[Show abstract][Hide abstract] ABSTRACT: Responding to the concern from our faculty that undergraduate students do not have robust laboratory skills, we designed and implemented a strategy to individually teach and assess the manipulative skills of students in first-year laboratories. Five core laboratory skills were selected for the course entitled Human Biology, a large, first-year class of students, most of whom were enrolled in Bachelor of Pharmacy and Human Movement Studies. Here, we report details for the 365 students enrolled primarily in Pharmacy and Human Movement Studies bachelor degree programs in semester 1 of 2006. We designed a specific strategy to assess five core laboratory skills: 1) accurate and precise use of a micropipette, 2) calculation of dilutions and preparation of diluted samples of saline, 3) accurate representation of data using a graph, 4) use of a light microscope, and 5) acquisition of digital data by measuring the latent period for the Achilles reflex. Graduate tutors were trained to teach and assess each student on each skill. The development of competency was tracked for all students across all five skills. Most students demonstrated proficiency on their first attempt. The development of proficiency across the core skills depended on both the skill and degree program. In semester 2 of 2006, 854 students mostly enrolled in the Bachelor of Science degree program and were similarly taught and assessed on the same five core skills. This approach was an effective teaching and assessment strategy that, when applied beyond first year, should increase the level of laboratory skills across undergraduate programs in physiology.
Full-text · Article · Oct 2007 · AJP Advances in Physiology Education
[Show abstract][Hide abstract] ABSTRACT: 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 'glomerular' 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.
No preview · Article · Aug 2007 · Current Opinion in Nephrology and Hypertension
[Show abstract][Hide abstract] ABSTRACT: We detail the design, implementation and evaluation of an eConference entitled "Biohorizons", using a presage-process-product model to describe the development of an eLearning community. Biohorizons was a summative learning and assessment task aiming to introduce large classes of first-year Human Biology students to the practices of professional scientists. It was implemented in semester 1 for students enrolled in Pharmacy and Human Movement Studies degree programs, and in semester 2, for Science students. Pairs of students selected a topic of interest in Human Biology, registered into on-line clusters, then developed and wrote a short scientific paper and accompanying PowerPoint presentation. They then individually participated in an online Discussion. All three tasks were assessed using standards-referenced assessment rubrics. Learning was supported by eTutors, working in asynchronous mode. Biohorizons was evaluated by analyzing student achievement data, surveys and focus group interviews. Most students were able to achieve high academic standards (global mean scores for semester 1, 85-96%; semester 2, 81-90%). Student evaluations support: 1) the successful integration of eLearning into large classes of Human Biology, 2) the engagement of first-year students through collaborative learning, and 3) the fostering of learning through challenging assessment relevant to the core practices of professional scientists.
[Show abstract][Hide abstract] ABSTRACT: The media role model was recently developed to frame how science faculty members can teach their students to write more effectively to lay audiences (14). An Opinion Editorial (Op-Ed) was introduced as a novel assignment for final-year physiology and pharmacology undergraduates. This second phase of this study, reported here, demonstrated the efficacy of explicit teaching of the Op-Ed, using a one-shot, pre-/posttest research design. Baseline writing skills of students were determined from a communication assignment. Students were then explicitly taught how to write an Op-Ed and subsequently wrote an Op-Ed based on a recent, relevant scientific article. Most students achieved higher grades for their Op-Ed following explicit teaching [mean (SD) = 84.4% (9.1%), n = 216] compared with their communication assignment [mean (SD) = 74.7% (11.9%), n = 218]. Improvement in student writing was also evident by an increase in text readability, which mirrored the features of Op-Eds written by professional journalists. A survey of students (n = 142) indicated that most believed that the assignments were valuable and that their ability to write to a lay audience had improved. Members of the lay public were then surveyed for their opinions on student writing. Two assignments were selected from one student whose grades had improved after explicit teaching. Respondents (n = 78) indicated that the Op-Ed was easier to read than the communication assignment. Thus, explicit teaching of the Op-Ed improved the ability of students to write to members of the lay public.
