RENAL SAFETY OF A SHORT COURSE OF GENTAMICIN IN ELDERLY PATIENTS (The REGENT study)

CSB SIA1, MR ANANDA-RAJAH1,2,3, NR ADLER1,4  Y-W BAEY1, D LIEW3, EY TONG1,5, AR KAR AUNG1,2,3

1Department of General Medicine, Alfred Hospital, Prahran, Victoria, Australia.  2Department of Infectious Diseases, Alfred Hospital, Prahran, Victoria, Australia; 3Monash University, Victoria, Australia; 4School of Public Health and Preventative Medicine, Monash University; 5Department of Pharmacy, Alfred Hospital, Prahran, Victoria, Australia

Aim: To determine the incidence of acute kidney injury (AKI) in elderly patients receiving a short course of gentamicin, clinical risk factors and outcomes related to AKI.

Background: A short course of gentamicin is recommended by the Australian Therapeutic Guidelines as empiric treatment for gram negative bacteria, however the incidence of nephrotoxicity in elderly patients has not been studied.

Methods: Retrospective cohort study from April 2013 to 2015 at the Alfred and Sandringham Hospital,  Melbourne Australia. Participants included elderly (aged ≥65 years) patients admitted to hospital from the ED who received gentamicin. The primary outcome, AKI was defined as a rise in serum creatinine by ≥50% and/or ≥26.5µmol/L within 14 days of first gentamicin dose.

Results: The incidence of AKI was 14.9% (36/242 patients). A composite serious adverse renal outcome (persistent kidney injury, requirement for renal replacement therapy or inpatient death in a patient with AKI) occurred in 10 (4.1%) patients. Patients who developed AKI were older (median 80.5 vs 78 years, p=0.026), had higher Charlson co-morbidity index (median 7 vs 5, p=0.0004), had more advanced chronic kidney disease (CKD) at baseline (Stage IV and V) (OR 4.38, 95% CI=1.45-13.2, p=0.013) and were more likely to have received diuretics within the first 72 hours (OR 2.35, 95% CI 1.13-4.92, p=0.025). Mortality was increased in those with AKI (13.9% vs. 4.4%, p=0.04). When adjusted for confounders, Stage IV CKD or worse remained associated with AKI.

Conclusions: In a cohort of elderly patients with predominantly Stage II CKD, in whom the majority received a single dose of gentamicin, the incidence of AKI was 14.9%. AKI was transient in the majority of patients.

ACTIVATED CD47 PROMOTES ACUTE KIDNEY INJURY BY LIMITING AUTOPHAGY

NM ROGERS1,2, B SANGANERIA1, M EL-RASHID1

1Westmead Institute for Medical Research, Sydney, NSW; 2University of Pittsburgh, Pittsburgh, PA, USA

Aim: To investigate mechanisms underlying cellular damage in acute kidney injury.

Background: Acute kidney injury (AKI) initiates a complex pathophysiological cascade leading to epithelial cell death. Recent studies identify autophagy, the mechanism of intracellular recycling of cytoplasmic constituents, as important in protection against AKI. We have reported that the protein thrombospondin-1 (TSP1), and its receptor CD47, are induced in AKI. However their role in regulating injury is unknown.

Methods: Age and gender-matched wild-type (WT) and CD47-/- mice were challenged with renal ischemia-reperfusion injury (IRI), a standard model of AKI. All animals underwent analysis of renal function and biomolecular phenotyping. Human and murine WT and CD47-/- renal tubular epithelial cells (rTEC) were studied in vitro.

Results: CD47-/- mice were resistant to renal IRI, with decreased serum urea and creatinine, and ameliorated histological changes compared to WT animals. CD47-/- mice demonstrated robust upregulation of key autophagy genes, including Atg5, Atg7, Beclin-1, and LC3 at baseline and post-IRI. WT mice demonstrated negligible autophagy expression at all time points. rTEC from CD47-/- mice displayed basal upregulation of autophagy that was preserved under exogenous stress (hypoxia, treatment with the CD47 ligand TSP1), and correlated with enhanced viability compared to WT cells. Treatment of WT rTEC with a CD47 antagonist antibody or oligonucleotide to block TSP1-CD47 signalling increased autophagy. Finally, in a syngeneic mouse kidney transplantation model, treatment with a CD47 blocking antibody improved renal function and decreased histologic damage compared to control mice, and this was associated with increased autophagy.

Conclusions: These data suggest that activated CD47 is a proximate promoter of AKI through inhibition of autophagy, and point to CD47 as a target to restore renal function following injury.

