Anne M. Durkan

Membrane Curvature Alters the Activation Kinetics of the Epithelial Na+/H+ Exchanger, NHE3

The epithelial Na+/H+ exchanger, NHE3, was found to activate slowly following an acute cytosolic acidification. The sigmoidal course of activation could not be explained by the conventional two-state model, which postulates that activation results from protonation of an allosteric modifier site. Instead, mathematical modeling predicted the existence of three distinct states of the exchanger: two different inactive states plus an active form. The interconversion of the inactive states is rapid and dependent on pH, whereas the conversion between the second inactive state and the active conformation is slow and pH-independent but subject to regulation by other stimuli. Accordingly, exposure of epithelial cells to hypoosmolar solutions activated NHE3 by accelerating this latter transition. The number of surface-exposed exchangers and their association with the cytoskeleton were not affected by hypoosmolarity. Instead, NHE3 is activated by the membrane deformation, a result of cell swelling. This was suggested by the stimulatory effects of amphiphiles that induce a comparable positive (convex) deformation of the membrane. We conclude that NHE3 exists in multiple states and that different physiological parameters control the transitions between them.

Expression and Targeting of CX3CL1 (Fractalkine) in Renal Tubular Epithelial Cells

The chemokine CX3CL1 plays a key role in glomerulonephritis and can act as both chemoattractant and adhesion molecule. CX3CL1 also is upregulated in tubulointerstitial injury, but little is known about the subcellular distribution and function of CX3CL1 in renal tubular epithelial cells (RTEC). Unexpectedly, it was found that CX3CL1 is expressed predominantly on the apical surface of tubular epithelium in human renal transplant biopsy specimens with acute rejection or acute tubular necrosis. For studying the targeting of CX3CL1 in polarized RTEC, MDCK cells that expressed untagged or green fluorescent protein-tagged CX3CL1 were generated. The chemokine was present on the apical membrane and in subapical vesicles. Apical targeting of CX3CL1 was not due to signals that were conferred by its intracellular domain, to associations with lipid rafts, or to O-glycosylation but, rather, depended on N-linked glycosylation of the protein. With the use of fluorescence recovery after photobleaching, it was found that CX3CL1 is immobile in the apical membrane. However, CX3CL1 partitioned with the triton-soluble rather than -insoluble cellular fraction, indicating that it is not associated directly with the actin cytoskeleton or with lipid rafts. Accordingly, disruption of rafts through cholesterol depletion did not render CX3CL1 mobile. For exploration of potential functions of apical CX3CL1, binding of CX3CR1-expressing leukocytes to polarized RTEC was examined. Leukocyte adhesion to the luminal surface was enhanced significantly when CX3CL1 was present. These data demonstrate that CX3CL1 is expressed preferentially on the apical membrane of RTEC and suggest a novel function for the chemokine in recruitment and retention of leukocytes in tubulointerstitial inflammation.

Thromboxane prostanoid receptor stimulation induces shedding of the transmembrane chemokine CX3CL1 yet enhances CX3CL1-dependent leukocyte adhesion

In atherosclerosis, chemokines recruit circulating mononuclear leukocytes to the vascular wall. A key factor is CX(3)CL1, a chemokine with soluble and transmembrane species that acts as both a chemoattractant and an adhesion molecule. Thromboxane A(2) and its receptor, TP, are also critical to atherogenesis by promoting vascular inflammation and consequent leukocyte recruitment. We examined the effects of TP stimulation on processing and function of CX(3)CL1, using CX(3)CL1-expressing human ECV-304 cells and primary human vascular endothelial cells. TP agonists promoted rapid shedding of cell surface CX(3)CL1, which was inhibited by pharmacological inhibitors or specific small interfering RNA targeting tumor necrosis factor-alpha-converting enzyme (TACE). Because it reduced cell surface CX(3)CL1, we predicted that TP stimulation would inhibit adhesion of leukocytes expressing the CX(3)CL1 cognate receptor but, paradoxically, saw enhanced adhesion. We questioned whether the enhanced ability of the remaining membrane-associated CX(3)CL1 to bind targets was caused by changes in its lateral mobility. Using fluorescence recovery after photobleaching, we found that plasmalemmal CX(3)CL1 was initially tethered but ultimately mobilized by TP agonists. TP stimulation provoked clustering of transmembrane CX(3)CL1 at sites of contact with adherent leukocytes. These data demonstrate that TP stimulation induces two distinct effects: a rapid cleavage of surface CX(3)CL1, thereby releasing the soluble chemoattractant, plus mobilization of the remaining transmembrane CX(3)CL1 to enhance the avidity of interactions with adherent leukocytes. The dual effect of TP allows CX(3)CL1 to recruit leukocytes to sites of vascular inflammation while enhancing their adhesion once recruited.

