philip poronnik | The University of Sydney (original) (raw)
Papers by philip poronnik
VRST 2019 - 25th ACM Symposium on Virtual Reality Software and Technology, 2019
The delivery of ongoing training and support to Advanced Life Support (ALS) teams poses significa... more The delivery of ongoing training and support to Advanced Life Support (ALS) teams poses significant resourcing and logistical challenges. A reduced exposure to cardiac arrests and mandated re-accreditation pose further challenges for educators to overcome. This work presents the ALS-SimVR (Advanced Life Support Simulation in VR) application. The application is intended for use as a supplementary training and refresher asset for ALS team leaders. The purpose of the application is to allow critical care clinicians to rehearse the role of ALS Team leader in their own time and location of choice. The application was developed for the Oculus-Go and ported to the Oculus-Quest. The application is also supported for a desktop and server based streaming release.
Electrophoresis, 1999
Human salivary gland epithelial cells, a continuous cell line derived from an irradiated human sa... more Human salivary gland epithelial cells, a continuous cell line derived from an irradiated human salivary gland and human embryonic kidney cell line human embryonic kidney (HEK)293 were examined for the purpose of establishing whether they expressed endogenous P2X ionotropic receptors at any stage in their cycles. HSG cells were found to express P2X1-6 subtypes using both Western blotting and immunofluorescence labeling. HEK293 cells had no detectable levels of P2X1-3 and P2X6 under normal circumstances along with very low levels of P2X4 and P2X5 but when the cells were grown past confluence then all subtypes were expressed on the surface membrane with the exception of P2X2. The results are discussed in terms of the likely influence of ATP acting as an intercellular signaling molecule.
Electrophoresis, 1999
Human salivary gland epithelial cells, a continuous cell line derived from an irradiated human sa... more Human salivary gland epithelial cells, a continuous cell line derived from an irradiated human salivary gland and human embryonic kidney cell line human embryonic kidney (HEK)293 were examined for the purpose of establishing whether they expressed endogenous P2X ionotropic receptors at any stage in their cycles. HSG cells were found to express P2X1-6 subtypes using both Western blotting and immunofluorescence labeling. HEK293 cells had no detectable levels of P2X1-3 and P2X6 under normal circumstances along with very low levels of P2X4 and P2X5 but when the cells were grown past confluence then all subtypes were expressed on the surface membrane with the exception of P2X2. The results are discussed in terms of the likely influence of ATP acting as an intercellular signaling molecule.
Neuropharmacology, 2000
The P2X 1 purinergic receptor subtype occurs on smooth muscle cells of the vas deferens and urina... more The P2X 1 purinergic receptor subtype occurs on smooth muscle cells of the vas deferens and urinary bladder where it is localized in two different size receptor clusters, with the larger beneath autonomic nerve terminal varicosities. We have sought to determine whether these synaptic-size clusters only form in the presence of varicosities and whether they are labile when exposed to agonists. P2X 1 and a chimera of P2X 1 and green fluorescent protein (GFP) were delivered into cells using microinjection, transient transfection or infection with a replication-deficient adenovirus. The P2X 1 -GFP chimera was used to study the time course of P2X 1 receptor clustering in plasma membranes and the internalization of the receptor following prolonged exposure to ATP. Both P2X 1 and P2X 1 -GFP clustered in the plasma membranes of Xenopus oocytes, forming patches 4-6 µm in diameter. Human embryonic kidney 293 (HEK293) cells, infected with the adenovirus, possessed P2X 1 antibody-labeled regions in the membrane colocalized with GFP fluorescence. The ED 50 for the binding of α,β-methylene adenosine triphosphate (α,β-meATP) to the P2X 1 -GFP chimera was similar to native P2X 1 receptors. ATP-generated whole-cell currents in oocytes or HEK293 cells expressing either P2X 1 or P2X 1 -GFP were similar. Exposure of HEK293 cells to α,β-meATP for 10-20 min in the presence of 5 µM monensin led to the disappearance of P2X 1 -GFP fluorescence from the surface of the cells. These observations using the P2X 1 -GFP chimera demonstrate that P2X 1 receptors spontaneously form synaptic-size clusters in the plasma membrane that are internalized on exposure to agonists.
Journal of Cellular Physiology, 1996
Leukocyte recruitment to inflammatory foci is generally associated with cellular activation. Rece... more Leukocyte recruitment to inflammatory foci is generally associated with cellular activation. Recent evidence suggests that chemotactic agents can be divided into two classes, "classical chemoattractants" such as FMLP, C5a, and IL-8, which stimulate directed migration and activation events and "pure chemoattractants" such as TGF-beta 1 which influence actin polymerisation and movement but not oxidative burst and associated granular enzyme release. The studies reported here demonstrate that the murine S100 chemoattractant protein, CP-10, belongs to the "non-classical" group. Despite its potent chemotactic activity for neutrophils and monocytes/macrophages, CP-10 failed to increase [Ca2+]i in human or mouse PMN, although chemotaxis was inhibited by pertussis toxin, confirming the suggestion of a novel Ca(2+)-independent G-protein-coupled pathway for post-receptor signal transduction triggered by "pure chemoattractants." The co-ordinated up-regulation of Mac-1 and down-regulation of L-selectin induced by FMLP on human PMN in vitro was not observed with CP-10. Quantitative changes in immediate (30 s) actin polymerisation occurred with FMLP and CP-10-treated human PMN. The relative F-actin increases induced in WEHI 265 monocytoid cells by FMLP and CP-10 was optimal at 60 s and declined over 120 s. F-actin changes reflected the concentration and potencies of the agonists required to provoke chemotaxis. After 90 min, CP-10 profoundly altered cell shape and increased both cell size and F-actin within pseudopodia. These changes are typical of those mediating leukocyte deformability, and CP-10 may mediate leukocyte retention within microcapillaries and thereby contribute to the initiation of inflammation in vascular beds.
Cell Calcium, 1998
Here we report on the feasibility of using replication deficient adenoviruses to modify signal tr... more Here we report on the feasibility of using replication deficient adenoviruses to modify signal transduction systems in epithelia. We constructed two viruses, one expressing a dominant negative mutant of the a-subunit of G, (Ad-EFl-dnGa,) and the other expressing the wild-type o-subunit of G, (Ad-EFl-wtGa,). We used an adenovirus expressing green fluorescent protein (Ad-EFl-GFP20) to show that infection of cultured cells with an adenovirus results in at least 95% expression of the transgene in both HSG and HT29 cells. We also used an adenovirus that expresses no transgene (Ad-MX17) to demonstrate that adenoviral infection itself does not affect the resting concentration of cytosolic Ca2+ ([Ca"],) or the carbachol responses in these cells. We further show that Ad-EFl-dnGa, inhibits the increase in [Ca2+], produced by muscarinic receptor activation in both the cell lines we studied. This inhibitory effect is not shared by Ad-EFl -v&Go,, which indicates that in both HSG and HT29 cells, the increase in [Ca2+li produced by muscarinic receptor activation is largely mediated by activation of G,. Neither virus affected the resting level of [Ca2+], in these cells. Our findings confirm the feasibility of using replication deficient adenoviruses expressing dominant negative mutants to investigate the role of G proteins in signal transduction systems.