RENAL TUBULAR EPITHELIAL CELL ISOMETRIC VACUOLATION ASSOCIATED WITH SODIUM-GLUCOSE CO-TRANSPORTER 2 INHIBITOR USE: A CASE REPORT

B ZAHOROWSKA1, S D ROGER2

1Liverpool Hospital, Liverpool, New South Wales, 2Gosford Hospital, Gosford, New South Wales

Background: Sodium-glucose co-transporter 2 (SGLT2) inhibitors have been available in Australia for 4 years as sole therapy or in combination therapy for type 2 diabetic patients with insufficient glycaemic control. SGLT2 inhibitors act by blocking glucose uptake by renal proximal tubular epithelial cells thus promoting glycosuria. To date, investigation into further effects of SGLT2 inhibitors has been performed in experimental animal diabetes models and in vitro human proximal tubular cell lines.

Case Report: We report the first known case of renal tubular epithelial cell isometric vacuolation associated with SGLT2 inhibitor use. A 56 year old female presented with acute kidney injury, creatinine increased from 75µmol/L in January 2016 to 130µmol/L in December 2016. Her background included recent diagnosis of type 2 diabetes mellitus and hypertension in August 2016 and hyperlipidaemia. She had been commenced on dapagliflozin and metformin hydrochloride (5mg/1000mg) daily alongside perindopril 5mg and pre-existing rosuvastatin 5mg daily. Investigations demonstrated normal sized kidneys and a bland urine sediment, only the expected glycosuria. Renal biopsy demonstrated acute tubular damage and fine isometric type vacuolation in tubular lining epithelial cells on light microscopy with membrane bound vacuoles of different sizes on electron microscopy. These changes cleared 3 months post drug cessation, but were replaced by an infiltrate with lymphocytes and eosinophils. Creatinine failed to improve post cessation of SGLT2 inhibitor.

Conclusions: The renal biopsy findings are suggestive of drug induced injury with proposed mechanism of dapagliflozin inducing an osmotic nephrosis, possibly secondary to carbohydrate malabsorption as has been previously described in a canagliflozin rat model. These new findings require further investigation particularly due to limited human renal biopsy data associated with SGLT2 inhibitors.

MODULATION OF UPREGULATED TRANSCRIPTIONAL SIGNALING PATHWAYS IN RODENT DIABETIC NEPHROPATHY AFTER TREATMENT WITH A NOVEL ANTI-FIBROTIC AGENT FT011: CORRELATES WITH HUMAN DIABETIC KIDNEY DISEASE

RG LANGHAM1,2, S MARTINI3, F EICHINGER3, V NAIR3, Y ZHANG1, A COX1, M KRETZLER3, D J KELLY1

1St. Vincent’s Hospital University of Melbourne Department of Medicine, Fitzroy, VIC; 2Monash University School of Rural Health, Clayton, VIC 3University of Michigan, Ann Arbour, MI, USA.

Background; Transcriptomic analysis of diabetic nephropathy (DN) biopsies has provided information into the molecular pathways that may drive disease. In this study, we aimed to measure the transcriptional changes of a new anti-fibrotic, FT011, in a rodent model of DN and to compare findings to that already described in human DN.

Method; Control and diabetic Ren(2) rats were treated with FT-011 (200mg/kg/day) or vehicle for 16 weeks (Early) or for the last 2 days of the 16-week period (Late). Total RNA was isolated from renal cortex, reverse transcribed, linearly amplified, and hybridized on Affymetrix microarrays. Identification of significantly differentially regulated genes between the cohorts for their enrichment in canonical pathways and functional groupings was done using the Ingenuity® Pathway Analysis Software Suite.

Results; Gene expression in DN showed early findings consistent with Type 1 diabetes.  Early FT011-treatment of DN showed an acute up-regulation of Fatty acid oxidation pathways, while the longer term changes after 16-week treatment showed suppression of activated inflammatory pathways, suppression of drivers of fibrosis and of transcriptional regulators known to be involved in progressive forms of DN (STAT1). Reduced inhibitory effect on inflammatory pathways was seen with Late treatment. Comparison with the human DN data demonstrated several overlapping key features and pathways of the human DN to be recapitulated in the animal model.

Conclusion; Analysis of transcriptional changes mediated by FT011 has demonstrated a sustained anti-inflammatory and anti-fibrotic action. Similarity of the changes seen in the human transcriptional data set highlights the ability of FT011 to modulate key signaling pathways identified as important in the pathogenesis of human DN, and further supports its potential utility as a new therapy for DN.