[Show abstract][Hide abstract] ABSTRACT: The muscarine-sensitive K+ current (M-current) stabilizes the resting membrane potential in neurons, thus limiting neuronal excitability. The M-current
is mediated by heteromeric channels consisting of KCNQ3 subunits in association with either KCNQ2 or KCNQ5 subunits. The role
of KCNQ2/3/5 in the regulation of neuronal excitability is well established; however, little is known about the mechanisms
that regulate the cell surface expression of these channels. Ubiquitination by the Nedd4/Nedd4-2 ubiquitin ligases is known
to regulate a number of membrane ion channels and transporters. In this study, we investigated whether Nedd4/Nedd4-2 could
regulate KCNQ2/3/5 channels. We found that the amplitude of the K+ currents mediated by KCNQ2/3 and KCNQ3/5 were reduced by Nedd4-2 (but not Nedd4) in a Xenopus oocyte expression system. Deletion experiments showed that the C-terminal region of the KCNQ3 subunit is required for the
Nedd4-2-mediated regulation of the heteromeric channels. Glutathione S-transferase fusion pulldowns and co-immunoprecipitations demonstrated a direct interaction between KCNQ2/3 and Nedd4-2. Furthermore,
Nedd4-2 could ubiquitinate KCNQ2/3 in transfected cells. Taken together, these data suggest that Nedd4-2 is potentially an
important regulator of M-current activity in the nervous system.
Full-text · Article · May 2007 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The differentiation of colon cancer cell lines is associated with changes in calcium homeostasis. Concomitantly there are changes in the expression of some calcium transporters and G-protein-coupled receptors, which are capable of altering cytosolic-free calcium levels. Recent studies associate alterations in calcium transporter expression with tumourigenesis, such as changes in specific isoforms of the plasma membrane calcium ATPase (PMCA) in breast cancer cell lines. In this study, we examined the expression of PMCA isoforms in the HT-29 colon cancer cell line using two methods of differentiation (sodium butyrate-mediated and spontaneous post-confluency induced differentiation). Our studies show that differentiation of HT-29 colon cancer cells is associated with the up-regulation of the PMCA isoform PMCA4 but no significant alteration in PMCA1. These results suggest that PMCA4 may be important and have a specific role in colon cells as well as being significant in colon cancer tumourigenesis.
No preview · Article · May 2007 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The teaching of highly valued scientific writing skills in the first year of university is challenging. This report describes the design, implementation, and evaluation of a novel written assignment, The Personal Response and accompanying Peer Review, in the course, Human Biology (BIOL1015) at The University of Queensland. These assignments were the first assessment tasks of the course and were set early in the first semester of university. BIOL1015 had a diverse cohort of 319 first year students from five bachelor degree programs, primarily from Pharmacy and Human Movement Studies. Audio files in the form of interviews with eminent biomedical scientists were obtained from a leading public radio program. Students used these files as triggers to submit a short but highly structured assignment written from a personal perspective and in an expressive style. Evaluations revealed that overall, students found the task interesting and challenging. Students performed well, regardless of their background knowledge, disciplinary interest, or preference for topics within human biology. This study demonstrated that The Personal Response was an appropriate task for these first year students of human biology. It represents an alternative to traditional essay writing.