Is laparoscopic donation safe for paediatric recipients? A study of 85 paediatric recipients comparing open and laparoscopic donor nephrectomy

INTRODUCTION The safety of adult laparoscopic donor nephrectomy remains controversial with respect to paediatric recipients with few data existing about its efficacy. Small studies have shown no difference in graft survival when compared with open techniques, but previous data from United Network for Organ Sharing suggests a higher incidence of rejection in laparoscopically procured kidneys. METHODS We examined the outcome in a total of 85 consecutive paediatric renal recipients, comparing 46 recipients of laparoscopically procured kidneys (performed over a 3-year period, 2004-07) to a historical control of 39 open donor recipients. Thirty-seven laparoscopic donors were by the hand-assisted technique. RESULTS Mean recipient age was 9.8 (SD 5.04) years in the laparoscopic group and 10.4 (SD 4.67) years in the open group (P = 0.617). Two patients had delayed graft function in the laparoscopic group (4.3%) and one (2.5%) in the open group (P = 0.562). At 1 year follow-up, there was 100% graft survival in the laparoscopic group compared to 92% (P = 0.093) in the open group. Incidence of biopsy-proven acute rejection within 1 year of transplant was 26% (16 episodes in 12 patients) in the laparoscopic group compared to 41% (29 episodes in 16 patients) in the open group (P = 0.219). There were no deaths in the laparoscopic group but there were three deaths (7.6%) in the open group (P = 0.093). CONCLUSIONS Our experience of laparoscopic kidney donation for paediatric recipients suggests excellent outcome with no difference in rejection rate or graft survival compared to open donation. Laparoscopic donation is the optimal method of kidney procurement for paediatric recipients.

The long-term outcomes of atypical haemolytic uraemic syndrome: a national surveillance study

Background Atypical haemolytic uraemic syndrome (aHUS) accounts for ∼10% of all cases of HUS and is often due to complement dysregulation. The short-term outcomes for this disease are established, but there are limited long-term data. The long-term outcomes of a comprehensive nationwide cohort of children with aHUS are presented here. Methods The Australian Paediatric Surveillance Unit prospectively collected data on all cases of HUS in children seen by paediatricians between 1994 and 2001. Patients with aHUS were followed-up with a written questionnaire to the treating clinician at 1 year and again before transition to adult services or at last known follow-up. Results There were 146 reported cases of HUS, of which 14 were aHUS. Ten children required dialysis at first presentation, including two who died and three who did not recover renal function. The disease was relapsing in all but one who survived the presenting episode, with most relapses occurring in the first 12 months. At 1 year, one child was lost to follow-up. Nine of the remaining 11 patients were dialysis dependent. Thirteen kidneys were transplanted into eight children. There was disease recurrence in eight kidneys, which resulted in graft loss in seven. There were three further deaths 1.7, 6.7 and 16.1 years after the initial presentation. Five children developed neurological complications and two had cardiac complications, largely at the time of onset of the disease. Conclusions aHUS is a rare but devastating disease with very high mortality and morbidity that extends beyond the initial presentation period.

Consensus opinion on diagnosis and management of thrombotic microangiopathy in Australia and New Zealand: Thrombotic microangiopathy in Aust/NZ

Thrombotic microangiopathy (TMA) arises in a variety of clinical circumstances with the potential to cause significant dysfunction of the kidneys, brain, gastrointestinal tract and heart. TMA should be considered in all patients with thrombocytopenia and anaemia, with an immediate request to the haematology laboratory to look for red cell fragments on a blood film. While TMA of any aetiology generally demands prompt treatment, this is especially so in thrombotic thrombocytopenic purpura (TTP) and atypical haemolytic uraemic syndrome (aHUS), where organ failure may be precipitous, irreversible and fatal. In all adults, urgent, empirical plasma exchange (PE) should be started within 4–8 h of presentation for a possible diagnosis of TTP, pending a result for ADAMTS13 activity (a disintegrin and metalloprotease thrombospondin, number 13). A sodium citrate plasma sample should be collected for ADAMTS13 testing prior to any plasma therapy. In children, Shiga toxin‐associated haemolytic uraemic syndrome due to infection with Escherichia coli (STEC‐HUS) is the commonest cause of TMA, and is managed supportively. If TTP and STEC‐HUS have been excluded, a diagnosis of aHUS should be considered, for which treatment is with the monoclonal complement C5 inhibitor, eculizumab. While early confirmation of aHUS is often not possible, except in the minority of patients in whom autoantibodies against factor H are identified, genetic testing ultimately reveals a complement‐related mutation in a significant proportion of aHUS cases. The presence of other TMA‐associated conditions (e.g. infection, pregnancy/postpartum and malignant hypertension) does not exclude TTP or aHUS as the underlying cause of TMA.