CHRONIC FETAL HYPOXIA LEADS TO LONG-TERM PROXIMAL TUBULE INJURY IN THE SHEEP KIDNEY

MR SUTHERLAND1,2, KL BRAIN1, KJ BOTTING1, SN AUSTIN-WILLIAMS1, EJ CAMM1, DA GIUSSANI1

1University of Cambridge, Cambridge, United Kingdom; 2Monash University, Clayton, VIC

Aim: To determine the impact of chronic fetal hypoxia, with and without maternal antioxidant treatment, on renal morphology in the adult sheep kidney.

Background: Chronic fetal hypoxia is a common outcome of pregnancy complications such as pre-eclampsia, as well as maternal smoking. The fetal adaptive response to hypoxia includes peripheral vasoconstriction, which reduces renal blood flow and may therefore impair kidney development. Maternal vitamin C (antioxidant) treatment can ameliorate fetal vasoconstriction in hypoxic pregnancies, and therefore has the potential to improve outcomes.

Methods: Welsh mountain ewes carrying singleton female fetuses were exposed to chronic hypoxia (10% O2; H) or normoxia (21% O2; N) from 105-138 days gestation (term=147 days). During this period, half of the ewes received a high dose (200 mg/kg/d) of vitamin C. Lambs (n=6/group) were delivered naturally and grown to 9 months of age (adulthood). Stereological methods were used to determine kidney volume and nephron number. Fibrosis, glomerulosclerosis, and renal tubule morphology were assessed histologically.

Results: Kidney volumes were significantly larger in the hypoxia-exposed sheep (N: 18,381±1,085; N+VitC: 18,741±991; H: 24,286±2,049; H+VitC: 25,097±1,762 mm3; p<0.001), mediated through a markedly increased volume of renal cortical tubules. Fetal hypoxia also resulted in severe proximal tubule injury (brush border loss), and increased levels of glomerulosclerosis and interstitial fibrosis; there was no difference in nephron number between groups. Vitamin C treatment ameliorated medullary fibrosis in the hypoxia-exposed sheep kidneys.

Conclusions: Chronic fetal hypoxia resulted in significant renal tubule pathology in adulthood; maternal vitamin C treatment, however, had little effect on kidney morphology. These findings have important implications for the long-term renal health of individuals born following hypoxic pregnancies.

ROLE OF NADPH-OXIDASE NOX5 IN HUMAN DIABETIC NEPHROPATHY

J C JHA1,2, M T COUGHLAN1,2, D A. POWER3, A SKENE4, E I. EKINCI5, M E. COOPER1,2, C R. KENNEDY6, K JANDELEIT-DAHM1,2.

1Department of Diabetes, Central Clinical School, Monash University, Melbourne, Australia; 2Baker IDI Heart and Diabetes Institute, Melbourne, Australia; 3Department of Nephrology and Institute of Breathing and Sleep, Austin Health, Heidelberg, Australia, 4Department of Anatomical Pathology, Austin Health, Heidelberg, Australia; 5Endocrine Centre, Austin Health, Repatriation Campus, Heidelberg, Australia and 6Department of Medicine, Ottawa Hospital Research Institute, Ottawa, Canada. 

Aim: We examined the role of Nox5 in human diabetic nephropathy (DN), in human renal cell populations and in a high fat fed rabbit model of kidney disease.

Background: Oxidative stress plays an important role in mediating kidney injury in diabetes. Evidences suggest that pro-oxidant enzyme, Nox5 plays a significant role in human DN. Nox5 is present in humans and rabbits but not in mice or rats. Thus, there is a paucity of information about Nox5 in conventional animal models of DN.

Methods: Protein expression of Nox5 was examined by immunostaining in human kidney biopsies obtained from non-diabetic and diabetic individuals. In vitro, Nox5 was silenced in human mesangial cells, podocytes and in proximal tubules and were exposed to high glucose, TGF-β and AngII. Cell morphology, gene and protein expression of markers of fibrosis and inflammation and the level of ROS were assessed. We also examined expression of pro-fibrotic gene in high fat fed rabbits by next generation sequencing (NGS) and renal injury by histochemistry.

Results: Expression of Nox5 was increased in both glomerular and tubular compartments of kidney biopsies obtained from diabetic versus non-diabetic individuals. Silencing of Nox5 resulted in reduced ROS production and decreased expression of pro-fibrotic and pro-inflammatory markers as well as putative elements that are implicated in DN. Moreover, increased expression of Nox5 in high fat fed rabbits versus normal diet fed rabbits was associated with increased expression of fibronectin, CTGF, collagen IV and VCAM-1 and increased mesangial expansion in the kidney.

Conclusions: Collectively, these findings suggest that Nox5 derived ROS accelerates renal injury in diabetes and provide proof of principle for the development of a new renoprotective agent in diabetes.