Full-text · Article · Mar 2007 · Biochemistry and Molecular Biology Education
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The undergraduate experience is arguably the most important in shaping the future career trajectories of students. It is here that early exposure to the widest possible range of disciplines and practical experiences will have the most impact. In the face of a reduction in the number of students entering both undergraduate science and research as a career option, we must urgently initiate strategies to engage and retain students in science. This can be achieved by a research experience in a ‘mentored apprenticeship model’ in the context of an authentic laboratory/field during their formative undergraduate years. It is widely acknowledged that an interactive, enquiry-based approach to learning provides the most meaningful and lasting learning experience for students. It is similarly accepted that, within science, undergraduate research experiences are pivotal in providing context to student learning and providing a true sense of what it means to be a 'scientist'. In this discussion forum we will summarise research-based experiences currently available for Bachelor of Science (BSc) students at The University of Queensland (UQ). We will then look in detail at a proposed new ‘mentored apprenticeship model’, being examined for introduction into the UQ BSc from 2008 following the recent major review. The proposed model builds on the existing UQ Advanced Study Program in Science combined with the University of Michigan’s Undergraduate Research Opportunity Program and aims to: • achieve an increased level of student engagement to complement other strategies for motivating students who are in large first year classes • show students the functional/practical relevance of the core content of their course material • provide students with a personal experience of doing science so that they can plan their future studies from a more informed perspective • minimize the attrition rate from the first year science cohort • provide a mentored cohort experience to engage and support under-represented groups such as indigenous and international students • actively build on the tremendous investment in institutes at UQ by increasing the direct involvement of these research academics in the undergraduate science program (for example, places for 25 students have already been committed by one of the UQ research institutes) • increase the number of students proceeding to postgraduate education as the next step to a worthwhile and personally rewarding career trajectory in science. The proposed ‘mentored apprenticeship model’ provides a step-wise approach to a student’s growth as an apprentice scientist. As undergraduates progress through their degree-program their learning experiences in science should also progress closer and closer to those of a ‘scientist’ until, on graduation, they are fully-prepared for their science-related career. The new model achieves this through establishing strong working partnerships between students and research groups, supplementing traditional practical components of undergraduate courses by ‘doing’ more science and providing students with an opportunity to talk more about science. Within this forum participants will be asked to explore: • How are the theoretical frameworks of enquiry-based learning being translated into practical applications? • What are the outcomes of an undergraduate research opportunity? • How do we assess this learning? • What are the experiences of other institutions – how have they met the challenge of an authentic research experience, in a research-intensive university, for large numbers of students? • Are there discipline-specific variations to these approaches?
[Show abstract][Hide abstract] ABSTRACT: Improving the public understanding of science is an important challenge for the future professional scientists who are our current undergraduates. In this paper, we present a conceptual model that explores the role of mass media as community gatekeepers of new scientific findings. This model frames the benefits for undergraduate science students to learn about media genres so that they can learn to communicate science more effectively to nonprofessional audiences. Informed by this Media Role model, we then detail a novel writing task for undergraduate physiology students, the Opinion Editorial (Op-Ed), and an accompanying Peer Review. The Op-Ed genre was directly taught to the students by a professional journalist. As an assessment task, students presented a recent, highly technical paper as an Op-Ed. This was assessed by both faculty members and peers using a detailed assessment rubric. Most students were able to replicate the features of Op-Eds and attained high grades on their writing tasks. Survey data from final-year physiology students (n = 230) were collected before and after the implementation of the Op-Ed/Peer Review. These indicated that most students were aware of the importance of scientists to effectively communicate their knowledge to nonprofessional audiences, that the Op-Ed writing task was challenging, and that they believed that their ability to write to nonprofessional audiences was improved after explicit teaching and feedback.
[Show abstract][Hide abstract] ABSTRACT: The constitutive reuptake of albumin from the glomerular filtrate by receptor-mediated endocytosis is a key function of the renal proximal tubules. Both the Cl- channel ClC-5 and the Na+-H+ exchanger isoform 3 are critical components of the macromolecular endocytic complex that is required for albumin uptake, and therefore the cell-surface levels of these proteins may limit albumin endocytosis. This study was undertaken to investigate the potential roles of the epithelial PDZ scaffolds, Na+-H+ exchange regulatory factors, NHERF1 and NHERF2, in albumin uptake by opossum kidney (OK) cells. We found that ClC-5 co-immunoprecipitates with NHERF2 but not NHERF1 from OK cell lysate. Experiments using fusion proteins demonstrated that this was a direct interaction between an internal binding site in the C terminus of ClC-5 and the PDZ2 module of NHERF2. In OK cells, NHERF2 is restricted to the intravillar region while NHERF1 is located in the microvilli. Silencing NHERF2 reduced both cell-surface levels of ClC-5 and albumin uptake. Conversely, silencing NHERF1 increased cell-surface levels of ClC-5 and albumin uptake, presumably by increasing the mobility of NHE3 in the membrane and its availability to the albumin uptake complex. Surface biotinylation experiments revealed that both NHERF1 and NHERF2 were associated with the plasma membrane and that NHERF2 was recruited to the membrane in the presence of albumin. The importance of the interaction between NHERF2 and the cytoskeleton was demonstrated by a significant reduction in albumin uptake in cells overexpressing an ezrin binding-deficient mutant of NHERF2. Thus NHERF1 and NHERF2 differentially regulate albumin uptake by mechanisms that ultimately alter the cell-surface levels of ClC-5.