We hypothesized that an atypical isoform of protein kinase (PK) C, PKC- z , is essential for prol... more We hypothesized that an atypical isoform of protein kinase (PK) C, PKC- z , is essential for proliferation of human airway smooth muscle (HASM) cells in primary culture. Recombinant replication-deficient E1-deleted adenoviruses (100 plaque-form- ing units (pfu)/cell) expressing the antisense of PKC- z and the wild-type PKC- z (Ad-CMV-PKC- z ) were added to actively growing cells that were subsequently
Glia, 2007
Glutamate is a key neurotransmitter and its levels in the synaptic cleft are tightly regulated by... more Glutamate is a key neurotransmitter and its levels in the synaptic cleft are tightly regulated by reuptake mechanisms that primarily involve transporters in astrocytes. This requires that the glutamate transporters be spatially constrained to effect maximum glutamate transport. GLAST (EAAT1) is the predominant astroglial transporter and contains a class I PDZ-binding consensus (ETKM) in its C-terminus. The epithelial Na+/H+ exchanger regulatory factors NHERF1 and NHERF2 are PDZ proteins that contain two tandem PDZ domains and a C-terminal domain that binds members of the ERM (ezrin–radixin–moesin) family of membrane-cytoskeletal adaptors. NHERF proteins have been extensively characterized in renal epithelia and their expression in brain has recently been reported; however, their function in the brain remains unknown. The aims of the current study were to (1) determine the distribution of NHERF1/2 in the rodent brain and (2) investigate whether GLAST was a physiological ligand for NHERF1/2. Immunohistochemistry revealed that NHERF1 expression was widespread in rat brain (abundant in cerebellum, cerebral cortex, hippocampus, and thalamus) and primarily restricted to astrocytes whereas NHERF2 expression was primarily restricted to endothelial cells of blood vessels and capillaries. Importantly, NHERF1 distribution closely matched that of GLAST and confocal imaging demonstrated co-localization of the two proteins. Co-immunoprecipitation demonstrated that GLAST, NHERF1, and ezrin associate in vivo. In vitro binding assays showed that GLAST bound directly to the PDZ1 domain of NHERF1 via the C-terminal ETKM motif of GLAST. These findings implicate the GLAST–NHERF1 complex in the regulation of glutamate homeostasis in astrocytes. © 2006 Wiley-Liss, Inc.
Pflugers Archiv-european Journal of Physiology, 1999
HSG and HT29 cells express muscarinic receptors that increase intracellular free Ca2+ ([Ca2+]i) ... more HSG and HT29 cells express muscarinic receptors that increase intracellular free Ca2+ ([Ca2+]i) by activating phospholipase Cβ. In the present study, we have used the measurement of [Ca2+]i with Fura-2 to show that these receptors are of the M3 sub-type and that the increase in [Ca2+]i triggered when they are activated is not sensitive to pertussis toxin. We have also used replication-deficient adenoviruses expressing wild-type and dominant-negative mutants of the α-subunits of the heterotrimeric G proteins, Gq and Gi2, to investigate the mechanisms by which these receptors control phospholipase Cβ. We find that the Ca2+ response to 100 µmol/l carbachol is not affected by increased expression of the wild-type α-subunit of Gq, but is blocked by the dominant-negative mutant of Gq and by both the wild-type and the dominant-negative mutant α-subunits of Gi2. Expression of α-subunits of Gi2 presumably blocks the response to carbachol by scavenging free βγ-subunits. We conclude that in HSG and HT29 cells, the Ca2+ response to M3 receptor activation is mediated by the βγ- rather than the α-subunits of Gq.
International Journal of Biochemistry & Cell Biology, 2008
Transforming growth factor-beta(1) (TGFbeta(1)) is recognized as both a fibrogenic and inflammato... more Transforming growth factor-beta(1) (TGFbeta(1)) is recognized as both a fibrogenic and inflammatory cytokine and plays a critical role in the kidney pathophysiology. The dysregulation of TGFbeta(1) has been linked with the development of diabetic nephropathy. Connective tissue growth factor (CTGF) is a fibrogenic cytokine and is recognized as a downstream mediator of TGFbeta(1) in kidney fibrosis. TGFbeta(1) is involved in immunomodulation and fibrosis in the kidney. However, CTGF plays a more specific role in the fibrogenic pathways in the kidney proximal tubule cells. Moreover, CTGF facilitates TGFbeta(1) signaling and promotes renal fibrosis. This suggests CTGF could be a potential target for kidney fibrosis. Long-term inhibition and targeting TGFbeta(1) directly is problematic, therefore, a more fruitful direction targeting diabetic nephropathy may involve the development of therapeutic strategies specifically targeting CTGF.
Nephrology, 2007
Summary: In order to establish an in vitro model for studying human proximal tubule transport, pr... more Summary: In order to establish an in vitro model for studying human proximal tubule transport, primary culture of human proximal tubule cells (PTC) was carried out using an improved technique and the properties of these cells were characterised in detail. Using a combination of collagenase treatment, mechanical sieving and isopycnic ultracentrifugation, large numbers of highly purified populations of PTC were isolated and propagated from histologically normal regions of human nephrectomy specimens. Cultured human PTC demonstrated typical histologic and ultrastructural morphologies, well-preserved brush border enzyme activities, and cyclic adenosine monophosphate (cAMP) production which was stimulated by parathyroid hormone (PTH) but not by vasopressin. Tight confluence, as evidenced by relative impermeability to the paracellular diffusion of inulin, was achieved on porous membrane inserts within 6–8 days. Confluent monolayers generated Na+, K+, Cl−, HCO3− and PO43- concentration gradients between apical and basolateral medium compartments, which correlated well with the reabsorption processes known to occur in human PTC in vivo. A number of polarised transport systems were demonstrated, including phlorizin-inhibitable apical Na+-glucose transport, PTH-inhibitable apical Na+-phosphate transport, probenecid-inhibitable organic anion transport and quinine-inhibitable organic cation transport. Using microspectrofluorimetric and 22Na+ uptake measurements, pharmacologically distinct apical and basolateral sodium-hydrogen exchangers (NHE) were identified. Apical NHE was significantly inhibited by micromolar concentrations of phorbol esters, ethylisopropylamiloride (EIPA) and 3-methylsulphonyl-4-piperidino-benzoylguanidine methanesulphonate (HOE694). the mean resting intracellular pH of human PTC was 7.23 ± 0.04 and the mean intrinsic buffering capacity following a 20 mmol/L NH4Cl prepulse was 28.45 ± 0.96 mmol/L/unit pH. the results suggest that human PTC, prepared for culture as described herein, maintain morphological and physiological properties characteristic of the segment in vivo. the method therefore provides a useful model for the study of highly polarised transport processes in the human proximal tubule.