EPIGENETIC MODIFICATIONS TO H3K9 IN RENAL TUBULOINTERSTITIAL CELLS AFTER UNILATERAL URETERIC OBSTRUCTION AND TGF-BETA1 STIMULATION

TD HEWITSON1,2, SG HOLT1,2, SJ TAN1, B WIGG1, CS SAMUEL3, ER SMITH1,2

1Department of Nephrology, The Royal Melbourne Hospital, Parkville, Melbourne, VIC; 2Department of Medicine-RMH, University of Melbourne, Parkville, Melbourne, VIC; 3Department of Pharmacology, Monash University, Clayton, Melbourne, VIC, Australia.

Aim: To examine the distribution and acquisition of epigenetic modifications to histone 3 Lysine 9 (H3K9) after injury and stimulation with the pro-fibrotic cytokine TGF-b1.

Background: Epigenetic regulation of fibrogenesis through post translational histone modifications (marks) may be a key determinant of progression in renal disease.

Methods: Immunofluorescent microscopy was used to examine global H3K9 acetylation (H3K9Ac) and tri-methylation (H3K9Me3) after unilateral ureteric obstruction (UUO) in mice. Confocal, super-resolution microscopy and flow cytometry were used to determine the in vitro effect of TGF-b1 on structural arrangement of these marks, and their relationship with a- smooth muscle actin (aSMA) expression, a marker of myofibroblasts and early epithelial mesenchymal transition (EMT).

Results: The number of individual histone marks was increased 10 days after UUO (p<0.05 vs control), with both clearly seen in various cell types. Sub-nuclear microscopy in primary rat renal fibroblasts and a proximal tubule cell line (NRK-52e) showed that H3K9Ac was co-localised with phosphorylated-Ser2 RNA polymerase II (pRNAPol II), while H3K9Me3 was not, consistent with permissive and repressive effects on gene expression respectively. In both cell types H3K9Ac was diffusely distributed throughout the nucleus, while H3K9Me3 was found in compartments resembling the nucleolus, and in the case of the fibroblast, also juxtapositioned with the nuclear membrane. TGF-b1 had no effect on H3K9Ac marks in either cell, but resulted in a redistribution of H3K9Me3 within the fibroblast nucleus. This was unrelated to mitogenesis, but was associated with increased aSMA expression.

Conclusions: These findings highlight why it is important to consider the epigenetics of each cell individually. Whilst no overall enrichment occurred, renal myofibroblast differentiation was accompanied by distinct changes in histone mark arrangements.

INCREASED FIBROSIS IN ACC KNOCKIN MICE FOLLOWING RENAL INJURY CONFIRMS A PATHOGENIC ROLE FOR REDUCED FATTY ACID OXIDATION

M LEE1,2, PF MOUNT1, M KATERELOS1, K GLEICH1, DA POWER1,2.

1Kidney Laboratory, Department of Nephrology, Austin Health, Heidelberg, VIC; 2Department of Medicine, The University of Melbourne, Heidelberg, VIC

Aim: To determine whether reduced fatty acid oxidation contributes to renal fibrosis.

Background: Expression of genes regulating fatty acid metabolism is reduced in kidneys with tubulointerstitial fibrosis. It is not known, however, whether this response contributes to fibrosis or is its consequence. To answer this question, we determined whether mice with knock-in mutations of regulatory phosphorylation sites in the major controllers of fatty acid oxidation, acetyl CoA carboxylase 1 and 2 (ACC1/2KI mice), have increased renal fibrosis following injury.

Methods: The folic acid nephropathy (FAN) and unilateral ureteric obstruction (UUO) models were induced in male ACC1/2KI mice and wild type (WT) controls.  Mice were sacrificed at 14 and 7 days, respectively. Samples were studied by histomorphometry, Western blot and qRT-PCR.

Results: There was no difference in the appearance or function of ACC1/2KI kidneys at 8-10 weeks of age compared with WT. Reduced expression of genes controlling fatty acid oxidation was confirmed in the FAN model. In both FAN and UUO models there was increased accumulation of lipid by Oil Red O staining in ACC1/2KI mice (p<0.05 and p<0.01, respectively). Sirius red staining demonstrated increased fibrosis in ACC1/2KI mice in both models (p<0.05 and p<0.001). This was associated with increased expression of α-smooth muscle actin by Western blot and qRT-PCR (p<0.01). Increased fibrosis in the ACC1/2KI mice was confirmed by assay of Collagen I mRNA transcripts by qRT-PCR (P<0.05).