No preview · Article · Jul 2006 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: ClC-5 is a chloride (Cl(-)) channel expressed in renal tubules and is critical for normal tubular function. Loss of function nonsense or missense mutations in ClC-5 are associated with Dent's disease, a condition in which patients present with low molecular weight (LMW) proteinuria (including albuminuria), hypercalciuria and nephrolithiasis. Several key studies in ClC-5 knockout mice have shown that the proteinuria results from defective tubular reabsorption of proteins. ClC-5 is typically regarded as an intracellular Cl(-) channel and thus the defect in this receptor-mediated uptake pathway was initially attributed to the failure of the early endosomes to acidify correctly. ClC-5 was postulated to play a key role in transporting the Cl(-) ions required to compensate for the movement of H(+) during endosomal acidification. However, more recent studies suggest additional roles for ClC-5 in the endocytosis of albumin. ClC-5 is now known to be expressed at low levels at the cell surface and appears to be a key component in the assembly of the macromolecular complex involved in protein endocytosis. Furthermore, mutations in ClC-5 affect the trafficking of v-H(+)-ATPase and result in decreased expression of the albumin receptor megalin/cubulin. Thus, the expression of ClC-5 at the cell surface as well as its presence in endosomes appears to be essential for normal protein uptake by the renal proximal tubule.
No preview · Article · Feb 2006 · The International Journal of Biochemistry & Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Sensory transduction in the mammalian cochlea requires the maintenance of specialized fluid compartments with distinct ionic compositions. This is achieved by the concerted action of diverse ion channels and transporters, some of which can interact with the PDZ scaffolds, Na(+)-H(+) exchanger regulatory factors 1 and 2 (NHERF-1, NHERF-2). Here, we report that NHERF-1 and NHERF-2 are widely expressed in the rat cochlea, and that their expression is developmentally regulated. Reverse transcription/polymerase chain reaction (RT-PCR) and Western blotting initially confirmed the RNA and protein expression of NHERFs. We then performed immunohistochemistry on cochlea during various stages of postnatal development. Prior to the onset of hearing (P8), NHERF-1 immunolabeling was prominently polarized to the apical membrane of cells lining the endolymphatic compartment, including the stereocilia and cuticular plates of the inner and outer hair cells, marginal cells of the stria vascularis, Reissner's epithelia, and tectorial membrane. With maturation (P21, P70), NHERF-1 immunolabeling was reduced in the above structures, whereas labeling increased in the apical membrane of the interdental cells of the spiral limbus and the inner and outer sulcus cells, Hensen's cells, the inner and outer pillar cells, Deiters cells, the inner border cells, spiral ligament fibrocytes, and spiral ganglion neurons (particularly type II). NHERF-1 expression in strial basal and intermediate cells was persistent. NHERF-2 immunolabeling was similar to that for NHERF-1 during postnatal development, with the exception of expression in the synaptic regions beneath the outer hair cells. NHERF-1 and NHERF-2 co-localized with glial fibrillary acidic protein and vimentin in glia. The cochlear localization of NHERF scaffolds suggests that they play important roles in the developmental regulation of ion transport, homeostasis, and auditory neurotransmission.
Full-text · Article · Feb 2006 · Cell and Tissue Research
[Show abstract][Hide abstract] ABSTRACT: Renal cortical fibroblasts have key roles in mediating intercellular communication with neighboring/infiltrating cells and extracellular matrix (ECM) and maintenance of renal tissue architecture. They express a variety of cytokines, chemokines, growth factors and cell adhesion molecules, playing an active role in paracrine and autocrine interactions and regulating both fibrogenesis and the interstitial inflammatory response. They additionally have an endocrine function in the production of epoetin. Tubulointerstitial fibrosis, the common pathological consequence of renal injury, is characterized by the accumulation of extracellular matrix largely due to excessive production in parallel with reduced degradation, and activated fibroblasts characterized by a myofibroblastic phenotype. Fibroblasts in the kidney may derive from resident fibroblasts, from the circulating fibroblast population or from haemopoetic progenitor or stromal cells derived from the bone marrow. Cells exhibiting a myofibroblastic phenotype may derive from these sources and from tubular cells undergoing epithelial to mesenchymal transformation in response to renal injury. The number of interstitial myofibroblasts correlates closely with tubulointerstitial fibrosis and progressive renal failure. Hence inhibiting myofibroblast formation may be an effective strategy in attenuating the development of renal failure in kidney disease of diverse etiology.
