid conditions of the child and caregivers contribute significantly to the association between low SES and poor health.

EFFECT OF PLACENTAL GROWTH FACTOR ON TROPHOBLAST INTEGRATION INTO ENDOTHELIAL CELL NETWORKS

K CHAU1,2,3, B XU1, A HENNESSY1,3,4, A MAKRIS1,5

1Vascular Immunology Group, Heart Research Institute, Newtown NSW; 2Renal Department, Blacktown Hospital, Blacktown NSW; 3Sydney Medical School University of Sydney, NSW; 4School of Medicine, Western Sydney University, Campbelltown NSW; 5The Renal Unit, Liverpool Hospital, Liverpool NSW

Aim: To investigate the effect of supplemental placental growth factor (PlGF) on the interaction between a first trimester human trophoblast cell line and capillary-like endothelial cell networks.

Background: PlGF is deficient in early pregnancy in women who subsequently develop preeclampsia. The process of maternal spiral arteriole invasion by trophoblast is vital for normal placentation. The role of PlGF in influencing trophoblast interactions with endothelial cells is not known.

Methods: 24-well tissue culture plates were coated with 300 uL of undiluted Matrigel and allowed to gelatinise at 37oC for 30 minutes. Fluorescent labelled HTR8/SVNeo cells and uterine myometrial microvascular endothelial cells were co-cultured (1 X 105 per well) for 20 hours at 21% or 2% oxygen (to replicate physiological oxygen conditions in first trimester pregnancy) treated with PlGF 10 ng/mL or 100 ng/mL.  HTR8/SVNeo cells were also cultured singly under the same conditions. Images were captured by fluorescence microscopy and analysed using ImageJ. Experiments were repeated 5 times, data was analysed using SPSS v24.

Results: Integration of HTR8/SVNeo cells into endothelial cell networks expressed as % of control was unaffected by addition of PlGF at both 10 ng/mL and 100 ng/mL concentrations [p = 0.61]. Network formation of trophoblast cells on matrigel was enhanced by addition of PlGF [p = 0.01].

Conclusions: PlGF improves trophoblast network formation but does not affect integration of HTR8/SVNeo cells into uterine endothelial cell networks in vitro.

PRE-ECLAMPSIA IS ASSOCIATED WITH CHANGES IN THE ABUNDANCE AND PHOSPHORYLATION OF SODIUM TRANSPORTERS IN URINARY EXOSOMES

C-C HU3, M KATERELOS4, PF MOUNT1,3,4, N COOK1,3, S-W CHOY1,2,3, A CROSTHWAITE1,2,3, SP WALKER2,3, G PELL2, K PAIZIS1,2, DA POWER1,3,4

1Department of Nephrology, Austin Health, Heidelberg, VIC; 2Mercy Hospital for Women, Heidelberg, VIC; 3University of Melbourne, Parkville, VIC; 4Kidney Laboratory, IBAS at Austin Health, Heidelberg, VIC

Aim: To characterise expression and phosphorylation of renal tubular sodium transporters in urinary exosomes from women with pre-eclampsia (PE) compared to normotensive pregnant (NP) and non-pregnant controls (NC).

Background: PE is characterised by hypertension, systemic vasoconstriction, proteinuria and renal sodium retention. To better understand the cause of sodium retention, we examined expression and phosphorylation of the major distal renal tubular salt transporters (bumetanide-sensitive cotransporter NKCC2, thiazide-sensitive cotransporter NCC, epithelial sodium channel ENaC) in women with PE, using urinary exosomes.

Methods: A cross-sectional study of 18 PE patients, 22 NP patients and 20 NC women was performed. Exosomes were isolated from urine by ultracentrifugation. Expression of sodium transporters was analysed by Western Blot corrected for expression of the exosome marker CD9.  Statistical comparisons were made using the Kruskal-Wallis test.

Results: Expression of NKCC2 was increased 1.6-fold in PE compared to NP (p=0.046). Phosphorylation on the SPAK/OSR1 activation site T101/105 was reduced 1.9-fold (p=0.030) while phosphorylation of the activating PKA S130 site was increased 2.8-fold (p<0.001) in PE compared to NP. There was no difference in expression of NCC but phosphorylation of the SPAK/OSR1 site T60 was reduced 2.5-fold (p=0.008). Expression of the alpha (p<0.001) and full-length gamma (p<0.001) subunits of ENaC was increased 6.0-fold and 2.7-fold, respectively, in PE compared to NP. There was a non-significant trend to increased expression of the cleaved 50 kD form of gamma ENaC (3.0; p=0.06).

Conclusions: This data suggests a role for increased activity of ENaC and NKCC2 in mediating sodium-retention in PE, occurring despite reduced signalling through the WNK/SPAK/OSR1 pathway. Factors responsible for up-regulation of ENaC and NKCC2 expression and phosphorylation of NKCC2 on S130 remain to be identified.