Kidney International, 2004
Background. Peroxisome proliferator-activated receptor gamma (PPARc) agonists, which are known to... more Background. Peroxisome proliferator-activated receptor gamma (PPARc) agonists, which are known to be critical factors in lipid metabolism, have also been reported to reduce proteinuria. The mechanism and its relevance to progressive nephropathy have not been determined. The aims of this study were to assess the direct effects of a PPARc agonist on tubular cell albumin uptake, proinflammatory and profibrotic markers of renal pathology, using an opossum kidney model of proximal tubular cells.
Kidney International, 1998
Stimulation of proximal tubule cell (PTC) growth in a variety of physiological and pathological r... more Stimulation of proximal tubule cell (PTC) growth in a variety of physiological and pathological renal conditions is preceded by increased renal production of transforming growth factor-beta 1 (TGF-beta 1) and by augmented tubular sodium transport via activated sodium hydrogen exchange (NHE). Since TGF-beta 1 has been shown to be an important paracrine and autocrine regulator of PTC growth, the hypothesis that TGF-beta 1 modulates basal and mitogen-stimulated PTC growth via an effect on NHE activity was examined. Confluent, quiescent, human PTC were incubated for 24 hours in serum-free media containing vehicle (control) or 1 ng/ml TGF-beta 1, in the presence or absence of 100 ng/ml insulin-like growth factor-1 (IGF-I). Under basal conditions, TGF-beta 1 inhibited thymidine incorporation (73.5 +/- 7.3% of control, P < 0.05), but exerted no effect on cellular protein content (97.4 +/- 10.7% of control), an index of hypertrophy. There was no significant alteration of NHE activity, measured as ethylisopropylamiloride (EIPA)-sensitive H+ efflux (2.72 +/- 0.50 vs. control 3.26 +/- 0.68 mmol/liter/min) or 22Na+ influx (2.20 +/- 0.23 vs. control 2.19 +/- 0.19 nmol/mg protein/min). When co-incubated with IGF-I. TGF-beta 1 induced significant PTC hypertrophy (116.9 +/- 8.2% of control, P < 0.05), which was not seen with either agent alone. TGF-beta 1 counteracted the stimulatory effect of IGF-I on DNA synthesis (TGF-beta 1 + IGF-I 103.0 +/- 7.3% vs. IGF-I alone 181.2 +/- 30.3% of control, P < 0.05), but did not affect IGF-I-stimulated EIPA-sensitive 22Na+ influx (3.63 +/- 0.63 vs. IGF-I alone 3.67 +/- 0.50 nmol/mg protein/min, P = NS, both vs. control 2.19 +/- 0.19 nmol/mg protein/min, P < 0.05). Similar results were obtained when NHE activity was measured as EIPA-sensitive H+ efflux. Moreover, the kinetics of NHE activation by the combination of TGF-beta 1 and IGF-I (involving an increase in Vmax) were identical to that previously found for PTC exposed to IGF-I alone. The study demonstrates that TGF-beta 1 elicits distinct PTC growth responses in the presence and absence of IGF-I, without modification of NHE activity. The combination of predominant PTC hypertrophy and enhanced proximal tubule Na+ reabsorption found in many conditions that are associated with renal growth is likely to require the integrated actions of both TGF-beta 1 and IGF-I.
Ajp: Renal Physiology, 2005
One key role of the renal proximal tubule is the reabsorption of proteins from the glomerular fil... more One key role of the renal proximal tubule is the reabsorption of proteins from the glomerular filtrate by constitutive receptor-mediated endocytosis. In the opossum kidney (OK) renal proximal tubule cell line, inhibition of protein kinase C (PKC) reduces albumin uptake, although the isoforms involved and mechanisms by which this occurs have not been identified. We used pharmacological and molecular approaches to investigate the role of PKCα in albumin endocytosis. We found that albumin uptake in OK cells was inhibited by the pan-PKC blocker BIM-1 and the isoform specific PKC blockers Gö6976 and HBDDE, indicating a role for PKCα. Over-expression of a kinase deficient PKCα(K368R) but not wild type PKCα significantly reduced albumin endocytosis. Western blot analysis of fractionated cells showed an increased association of PKCα -GFP with the membrane fraction within 10-20 min of exposure to albumin. We used phalloidin to demonstrate that albumin induces the formation of clusters of actin at the apical surface of OK cells and that these clusters correspond to the location of albumin uptake. These clusters were not present in cells grown in the absence of albumin. In cells treated either with PKC inhibitors or over-expressing kinase deficient PKCα(K368R) this actin cluster formation was significantly reduced. This study identifies a role for PKCα in constitutive albumin uptake in OK cells by mediating assembly of actin microfilaments at the apical membrane.
Clinical and Experimental Pharmacology and Physiology, 2004
1. Diabetic kidney disease is initially associated with hypertension and increased urinary albumi... more 1. Diabetic kidney disease is initially associated with hypertension and increased urinary albumin excretion. The hypertension is mediated by enhanced volume expansion due to enhanced salt and water retention by the kidney. The increased urinary albumin is not only due to increased glomerular leak, but also to a decrease in albumin reabsorption by the proximal tubule. The precise molecular mechanisms underlying these two phenomena and whether there is any link between the increase in Na + retention and proteinuria remain unresolved.