No preview · Article · Feb 2006 · The International Journal of Biochemistry & Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Serum glucocorticoid regulated kinase (SGK-1) is induced in the kidney in diabetes mellitus. However, its role in the proximal tubule is unclear. This study determined the expression and functional role of SGK-1 in PTCs in high glucose conditions. As the epidermal growth factor (EGF) receptor is activated by both EGF and other factors implicated in diabetic nephropathy, the relationship of SGK-1 with EGFR activity was assessed.
mRNA and protein expression of SGK-1 and mRNA expression of the sodium hydrogen exchanger NHE3 were measured in human PTCs exposed to 5 mmol/L (control) and 25 mmol/L (high) glucose. The effects of SGK-1 on cell growth, apoptosis, and progression through the cell cycle and NHE3 mRNA were examined following overexpression of SGK-1 in PTCs. The role of EGFR activation in observed changes was assessed by phospho-EGFR expression, and response to the EGFR blocker PKI166. SGK-1 expression was then assessed in vivo in a model of streptozotocin-induced diabetes mellitus type 2.
A total of 25 mmol/L glucose and EGF (10 ng/mL) increased SGK-1 mRNA (P < 0.005 and P < 0.002, respectively) and protein (both P < 0.02) expression. High glucose and overexpression of SGK-1 increased NHE3 mRNA (P < 0.05) and EGFR phosphorylation (P < 0.01), which were reversed by PKI166. SGK-1 overexpression increased PTC growth (P < 0.0001), progression through the cell cycle (P < 0.001), and increased NHE3 mRNA (P < 0.01), which were all reversed with PKI166. Overexpression of SGK-1 also protected against apoptosis induced in the PTCs (P < 0.0001). Up-regulation of tubular SGK-1 mRNA in diabetes mellitus was confirmed in vivo. Oral treatment with PKI166 attenuated this increase by 51%. No EGF protein was detectable in PTCs, suggestive of phosphorylation of the EGFR by high glucose and downstream induction of SGK-1.
The effects of high glucose on PTC proliferation, reduced apoptosis and increased NHE3 mRNA levels are mediated by EGFR-dependent up-regulation of SGK-1.
Full-text · Article · Oct 2005 · Kidney International
[Show abstract][Hide abstract] ABSTRACT: 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 PKC-alpha in albumin endocytosis. We found that albumin uptake in OK cells was inhibited by the pan-PKC blocker bisindolylmaleimide-1 and the isoform-specific PKC blockers Go-6976 and 2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanol dimethyl ether, indicating a role for PKC-alpha. Overexpression of a kinase deficient PKC-alpha(K368R) but not wild-type PKC-alpha significantly reduced albumin endocytosis. Western blot analysis of fractionated cells showed an increased association of PKC-alpha-green fluorescent protein 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 overexpressing kinase-deficient PKC-alpha(K368R) this actin cluster formation was significantly reduced. This study identifies a role for PKC-alpha in constitutive albumin uptake in OK cells by mediating assembly of actin microfilaments at the apical membrane.
Preview · Article · Jul 2005 · American journal of physiology. Renal physiology
[Show abstract][Hide abstract] ABSTRACT: There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA(A), GABA(C) and glycine receptor Cl- channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA(A), GABA(C) and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I- ions, driven by an external I- concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration-responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels.
[Show abstract][Hide abstract] ABSTRACT: Fibrin deposition is frequently observed within the tubulointerstitium in various forms of chronic renal disease. This suggests the presence of active components of the coagulation pathway, which may contribute to the progressive deterioration in renal function. The aim of this study was to investigate the proinflammatory and fibroproliferative effects of the coagulation protease thrombin on human proximal tubular cells (PTC) in culture.