NEUROCOGNITIVE AND EDUCATIONAL OUTCOMES IN CHILDREN AND ADOLESCENTS WITH CHRONIC KIDNEY DISEASE: A SYSTEMATIC REVIEW AND META-ANALYSIS

K CHEN1,2, M DIDSBURY1,2, A V ZWIETEN1,2, M HOWELL1,2, S KIM1, A TONG1,2, B BARTON3, S LAH4, J LORENZO3, G STRIPPOLI5, S PALMER6, A TEIXERA-PINTO2, F MACKIE7, S MCTAGGART8, A WALKER9, T KARA10, J CRAIG1,2, G WONG1,2,11

1Centre for Kidney Research, Kids Research Institute, The Children’s Hospital at Westmead, Sydney, Australia; 2Sydney School of Public Health, The University of Sydney, Sydney, Australia; 3Children’s Hospital Education Research Institute, The Children’s Hospital at Westmead, Sydney, Australia; 4School of Psychology, The University of Sydney, Sydney, Australia, and the ARC Centre of Excellence in Cognition and its Disorders, Macquarie University, Sydney, Australia; 5Department of Emergency and Organ Transplantation, University of Bari, Italy; 6Department of Medicine, University of Otago Christchurch, New Zealand; 7Department of Renal Medicine, Sydney Children’s Hospital at Randwick, Sydney, Australia; 8Child & Adolescent Renal Service, Children’s Health Queensland, Brisbane, Australia; 9Department of Renal Medicine, The Royal Children’s Hospital, Melbourne, Australia; 10Department of Nephrology, Starship Children’s Hospital, Auckland, New Zealand; 11Centre for Transplant and Renal Research, Westmead Hospital, Sydney, Australia

Aim: To examine global and domain-specific cognition and educational outcomes in children and adolescents with chronic kidney disease (CKD), and whether these outcomes vary with CKD stage.

Background: An association has been established between cognitive function and CKD stage in adults, but not in children, though reduced cognition may negatively impact on educational outcomes.

Methods: We conducted a systematic review comprising observational studies of patients with CKD aged 21 years or younger, and standardised measures of neurocognitive or educational outcomes, including intelligence, attention, executive function, language, memory, and academic skills. MEDLINE, Embase, and PsycINFO were searched from database inception to December 2016, with no language restrictions.

Results: Thirty-four studies (25 cross-sectional, 9 cohort; n=3,086) were eligible. The global cognition (full-scale intelligence quotient, FSIQ) of children with CKD was classified as average with an overall mean 92.7 (standard deviation 16.2). Compared to the general population, the mean differences (95%CI) in FSIQ were -10.5 (-13.2, -7.72) (all CKD stages, n=758), -9.39 (-12.6, -6.18) (pre-dialysis, n=582), -16.2 (-33.2, 0.86) (dialysis, n=23) and -11.2 (-17.8, -4.50) (transplant, n=153). Children with CKD also had lower scores in the domains of executive function and memory (verbal and visual), compared to the general population. Compared to children without CKD, the mean difference in academic skills ranged from -15.7 to 3.50 for mathematics, -15.5 to 3.10 for reading, and -15.0 to -1.68 for spelling.

Conclusions: Children with CKD may have below average intellectual functioning and academic skills compared to the general population, with mild deficits observed across executive function, visual and verbal memory. While patterns of impairment are unclear across cognitive domains, it appears that children on dialysis may be at greatest disadvantage.

SCOPE AND CONSISTENCY OF OUTCOMES REPORTED IN RANDOMISED TRIALS CONDUCTED IN CHILDREN WITH CHRONIC KIDNEY DISEASE

L SH CHONG1,2, B SAUTENET1,2, C S HANSON1,2, A TONG1,2, J C CRAIG1,2, ON BEHALF OF SONG-KIDS STEERING GROUP

1Sydney School of Public Health, The University of Sydney, Sydney, New South Wales; 2Centre for Kidney Research, The Children’s Hospital at Westmead, Westmead, Sydney, New South Wales

Aim: To assess the scope and consistency of outcomes reported in randomised controlled trials (RCTs) of interventions for children in any stage of chronic kidney disease (CKD).

Background: RCTs have been conducted to improve outcomes in children with CKD, but the outcomes reported may not be relevant to children, their families or clinicians. The variability in outcome domains and measures also makes it impossible to compare the effectiveness of interventions.

Methods: The Cochrane Renal Register of Controlled Trials was searched for all RCTs involving children across all stages of CKD published before March 2016. The frequency of reporting of each outcome domain, and the measurement characteristics were evaluated.

Results: From the 205 trials, 99 different outcome domains were reported. Across all outcome domains, 50 (51%) were surrogate, 40 (40%) were clinical, and 9 (9%) were patient-reported. The three most commonly reported domains were: blood pressure (75 [37%] trials), medication use/duration (73 [36%] trials) and relapse/remission (72 [35%]). Mortality was reported in 28 (14%) of trials. Cardiovascular disease and quality of life were reported in 8 (4%) and 2 (1%) trials, respectively. There was inconsistency of measures and time points used across all trials. Across the 99 outcome domains, 1671 different measurements were reported.

Conclusions: RCTs involving children with CKD across all disease stages and treatment modalities primarily report surrogate outcomes, rather than focusing on clinical and patient-centred outcomes such as mortality, cardiovascular disease, quality of life and cognition. Furthermore, the multiplicity and heterogeneity of outcomes at all levels – domain, measurement, threshold, and time points – deters efforts to evaluate the comparative effectiveness of interventions and the ability to use trial evidence in decision-making.

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.

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