Conclusions: These data indicate that a reduced ability to regulate fatty acid oxidation in response to renal injury contributes to the development of renal fibrosis, and is not simply a consequence of injury. Regulation of fatty acid oxidation may be a potential therapeutic target in renal fibrosis.

PD BUDDY: USING SMARTPHONE TECHNOLOGY TO IMPROVE PATIENT CARE.

M BUDD1, R ROGERS1 K SOON-TAN1, GHELLEN2, M VARNFIELD3

1Metro South Health Service District, Logan Hospital, Brisbane, Queensland; 2Metro South Health Service District, Transformation and Innovation Collaborative; 3The Australian e-Health Research Centre, Health and Biosecurity, CSIRO, Brisbane, Queensland

Aim: To develop and research the effectiveness of a peritoneal dialysis (PD) app (PD-By) and web portal for patients and clinicians. This smartphone app will allow patients to record vital information such as prescription, ultrafiltration, blood pressure and weight. This information will be automatically uploaded to the web-portal for clinician access. With more accurate records it is envisioned that we will see more efficient and effective patient clinics, improved peritonitis rates and will help patients better manage their health in their own environment.

Background: Online health tracking is a relatively new innovation used to empower patients. Currently PD patients in our unit document vital health information in a simple exercise book, which is often incomplete or forgotten when a patient attends a PD clinic appointment. PD-BUDDy is an innovation being developed locally in collaboration with CSIRO to create the platform for patients on PD and their clinicians.

Methods: Pilot trial underway for six months using the app to determine the ease and feasibility of a smartphone application. 38 patients with a mean age of 56 years have been identified as suitable, 27% of participants are on CAPD with 73% on APD. On Average patients spend 113 minutes at each clinic appointment. Primary endpoints are reduction in consultation time by one third without increase in complication rates. Secondary endpoints will be patient satisfaction evaluated through questionnaire responses.

Conclusions: Innovation and communication in health care are essential. PD-BUDDy will streamline the way patients record and store information and will provide better communication and management of PD patients, potentially leading to improved health outcomes and extending their adverse event free time on the home-based therapy.

ASEPTIC TECHNIQUE MAINTAINS STERILITY OF ANTIBIOTIC-LOADED PERITONEAL DIALYSIS FLUID

L HUANG1,2, E RAMAS1, P PRASAD3, J CATANIA4, P MEADE4, E BUTLER3, LP MCMAHON1,2

1Department of Renal Medicine, Eastern Health, Box Hill, Victoria; 2Department of Medicine, Eastern Health Clinical School, Box Hill, Victoria; 3Department of Pharmacy, Eastern Health, Box Hill, Victoria; 4Department of Pathology and Microbiology, Eastern Health, Box Hill, Victoria

Aim: To compare the sterility of antibiotic-loaded peritoneal dialysis fluid (PDF) admixed using sterile-technique versus non-touch aseptic technique (NTAT).

Background: There is a paucity of data on the sterility of PDF after drug admixture. ISPD guidelines suggest using sterile-technique when admixing IP antibiotics, however the degree of sterility remain unclear. This issue is most pertinent when preparing take-home PDF for the outpatient treatment of peritonitis.

Methods: Groups of n=8 PDF (1.5% Dianeal or Extraneal) were admixed with antibiotics (ceftazidime and vancomycin, 1g/L) or 20mL saline, either by a pharmacist using sterile-technique in a sterile-suite (sterile gloves, filtered air, laminar flow hood), or a nurse in a clinical room utilising NTAT. PDF inoculated with 1×106 CFU/L of Coagulase-negative Staphylococcus (CNS, S. epidermidis or S. haemolyticus) with or without antibiotics, served as positive controls. Admixed PDF were left in room temperature for 72-hours, before being cultured using the BacT/ALERT system. A positive culture by day-5 constitutes a contamination. Differences in proportion of contamination between groups were assessed by the Χ2 test.

Results: Eighty PDF-bags completed microbiological testing. PDF sterility was maintained in all bags, independent of technique (sterile-technique vs. NTAT), type of PDF (Dianeal vs. Extraneal), or whether antibiotics were admixed (antibiotics vs. saline). Of the positive controls, CNS inoculated bags without antibiotics were all culture positive (8/8 for both S. epidermidis and S. haemolyticus); however, when inoculated into antibiotic loaded-PDF, only S. haemolyticus remained culture-positive (Χ2,df=27.67, 3, p<0.0001).

Conclusions: PDF sterility can be maintained using non-touch aseptic technique, for up to 3-days at room temperature. Currently, there is insufficient evidence to utilise a sterile-suite when admixing take-home PDF.

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