Primary cultures of PTC were established from normal kidney tissue and grown under serum-free conditions with or without thrombin or the protease-activated receptor (PAR) activating peptides TFLLRN-NH(2), SLIGKV-NH(2), and SFLLRN-NH(2) (100 to 400 micromol/L). DNA synthesis (thymidine incorporation), intracellular Ca(2+) mobilization (fura-2 fluorimetry), fibronectin secretion [enzyme-linked immunosorbent assay (ELISA), immunoblotting], monocyte chemoattractant protein-1 (MCP-1) secretion (ELISA), and transforming growth factor-beta1 (TGF-beta1) secretion (ELISA) were measured. Reverse transcription-polymerase chain reaction (RT-PCR) was used to assess PAR mRNA expression in these cells.
Thrombin enhanced DNA synthesis, fibronectin secretion, MCP-1 secretion, and TGF-beta1 secretion in a concentration-dependent manner. Cell injury [lactate dehydrogenase (LDH) release] and cellular protein levels were unaffected. RT-PCR showed that cultures of PTC expressed mRNA transcripts for the thrombin receptors PAR-1 and PAR-3, but not PAR-4. Thrombin and each of the PAR activating peptides enhanced intracellular calcium mobilization. However, the other effects of thrombin were only fully reproduced by the PAR-2-specific peptide, SLIGKV-NH(2), only partially by SFLLRN-NH(2), (a PAR-1 peptide that can activate PAR-2), and not at all by the PAR-1-specific peptide, TFLLRN-NH(2). Thrombin-induced DNA synthesis, fibronectin, and MCP-1 secretion were unaffected by a TGF-beta neutralizing antibody, the matrix metalloproteinase (MMP) inhibitor, GM6001 and the epidermal growth factor (EGF) receptor kinase inhibitor AG1478.
Thrombin initiates both proinflammatory and fibroproliferative responses in human PTC. These responses which are dependent on its protease activity appear not to be mediated by PAR-1 activation, the autocrine action of thrombin-induced TGF-beta1 secretion, MMP activation, or EGF receptor transactivation. The proinflammatory and fibroproliferative actions of thrombin on human PTC may help explain the extent of tubulointerstitial fibrosis observed in kidney diseases where fibrin deposition is evident.
Full-text · Article · May 2005 · Kidney International
[Show abstract][Hide abstract] ABSTRACT: Matrix accumulation in the renal tubulointerstitium is predictive of a progressive decline in renal function. Transforming growth factor-beta(1) (TGF-beta(1)) and, more recently, connective tissue growth factor (CTGF) are recognized to play key roles in mediating the fibrogenic response, independently of the primary renal insult. Further definition of the independent and interrelated effects of CTGF and TGF-beta(1) is critical for the development of effective antifibrotic strategies. CTGF (20 ng/ml) induced fibronectin and collagen IV secretion in primary cultures of human proximal tubule cells (PTC) and cortical fibroblasts (CF) compared with control values (P < 0.005 in all cases). This effect was inhibited by neutralizing antibodies to either TGF-beta or to the TGF-beta type II receptor (TbetaRII). TGF-beta(1) induced a greater increase in fibronectin and collagen IV secretion in both PTC (P < 0.01) and CF (P < 0.01) compared with that observed with CTGF alone. The combination of TGF-beta(1) and CTGF was additive in their effects on both PTC and CF fibronectin and collagen IV secretion. TGF-beta(1) (2 ng/ml) stimulated CTGF mRNA expression within 30 min, which was sustained for up to 24 h, with a consequent increase in CTGF protein (P < 0.05), whereas CTGF had no effect on TGF-beta(1) mRNA or protein expression. TGF-beta(1) (2 ng/ml) induced phosphorylated (p)Smad-2 within 15 min, which was sustained for up to 24 h. CTGF had a delayed effect on increasing pSmad-2 expression, which was evident at 24 h. In conclusion, this study has demonstrated the key dependence of the fibrogenic actions of CTGF on TGF-beta. It has further uniquely demonstrated that CTGF requires TGF-beta, signaling through the TbetaRII in both PTCs and CFs, to exert its fibrogenic response in this in vitro model.
No preview · Article · Apr 2005 · American journal of physiology. Renal physiology