The distribution of P2X receptors on neurons in rat superior cervical ganglia and lability of P2X... more The distribution of P2X receptors on neurons in rat superior cervical ganglia and lability of P2X receptors on exposure to agonists were determined. Antibody labeling of each P2X subtype P2X(1)-P2X(7) showed neurons isolated into culture possessed primarily P2X(2) subunits with others occurring in order P2X(7) > P2X(6) > P2X(3) > P2X(1) > P2X(5) > P2X(4). Application of ATP and alpha,beta-meATP to neurons showed they possessed a predominantly nondesensitizing P2X receptor type insensitive to alpha,beta-meATP, consistent with immunohistochemical observations. P2X(1)-green fluorescent protein (GFP) was used to study the time course of P2X(1) receptor clustering in plasma membranes of neurons and internalization of receptors following prolonged exposure to ATP. At 12-24 h after adenoviral infection, P2X(1)-GFP formed clusters about 1 microm diameter in the neuron membrane. Application of ATP and alpha,beta-meATP showed these neurons possessed a predominantly desensitizing P2X receptor type sensitive to alpha,beta-meATP. Infection converted the major functional P2X receptor type in the membrane to P2X(1). Exposure of infected neurons to alpha,beta-meATP for less than 60 s led to the disappearance of P2X(1)-GFP fluorescence from the cell surface that was blocked by monensin, indicating the chimera is normally endocytosed into these organelles on exposure to agonist.
Protein Expression and Purification, 1999
Insulin-like growth factors (IGFs) in the circulation are predominantly sequestered into ternary ... more Insulin-like growth factors (IGFs) in the circulation are predominantly sequestered into ternary complexes comprising IGF, IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS). Besides its role in regulating IGF bioavailability in the circulation, IGF-BP-3 has both IGF-dependent and IGF-independent actions on cell proliferation. As part of our studies into the structure-function relationships of the multifunctional IGFBP-3, we have evaluated the efficiency of an adenovirus-mediated expression system for rapid, medium-scale production of functional, glycosylated IGF-BP-3. Replication-deficient adenovirus containing human IGFBP-3 cDNA was generated using standard techniques. Secreted, recombinant IGFBP-3 (IGFBP-3 Ad ) was purified from the culture medium of virusinfected cells by IGF-I affinity chromatography followed by reverse-phase HPLC. When analyzed by SDS-PAGE, IGFBP-3 Ad was similar in size (43-to 45-kDa glycoform doublet) to IGFBP-3 Pl derived from plasma. In addition, IGFBP-3 Ad was detected by immunoblot using an antibody specific for human IGFBP-3 and by ligand blot using radiolabeled IGF-I. IGFBP-3 Ad had similar affinities for IGF-I and ALS and an approximately 25% decreased affinity for IGF-II compared to IGFBP-3 Pl . IGFBP-3 Ad showed no significant difference in its susceptibility to an IGFBP-3 protease present in medium conditioned by MCF-7 breast cancer cells compared to IGFBP-3 Pl , but appeared more resistant to the IGFBP-3 protease present in pregnancy serum. IGFBP-3 Ad also exhibited increased binding to T47D cells which may be related to the glycosylation state of the protein.
Kidney International, 1997
Human renal fibroblasts modulate proximal tubule cell growth and transport via the IGF-I axis. To... more Human renal fibroblasts modulate proximal tubule cell growth and transport via the IGF-I axis. To determine the paracrine interactions involved in the tubulointerstitial response to progressive renal disease, the role of insulin-like growth factor-I (IGF-I) and its binding proteins (IGFBP5) in in Vitro interactions between human proximal tubule cells (PTC) and renal cortical fibroblasts (CF) were studied in primary cell culture. PTC growth and transport were increased in the presence of CF-conditioned media (CF-CM), as shown by increased thymidine incorporation, cellular protein content and sodium-hydrogen exchange (NHE) activity, to 185 31% (P < 0.01), 150 18% (P < 0.05) and 195 27% (P < 0.01) of the control values, respectively. IGF-I was produced by cultured CF at a rate of 64.6 7.5 ng/mg protein/day. Exogenous IGF-I
Cellular Physiology and Biochemistry, 2003
The degree of albuminuria, the presence of sodium-dependent hypertension, and histological eviden... more The degree of albuminuria, the presence of sodium-dependent hypertension, and histological evidence of both tubular and interstitial pathology correlate with the progression of diabetic nephropathy. The sodium-hydrogen exchanger NHE-3 plays an integral role in both sodium reabsorption and receptor-mediated albumin endocytosis in proximal tubular cells (PTCs). The aim of this study was to investigate the direct effects of hyperglycemia and albumin on cell growth parameters, NHE-3 protein expression and albumin uptake in an IN VITRO model of PTCs. Opossum kidney (OK) cells were exposed to 5 mmol/l glucose (control) or 25 mmol/l (high) glucose in the presence or absence of either 0.1 or 1.0 g/l bovine serum albumin (BSA) for up to 72 hrs prior to study. 20 mmol/l mannitol + 5 mmol/l glucose was used as a control for hyperosmolality. The cell number, the degree of cell swelling, cell protein content and NHE-3 protein expression were assessed. Cellular albumin uptake and the role of NHE in both control and high glucose conditions were determined by FITC-BSA +/- NHE-inhibitor ethyl isopropyl amiloride (EIPA). High glucose and the hyperosmolar control induced cellular hypertrophy, which was not modified in the presence of albumin. Cell volume was initially increased by 1.0 g/l BSA, +/-high glucose, which normalized over 48-72 hrs. All experimental conditions induced an early and sustained increase in NHE-3 protein expression. High glucose increased albumin uptake, independent of changes in osmolality. EIPA reduced the albumin uptake in PTCs with kinetics supporting the role of NHE-3 in this process. These results suggest that exposure of PTCs to high glucose concentrations promotes osmolality mediated cell hypertrophy and increased tubular albumin reabsorption linked to an increase in NHE-3 expression. It is postulated that this increase in albumin uptake due to high glucose exposure may lead to proinflammatory protein overload of PTCs, ultimately impairing the compensatory increase in tubular albumin reabsorption.
Current Opinion in Nephrology and Hypertension, 2007
Significant epidemiological and clinical trial evidence supports the association between increase... more Significant epidemiological and clinical trial evidence supports the association between increased urinary albumin excretion, cardiovascular events and renal failure. An increase in albumin excretion has traditionally been considered to reflect a &amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;glomerular&amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; leak of protein; however, it is now recognized that significant tubular reabsorption of albumin occurs under physiological conditions that may be modified by genetic determinants, systemic disease and drug therapies. The endocytosis of albumin by the proximal tubule is a highly regulated process depending on protein-protein interactions between several membrane proteins and scaffolding and regulatory molecules. The elucidation of these interactions is an ongoing research focus. There is also mounting evidence for a transcytotic pathway for retrieval of albumin from the tubular filtrate. The molecular basis for the role of albuminuria in both interstitial renal disease and cardiovascular pathology continues to be defined. The clinical implications of albuminuria due to a glomerular leak vs. reduced tubular reabsorption of albumin are, however, now under consideration. In particular, the prognostic implication of microalbuminuria induced by the more potent 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors is under study. The currently defined mechanisms underpinning the tubular reabsorption of albumin, how these are modified by pathology and pharmacology, and the clinical implications are the subject of this review.
VRST 2019 - 25th ACM Symposium on Virtual Reality Software and Technology, 2019
The delivery of ongoing training and support to Advanced Life Support (ALS) teams poses significa... more The delivery of ongoing training and support to Advanced Life Support (ALS) teams poses significant resourcing and logistical challenges. A reduced exposure to cardiac arrests and mandated re-accreditation pose further challenges for educators to overcome. This work presents the ALS-SimVR (Advanced Life Support Simulation in VR) application. The application is intended for use as a supplementary training and refresher asset for ALS team leaders. The purpose of the application is to allow critical care clinicians to rehearse the role of ALS Team leader in their own time and location of choice. The application was developed for the Oculus-Go and ported to the Oculus-Quest. The application is also supported for a desktop and server based streaming release.
Electrophoresis, 1999
Human salivary gland epithelial cells, a continuous cell line derived from an irradiated human sa... more Human salivary gland epithelial cells, a continuous cell line derived from an irradiated human salivary gland and human embryonic kidney cell line human embryonic kidney (HEK)293 were examined for the purpose of establishing whether they expressed endogenous P2X ionotropic receptors at any stage in their cycles. HSG cells were found to express P2X1-6 subtypes using both Western blotting and immunofluorescence labeling. HEK293 cells had no detectable levels of P2X1-3 and P2X6 under normal circumstances along with very low levels of P2X4 and P2X5 but when the cells were grown past confluence then all subtypes were expressed on the surface membrane with the exception of P2X2. The results are discussed in terms of the likely influence of ATP acting as an intercellular signaling molecule.
Electrophoresis, 1999
Human salivary gland epithelial cells, a continuous cell line derived from an irradiated human sa... more Human salivary gland epithelial cells, a continuous cell line derived from an irradiated human salivary gland and human embryonic kidney cell line human embryonic kidney (HEK)293 were examined for the purpose of establishing whether they expressed endogenous P2X ionotropic receptors at any stage in their cycles. HSG cells were found to express P2X1-6 subtypes using both Western blotting and immunofluorescence labeling. HEK293 cells had no detectable levels of P2X1-3 and P2X6 under normal circumstances along with very low levels of P2X4 and P2X5 but when the cells were grown past confluence then all subtypes were expressed on the surface membrane with the exception of P2X2. The results are discussed in terms of the likely influence of ATP acting as an intercellular signaling molecule.
Neuropharmacology, 2000
The P2X 1 purinergic receptor subtype occurs on smooth muscle cells of the vas deferens and urina... more The P2X 1 purinergic receptor subtype occurs on smooth muscle cells of the vas deferens and urinary bladder where it is localized in two different size receptor clusters, with the larger beneath autonomic nerve terminal varicosities. We have sought to determine whether these synaptic-size clusters only form in the presence of varicosities and whether they are labile when exposed to agonists. P2X 1 and a chimera of P2X 1 and green fluorescent protein (GFP) were delivered into cells using microinjection, transient transfection or infection with a replication-deficient adenovirus. The P2X 1 -GFP chimera was used to study the time course of P2X 1 receptor clustering in plasma membranes and the internalization of the receptor following prolonged exposure to ATP. Both P2X 1 and P2X 1 -GFP clustered in the plasma membranes of Xenopus oocytes, forming patches 4-6 µm in diameter. Human embryonic kidney 293 (HEK293) cells, infected with the adenovirus, possessed P2X 1 antibody-labeled regions in the membrane colocalized with GFP fluorescence. The ED 50 for the binding of α,β-methylene adenosine triphosphate (α,β-meATP) to the P2X 1 -GFP chimera was similar to native P2X 1 receptors. ATP-generated whole-cell currents in oocytes or HEK293 cells expressing either P2X 1 or P2X 1 -GFP were similar. Exposure of HEK293 cells to α,β-meATP for 10-20 min in the presence of 5 µM monensin led to the disappearance of P2X 1 -GFP fluorescence from the surface of the cells. These observations using the P2X 1 -GFP chimera demonstrate that P2X 1 receptors spontaneously form synaptic-size clusters in the plasma membrane that are internalized on exposure to agonists.
Journal of Cellular Physiology, 1996
Leukocyte recruitment to inflammatory foci is generally associated with cellular activation. Rece... more Leukocyte recruitment to inflammatory foci is generally associated with cellular activation. Recent evidence suggests that chemotactic agents can be divided into two classes, "classical chemoattractants" such as FMLP, C5a, and IL-8, which stimulate directed migration and activation events and "pure chemoattractants" such as TGF-beta 1 which influence actin polymerisation and movement but not oxidative burst and associated granular enzyme release. The studies reported here demonstrate that the murine S100 chemoattractant protein, CP-10, belongs to the "non-classical" group. Despite its potent chemotactic activity for neutrophils and monocytes/macrophages, CP-10 failed to increase [Ca2+]i in human or mouse PMN, although chemotaxis was inhibited by pertussis toxin, confirming the suggestion of a novel Ca(2+)-independent G-protein-coupled pathway for post-receptor signal transduction triggered by "pure chemoattractants." The co-ordinated up-regulation of Mac-1 and down-regulation of L-selectin induced by FMLP on human PMN in vitro was not observed with CP-10. Quantitative changes in immediate (30 s) actin polymerisation occurred with FMLP and CP-10-treated human PMN. The relative F-actin increases induced in WEHI 265 monocytoid cells by FMLP and CP-10 was optimal at 60 s and declined over 120 s. F-actin changes reflected the concentration and potencies of the agonists required to provoke chemotaxis. After 90 min, CP-10 profoundly altered cell shape and increased both cell size and F-actin within pseudopodia. These changes are typical of those mediating leukocyte deformability, and CP-10 may mediate leukocyte retention within microcapillaries and thereby contribute to the initiation of inflammation in vascular beds.
Cell Calcium, 1998
Here we report on the feasibility of using replication deficient adenoviruses to modify signal tr... more Here we report on the feasibility of using replication deficient adenoviruses to modify signal transduction systems in epithelia. We constructed two viruses, one expressing a dominant negative mutant of the a-subunit of G, (Ad-EFl-dnGa,) and the other expressing the wild-type o-subunit of G, (Ad-EFl-wtGa,). We used an adenovirus expressing green fluorescent protein (Ad-EFl-GFP20) to show that infection of cultured cells with an adenovirus results in at least 95% expression of the transgene in both HSG and HT29 cells. We also used an adenovirus that expresses no transgene (Ad-MX17) to demonstrate that adenoviral infection itself does not affect the resting concentration of cytosolic Ca2+ ([Ca"],) or the carbachol responses in these cells. We further show that Ad-EFl-dnGa, inhibits the increase in [Ca2+], produced by muscarinic receptor activation in both the cell lines we studied. This inhibitory effect is not shared by Ad-EFl -v&Go,, which indicates that in both HSG and HT29 cells, the increase in [Ca2+li produced by muscarinic receptor activation is largely mediated by activation of G,. Neither virus affected the resting level of [Ca2+], in these cells. Our findings confirm the feasibility of using replication deficient adenoviruses expressing dominant negative mutants to investigate the role of G proteins in signal transduction systems.
We hypothesized that an atypical isoform of protein kinase (PK) C, PKC- z , is essential for prol... more We hypothesized that an atypical isoform of protein kinase (PK) C, PKC- z , is essential for proliferation of human airway smooth muscle (HASM) cells in primary culture. Recombinant replication-deficient E1-deleted adenoviruses (100 plaque-form- ing units (pfu)/cell) expressing the antisense of PKC- z and the wild-type PKC- z (Ad-CMV-PKC- z ) were added to actively growing cells that were subsequently
Glia, 2007
Glutamate is a key neurotransmitter and its levels in the synaptic cleft are tightly regulated by... more Glutamate is a key neurotransmitter and its levels in the synaptic cleft are tightly regulated by reuptake mechanisms that primarily involve transporters in astrocytes. This requires that the glutamate transporters be spatially constrained to effect maximum glutamate transport. GLAST (EAAT1) is the predominant astroglial transporter and contains a class I PDZ-binding consensus (ETKM) in its C-terminus. The epithelial Na+/H+ exchanger regulatory factors NHERF1 and NHERF2 are PDZ proteins that contain two tandem PDZ domains and a C-terminal domain that binds members of the ERM (ezrin–radixin–moesin) family of membrane-cytoskeletal adaptors. NHERF proteins have been extensively characterized in renal epithelia and their expression in brain has recently been reported; however, their function in the brain remains unknown. The aims of the current study were to (1) determine the distribution of NHERF1/2 in the rodent brain and (2) investigate whether GLAST was a physiological ligand for NHERF1/2. Immunohistochemistry revealed that NHERF1 expression was widespread in rat brain (abundant in cerebellum, cerebral cortex, hippocampus, and thalamus) and primarily restricted to astrocytes whereas NHERF2 expression was primarily restricted to endothelial cells of blood vessels and capillaries. Importantly, NHERF1 distribution closely matched that of GLAST and confocal imaging demonstrated co-localization of the two proteins. Co-immunoprecipitation demonstrated that GLAST, NHERF1, and ezrin associate in vivo. In vitro binding assays showed that GLAST bound directly to the PDZ1 domain of NHERF1 via the C-terminal ETKM motif of GLAST. These findings implicate the GLAST–NHERF1 complex in the regulation of glutamate homeostasis in astrocytes. © 2006 Wiley-Liss, Inc.
Pflugers Archiv-european Journal of Physiology, 1999
HSG and HT29 cells express muscarinic receptors that increase intracellular free Ca2+ ([Ca2+]i) ... more HSG and HT29 cells express muscarinic receptors that increase intracellular free Ca2+ ([Ca2+]i) by activating phospholipase Cβ. In the present study, we have used the measurement of [Ca2+]i with Fura-2 to show that these receptors are of the M3 sub-type and that the increase in [Ca2+]i triggered when they are activated is not sensitive to pertussis toxin. We have also used replication-deficient adenoviruses expressing wild-type and dominant-negative mutants of the α-subunits of the heterotrimeric G proteins, Gq and Gi2, to investigate the mechanisms by which these receptors control phospholipase Cβ. We find that the Ca2+ response to 100 µmol/l carbachol is not affected by increased expression of the wild-type α-subunit of Gq, but is blocked by the dominant-negative mutant of Gq and by both the wild-type and the dominant-negative mutant α-subunits of Gi2. Expression of α-subunits of Gi2 presumably blocks the response to carbachol by scavenging free βγ-subunits. We conclude that in HSG and HT29 cells, the Ca2+ response to M3 receptor activation is mediated by the βγ- rather than the α-subunits of Gq.
International Journal of Biochemistry & Cell Biology, 2008
Transforming growth factor-beta(1) (TGFbeta(1)) is recognized as both a fibrogenic and inflammato... more Transforming growth factor-beta(1) (TGFbeta(1)) is recognized as both a fibrogenic and inflammatory cytokine and plays a critical role in the kidney pathophysiology. The dysregulation of TGFbeta(1) has been linked with the development of diabetic nephropathy. Connective tissue growth factor (CTGF) is a fibrogenic cytokine and is recognized as a downstream mediator of TGFbeta(1) in kidney fibrosis. TGFbeta(1) is involved in immunomodulation and fibrosis in the kidney. However, CTGF plays a more specific role in the fibrogenic pathways in the kidney proximal tubule cells. Moreover, CTGF facilitates TGFbeta(1) signaling and promotes renal fibrosis. This suggests CTGF could be a potential target for kidney fibrosis. Long-term inhibition and targeting TGFbeta(1) directly is problematic, therefore, a more fruitful direction targeting diabetic nephropathy may involve the development of therapeutic strategies specifically targeting CTGF.
Nephrology, 2007
Summary: In order to establish an in vitro model for studying human proximal tubule transport, pr... more Summary: In order to establish an in vitro model for studying human proximal tubule transport, primary culture of human proximal tubule cells (PTC) was carried out using an improved technique and the properties of these cells were characterised in detail. Using a combination of collagenase treatment, mechanical sieving and isopycnic ultracentrifugation, large numbers of highly purified populations of PTC were isolated and propagated from histologically normal regions of human nephrectomy specimens. Cultured human PTC demonstrated typical histologic and ultrastructural morphologies, well-preserved brush border enzyme activities, and cyclic adenosine monophosphate (cAMP) production which was stimulated by parathyroid hormone (PTH) but not by vasopressin. Tight confluence, as evidenced by relative impermeability to the paracellular diffusion of inulin, was achieved on porous membrane inserts within 6–8 days. Confluent monolayers generated Na+, K+, Cl−, HCO3− and PO43- concentration gradients between apical and basolateral medium compartments, which correlated well with the reabsorption processes known to occur in human PTC in vivo. A number of polarised transport systems were demonstrated, including phlorizin-inhibitable apical Na+-glucose transport, PTH-inhibitable apical Na+-phosphate transport, probenecid-inhibitable organic anion transport and quinine-inhibitable organic cation transport. Using microspectrofluorimetric and 22Na+ uptake measurements, pharmacologically distinct apical and basolateral sodium-hydrogen exchangers (NHE) were identified. Apical NHE was significantly inhibited by micromolar concentrations of phorbol esters, ethylisopropylamiloride (EIPA) and 3-methylsulphonyl-4-piperidino-benzoylguanidine methanesulphonate (HOE694). the mean resting intracellular pH of human PTC was 7.23 ± 0.04 and the mean intrinsic buffering capacity following a 20 mmol/L NH4Cl prepulse was 28.45 ± 0.96 mmol/L/unit pH. the results suggest that human PTC, prepared for culture as described herein, maintain morphological and physiological properties characteristic of the segment in vivo. the method therefore provides a useful model for the study of highly polarised transport processes in the human proximal tubule.
Kidney International, 2004
Background. Peroxisome proliferator-activated receptor gamma (PPARc) agonists, which are known to... more Background. Peroxisome proliferator-activated receptor gamma (PPARc) agonists, which are known to be critical factors in lipid metabolism, have also been reported to reduce proteinuria. The mechanism and its relevance to progressive nephropathy have not been determined. The aims of this study were to assess the direct effects of a PPARc agonist on tubular cell albumin uptake, proinflammatory and profibrotic markers of renal pathology, using an opossum kidney model of proximal tubular cells.
Kidney International, 1998
Stimulation of proximal tubule cell (PTC) growth in a variety of physiological and pathological r... more Stimulation of proximal tubule cell (PTC) growth in a variety of physiological and pathological renal conditions is preceded by increased renal production of transforming growth factor-beta 1 (TGF-beta 1) and by augmented tubular sodium transport via activated sodium hydrogen exchange (NHE). Since TGF-beta 1 has been shown to be an important paracrine and autocrine regulator of PTC growth, the hypothesis that TGF-beta 1 modulates basal and mitogen-stimulated PTC growth via an effect on NHE activity was examined. Confluent, quiescent, human PTC were incubated for 24 hours in serum-free media containing vehicle (control) or 1 ng/ml TGF-beta 1, in the presence or absence of 100 ng/ml insulin-like growth factor-1 (IGF-I). Under basal conditions, TGF-beta 1 inhibited thymidine incorporation (73.5 +/- 7.3% of control, P < 0.05), but exerted no effect on cellular protein content (97.4 +/- 10.7% of control), an index of hypertrophy. There was no significant alteration of NHE activity, measured as ethylisopropylamiloride (EIPA)-sensitive H+ efflux (2.72 +/- 0.50 vs. control 3.26 +/- 0.68 mmol/liter/min) or 22Na+ influx (2.20 +/- 0.23 vs. control 2.19 +/- 0.19 nmol/mg protein/min). When co-incubated with IGF-I. TGF-beta 1 induced significant PTC hypertrophy (116.9 +/- 8.2% of control, P < 0.05), which was not seen with either agent alone. TGF-beta 1 counteracted the stimulatory effect of IGF-I on DNA synthesis (TGF-beta 1 + IGF-I 103.0 +/- 7.3% vs. IGF-I alone 181.2 +/- 30.3% of control, P < 0.05), but did not affect IGF-I-stimulated EIPA-sensitive 22Na+ influx (3.63 +/- 0.63 vs. IGF-I alone 3.67 +/- 0.50 nmol/mg protein/min, P = NS, both vs. control 2.19 +/- 0.19 nmol/mg protein/min, P < 0.05). Similar results were obtained when NHE activity was measured as EIPA-sensitive H+ efflux. Moreover, the kinetics of NHE activation by the combination of TGF-beta 1 and IGF-I (involving an increase in Vmax) were identical to that previously found for PTC exposed to IGF-I alone. The study demonstrates that TGF-beta 1 elicits distinct PTC growth responses in the presence and absence of IGF-I, without modification of NHE activity. The combination of predominant PTC hypertrophy and enhanced proximal tubule Na+ reabsorption found in many conditions that are associated with renal growth is likely to require the integrated actions of both TGF-beta 1 and IGF-I.
Ajp: Renal Physiology, 2005
One key role of the renal proximal tubule is the reabsorption of proteins from the glomerular fil... more One key role of the renal proximal tubule is the reabsorption of proteins from the glomerular filtrate by constitutive receptor-mediated endocytosis. In the opossum kidney (OK) renal proximal tubule cell line, inhibition of protein kinase C (PKC) reduces albumin uptake, although the isoforms involved and mechanisms by which this occurs have not been identified. We used pharmacological and molecular approaches to investigate the role of PKCα in albumin endocytosis. We found that albumin uptake in OK cells was inhibited by the pan-PKC blocker BIM-1 and the isoform specific PKC blockers Gö6976 and HBDDE, indicating a role for PKCα. Over-expression of a kinase deficient PKCα(K368R) but not wild type PKCα significantly reduced albumin endocytosis. Western blot analysis of fractionated cells showed an increased association of PKCα -GFP with the membrane fraction within 10-20 min of exposure to albumin. We used phalloidin to demonstrate that albumin induces the formation of clusters of actin at the apical surface of OK cells and that these clusters correspond to the location of albumin uptake. These clusters were not present in cells grown in the absence of albumin. In cells treated either with PKC inhibitors or over-expressing kinase deficient PKCα(K368R) this actin cluster formation was significantly reduced. This study identifies a role for PKCα in constitutive albumin uptake in OK cells by mediating assembly of actin microfilaments at the apical membrane.
Clinical and Experimental Pharmacology and Physiology, 2004
1. Diabetic kidney disease is initially associated with hypertension and increased urinary albumi... more 1. Diabetic kidney disease is initially associated with hypertension and increased urinary albumin excretion. The hypertension is mediated by enhanced volume expansion due to enhanced salt and water retention by the kidney. The increased urinary albumin is not only due to increased glomerular leak, but also to a decrease in albumin reabsorption by the proximal tubule. The precise molecular mechanisms underlying these two phenomena and whether there is any link between the increase in Na + retention and proteinuria remain unresolved.
The distribution of P2X receptors on neurons in rat superior cervical ganglia and lability of P2X... more The distribution of P2X receptors on neurons in rat superior cervical ganglia and lability of P2X receptors on exposure to agonists were determined. Antibody labeling of each P2X subtype P2X(1)-P2X(7) showed neurons isolated into culture possessed primarily P2X(2) subunits with others occurring in order P2X(7) > P2X(6) > P2X(3) > P2X(1) > P2X(5) > P2X(4). Application of ATP and alpha,beta-meATP to neurons showed they possessed a predominantly nondesensitizing P2X receptor type insensitive to alpha,beta-meATP, consistent with immunohistochemical observations. P2X(1)-green fluorescent protein (GFP) was used to study the time course of P2X(1) receptor clustering in plasma membranes of neurons and internalization of receptors following prolonged exposure to ATP. At 12-24 h after adenoviral infection, P2X(1)-GFP formed clusters about 1 microm diameter in the neuron membrane. Application of ATP and alpha,beta-meATP showed these neurons possessed a predominantly desensitizing P2X receptor type sensitive to alpha,beta-meATP. Infection converted the major functional P2X receptor type in the membrane to P2X(1). Exposure of infected neurons to alpha,beta-meATP for less than 60 s led to the disappearance of P2X(1)-GFP fluorescence from the cell surface that was blocked by monensin, indicating the chimera is normally endocytosed into these organelles on exposure to agonist.
Protein Expression and Purification, 1999
Insulin-like growth factors (IGFs) in the circulation are predominantly sequestered into ternary ... more Insulin-like growth factors (IGFs) in the circulation are predominantly sequestered into ternary complexes comprising IGF, IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS). Besides its role in regulating IGF bioavailability in the circulation, IGF-BP-3 has both IGF-dependent and IGF-independent actions on cell proliferation. As part of our studies into the structure-function relationships of the multifunctional IGFBP-3, we have evaluated the efficiency of an adenovirus-mediated expression system for rapid, medium-scale production of functional, glycosylated IGF-BP-3. Replication-deficient adenovirus containing human IGFBP-3 cDNA was generated using standard techniques. Secreted, recombinant IGFBP-3 (IGFBP-3 Ad ) was purified from the culture medium of virusinfected cells by IGF-I affinity chromatography followed by reverse-phase HPLC. When analyzed by SDS-PAGE, IGFBP-3 Ad was similar in size (43-to 45-kDa glycoform doublet) to IGFBP-3 Pl derived from plasma. In addition, IGFBP-3 Ad was detected by immunoblot using an antibody specific for human IGFBP-3 and by ligand blot using radiolabeled IGF-I. IGFBP-3 Ad had similar affinities for IGF-I and ALS and an approximately 25% decreased affinity for IGF-II compared to IGFBP-3 Pl . IGFBP-3 Ad showed no significant difference in its susceptibility to an IGFBP-3 protease present in medium conditioned by MCF-7 breast cancer cells compared to IGFBP-3 Pl , but appeared more resistant to the IGFBP-3 protease present in pregnancy serum. IGFBP-3 Ad also exhibited increased binding to T47D cells which may be related to the glycosylation state of the protein.
Kidney International, 1997
Human renal fibroblasts modulate proximal tubule cell growth and transport via the IGF-I axis. To... more Human renal fibroblasts modulate proximal tubule cell growth and transport via the IGF-I axis. To determine the paracrine interactions involved in the tubulointerstitial response to progressive renal disease, the role of insulin-like growth factor-I (IGF-I) and its binding proteins (IGFBP5) in in Vitro interactions between human proximal tubule cells (PTC) and renal cortical fibroblasts (CF) were studied in primary cell culture. PTC growth and transport were increased in the presence of CF-conditioned media (CF-CM), as shown by increased thymidine incorporation, cellular protein content and sodium-hydrogen exchange (NHE) activity, to 185 31% (P < 0.01), 150 18% (P < 0.05) and 195 27% (P < 0.01) of the control values, respectively. IGF-I was produced by cultured CF at a rate of 64.6 7.5 ng/mg protein/day. Exogenous IGF-I
Cellular Physiology and Biochemistry, 2003
The degree of albuminuria, the presence of sodium-dependent hypertension, and histological eviden... more The degree of albuminuria, the presence of sodium-dependent hypertension, and histological evidence of both tubular and interstitial pathology correlate with the progression of diabetic nephropathy. The sodium-hydrogen exchanger NHE-3 plays an integral role in both sodium reabsorption and receptor-mediated albumin endocytosis in proximal tubular cells (PTCs). The aim of this study was to investigate the direct effects of hyperglycemia and albumin on cell growth parameters, NHE-3 protein expression and albumin uptake in an IN VITRO model of PTCs. Opossum kidney (OK) cells were exposed to 5 mmol/l glucose (control) or 25 mmol/l (high) glucose in the presence or absence of either 0.1 or 1.0 g/l bovine serum albumin (BSA) for up to 72 hrs prior to study. 20 mmol/l mannitol + 5 mmol/l glucose was used as a control for hyperosmolality. The cell number, the degree of cell swelling, cell protein content and NHE-3 protein expression were assessed. Cellular albumin uptake and the role of NHE in both control and high glucose conditions were determined by FITC-BSA +/- NHE-inhibitor ethyl isopropyl amiloride (EIPA). High glucose and the hyperosmolar control induced cellular hypertrophy, which was not modified in the presence of albumin. Cell volume was initially increased by 1.0 g/l BSA, +/-high glucose, which normalized over 48-72 hrs. All experimental conditions induced an early and sustained increase in NHE-3 protein expression. High glucose increased albumin uptake, independent of changes in osmolality. EIPA reduced the albumin uptake in PTCs with kinetics supporting the role of NHE-3 in this process. These results suggest that exposure of PTCs to high glucose concentrations promotes osmolality mediated cell hypertrophy and increased tubular albumin reabsorption linked to an increase in NHE-3 expression. It is postulated that this increase in albumin uptake due to high glucose exposure may lead to proinflammatory protein overload of PTCs, ultimately impairing the compensatory increase in tubular albumin reabsorption.
Current Opinion in Nephrology and Hypertension, 2007
Significant epidemiological and clinical trial evidence supports the association between increase... more Significant epidemiological and clinical trial evidence supports the association between increased urinary albumin excretion, cardiovascular events and renal failure. An increase in albumin excretion has traditionally been considered to reflect a &amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;glomerular&amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; leak of protein; however, it is now recognized that significant tubular reabsorption of albumin occurs under physiological conditions that may be modified by genetic determinants, systemic disease and drug therapies. The endocytosis of albumin by the proximal tubule is a highly regulated process depending on protein-protein interactions between several membrane proteins and scaffolding and regulatory molecules. The elucidation of these interactions is an ongoing research focus. There is also mounting evidence for a transcytotic pathway for retrieval of albumin from the tubular filtrate. The molecular basis for the role of albuminuria in both interstitial renal disease and cardiovascular pathology continues to be defined. The clinical implications of albuminuria due to a glomerular leak vs. reduced tubular reabsorption of albumin are, however, now under consideration. In particular, the prognostic implication of microalbuminuria induced by the more potent 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors is under study. The currently defined mechanisms underpinning the tubular reabsorption of albumin, how these are modified by pathology and pharmacology, and the clinical implications are the subject of this review.