Acute Allograft Dysfunction

With improved screening for immunological sensitization and newer immunosuppressants the early renal allograft survival rates have increased considerably over the past two decades. However a significant number of transplanted children will still encounter episodes of acute graft dysfunction due to a wide variety of aetiologies. This chapter will discuss the causes of allograft dysfunction seen in the first 6 months post-transplant. The investigations and management of these conditions will be considered with particular emphasis on the diagnosis and treatment of both acute cellular and antibody mediated rejection. Finally recurrence of renal diseases post-transplantation will be discussed along with possible therapeutic interventions for these diseases.

DEC205-DC targeted DNA vaccine against CX3CR1 protects against atherogenesis in mice

Studies disrupting the chemokine pathway CX3CL1 (fractalkine)/ CX3CR1 have shown decreased atherosclerosis in animal models but the techniques used to interrupt the pathway have not been easily translatable into human trials. DNA vaccination potentially overcomes the translational difficulties. We evaluated the effect of a DNA vaccine, targeted to CX3CR1, on atherosclerosis in a murine model and examined possible mechanisms of action. DNA vaccination against CX3CR1, enhanced by dendritic cell targeting using DEC-205 single chain variable region fragment (scFv), was performed in 8 week old ApoE-/- mice, fed a normal chow diet. High levels of anti-CX3CR1 antibodies were induced in vaccinated mice. There were no apparent adverse reactions to the vaccine. Arterial vessels of 34 week old mice were examined histologically for atherosclerotic plaque size, macrophage infiltration, smooth muscle cell infiltration and lipid deposition. Vaccinated mice had significantly reduced atherosclerotic plaque in the brachiocephalic artery. There was less macrophage infiltration but no significant change to the macrophage phenotype in the plaques. There was less lipid deposition in the lesions, but there was no effect on smooth muscle cell migration. Targeted DNA vaccination to CX3CR1 was well tolerated, induced a strong immune response and resulted in attenuated atherosclerotic lesions with reduced macrophage infiltration. DNA vaccination against chemokine pathways potentially offers a potential therapeutic option for the treatment of atherosclerosis.

Consensus opinion on diagnosis and management of thrombotic microangiopathy in Australia and New Zealand: Thrombotic microangiopathy in Australia/New Zealand

Thrombotic microangiopathy (TMA) arises in a variety of clinical circumstances with the potential to cause significant dysfunction of the kidneys, brain, gastrointestinal tract and heart. TMA should be considered in all patients with thrombocytopenia and anaemia, with an immediate request to the haematology laboratory to look for red cell fragments on a blood film. Although TMA of any aetiology generally demands prompt treatment, this is especially so in thrombotic thrombocytopenic purpura (TTP) and atypical haemolytic uraemic syndrome (aHUS), where organ failure may be precipitous, irreversible and fatal. In all adults, urgent, empirical plasma exchange (PE) should be started within 4–8 h of presentation for a possible diagnosis of TTP, pending a result for ADAMTS13 (a disintegrin and metalloprotease thrombospondin, number 13) activity. A sodium citrate plasma sample should be collected for ADAMTS13 testing prior to any plasma therapy. In children, Shiga toxin‐associated haemolytic uraemic syndrome due to infection with Escherichia coli (STEC‐HUS) is the commonest cause of TMA, and is managed supportively. If TTP and STEC‐HUS have been excluded, a diagnosis of aHUS should be considered, for which treatment is with the monoclonal complement C5 inhibitor, eculizumab. Although early confirmation of aHUS is often not possible, except in the minority of patients in whom auto‐antibodies against factor H are identified, genetic testing ultimately reveals a complement‐related mutation in a significant proportion of aHUS cases. The presence of other TMA‐associated conditions (e.g. infection, pregnancy/postpartum and malignant hypertension) does not exclude TTP or aHUS as the underlying cause of TMA.

Toddler with a time bomb: The challenges of homozygous familial hypercholesterolaemia.

S S15 department. An investigation of reported clinical incidents undertaken by the NSW Clinical Excellence Commission in 2012 revealed that 11% (3/27) of incidents resulting in serious harm (e.g., patient death) and 32% (24/75) of incidents with major consequences for patients were related to poor test result followup. Health information technology (IT) has a key role to play in the communication and follow-up of test results. Effective solutions must engage all stakeholders to arrive at decisions about who needs to receive the test results, how and when the results are communicated, and how they are acknowledged and acted upon. Reference 1. Georgiou A, Lymer S, Forster M, et al. Lessons learned from the introduction of an electronic safety net to enhance test result management in an Australian mothers’ hospital. J Am Med Inform Assoc 2014; 21: