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Peter W Mathieson

Publications and source records attributed to Peter W Mathieson.

At least 19 recordsLinked to original sources
PubMed2006

Statin-sensitive endocytosis of albumin by glomerular podocytes.

Glomerular podocytes are critical regulators of glomerular permeability via the slit diaphragm and may play a role in cleaning the glomerular filter. Whether podocytes are able to endocytose proteins is uncertain. We studied protein endocytosis in conditionally immortalized mouse and human podocytes using FITC-albumin by direct quantitative assay and by fluorescence microscopy and electron microscopy in mouse podocytes. Furthermore, in vivo uptake was studied in human, rat, and mouse podocytes. Both mouse and human podocytes displayed specific one-site binding for FITC-albumin with K(d) of 0.91 or 0.44 mg/ml and B(max) of 3.15 or 0.81 microg/mg cell protein, respectively. In addition, they showed avid endocytosis of FITC-albumin with K(m) of 9.48 or 4.5 mg/ml and V(max) of 474.3 or 97.4 microg.mg cell protein(-1).h(-1), respectively. Immunoglobulin and transferrin were inefficient competitors of this process, indicating some specificity for albumin. Accumulation of endocytosed albumin could be demonstrated in intracellular vesicles by fluorescence confocal microscopy and electron microscopy. Endocytosis was sensitive to pretreatment with simvastatin. In vivo accumulation of albumin was found in all three species but was most pronounced in the rat. We conclude that podocytes are able to endocytose protein in a statin-sensitive manner. This function is likely to be highly significant in health and disease. In addition, protein endocytosis by podocytes may represent a useful, measurable phenotypic characteristic against which potentially injurious or beneficial interventions can be assessed.

Animals↗
PubMed2006

Glomerulonephritis in the tropics: who are the culprits?

Glomerular disease is common in tropical regions and may complicate many locally prevalent infections by a variety of mechanisms. In areas where HIV and malaria are common, these may be factors predisposing to renal disease. Primary glomerulonephritis also occurs in these regions, and the nature of the renal lesion can only reliably be determined if renal biopsy and expert histologic analysis are available. Assistance to the developing world in provision of these skills and resources is a major priority for the International Society of Nephrology. This article highlights some general principles regarding glomerular disease in the developing world, illustrated by the author's experience in rural Uganda.

AIDS-Associated Nephropathy↗
PubMed2005

Functional expression of the renin-angiotensin system in human podocytes.

Experimental and clinical studies impressively demonstrate that angiotensin-converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB) significantly reduce proteinuria and retard progression of glomerular disease. The underlying intraglomerular mechanisms are not yet fully elucidated. As podocyte injury constitutes a critical step in the pathogenesis of glomerular proteinuria, beneficial effects of ACEI and ARB may partially result from interference with a local renin-angiotensin system (RAS) in podocytes. The knowledge of expression and function of a local RAS in podocytes is limited. In this study, we demonstrate functional expression of key components of the RAS in differentiated human podocytes: podocytes express mRNA for angiotensinogen, renin, ACE type 1, and the AT(1) and AT(2) angiotensin receptor subtypes. In Western blot experiments and immunostainings, expression of the AT(1) and AT(2) receptor was demonstrated both in differentiated human podocytes and in human kidney cortex. ANG II induced a concentration-dependent increase in cytosolic Ca(2+) concentration via AT(1) receptors in differentiated human podocytes, whereas it did not increase cAMP. Furthermore, ANG II secretion was detected, which was blocked by neither the ACEI captopril nor the renin inhibitor remikiren nor the chymase inhibitor chymostatin. ANG II secretion of podocytes was not increased by mechanical stress. Finally, ANG II was found to increase staurosporine-induced apoptosis in podocytes. We speculate that ACEI and ARB exert their beneficial effects, in part, by interfering with a local RAS in podocytes. Further experiments are required to identify the underlying molecular mechanism(s) of podocyte protection.

Angiotensinogen↗
PubMed2005

Sam68-like mammalian protein 2, identified by digital differential display as expressed by podocytes, is induced in proteinuria and involved in splice site selection of vascular endothelial growth factor.

Podocytes, the glomerular epithelial cells of the kidney, share important features with neuronal cells. In addition to phenotypical and functional similarities, a number of gene products have been found to be expressed exclusively or predominantly by both cell types. With the hypothesis of a common transcriptome shared by podocytes and neurons, digital differential display was used to identify novel podocyte-expressed gene products. Comparison of brain and kidney cDNA libraries with those of other organs identified Sam68-like mammalian protein 2 (SLM-2), a member of the STAR family of RNA processing proteins, as expressed by podocytes. SLM-2 expression was found to be restricted in the kidney to podocytes. In proteinuric diseases, SLM-2, a known regulator of neuronal mRNA splice site selection, was found significantly upregulated on mRNA and protein levels. Knockdown of SLM-2 by short interfering RNA in podocytes was performed to evaluate its biologic role. RNA splicing of vascular endothelial growth factor (VEGF), a key regulator of the filtration barrier and expressed as functionally distinct splice isoforms, was evaluated. VEGF(165) expression was found to be reduced by 25% after SLM-2 knockdown. In vivo, the glomerular expression of SLM-2 correlated with the mRNA levels of VEGF(165). This study demonstrates the power of digital differential display to predict cell type-specific gene expression by hypothesis-driven analysis of tissue cDNA libraries. SLM-2-dependent VEGF splicing indicates the importance of mRNA splice site selection for glomerular filtration barrier function.

Animals↗
PubMed2005

Nephrotic plasma alters slit diaphragm-dependent signaling and translocates nephrin, Podocin, and CD2 associated protein in cultured human podocytes.

Podocytes are critical in maintaining the filtration barrier of the glomerulus and are dependent on the slit diaphragm (SD) proteins nephrin, podocin, and CD2-associated protein (CD2AP) to function optimally. The effects of normal human plasma and nephrotic plasma on podocytes were tested, focusing particularly on the SD complex. With the use of a conditionally immortalized human podocyte cell line, it first was shown that exposure to normal and non-nephrotic human plasma leads to a concentration of nephrin, podocin, CD2AP, and actin at the cell surface. Next, the effects of plasma from patients with nephrotic conditions to non-nephrotic conditions were compared. When exposed to all nephrotic plasma samples (and a non-human serum control), nephrin podocin and CD2AP assumed a cytoplasmic distribution; nephrin and synaptopodin were selectively downregulated, and the relocation of nephrin induced by nephrotic plasma could be rescued back to the plasma membrane by co-incubation with non-nephrotic plasma. Furthermore, intracellular calcium signaling was altered by nephrotic plasma, which was mediated by tyrosine kinase phosphorylation. With the use of nephrin mutant human cell lines, it was shown that this signaling and translocation response to normal plasma is nephrin dependent. This work demonstrates that nephrotic plasma seems to be deficient in factors that act via the podocyte SD complex, which are essential in maintaining its physiologic function.

Actins↗
PubMed2005

Mechanism of perturbation of integrin-mediated cell-matrix interactions by reactive carbonyl compounds and its implication for pathogenesis of diabetic nephropathy.

Perturbation of interactions between cells and the extracellular matrix (ECM) of renal glomeruli may contribute to characteristic histopathological lesions found in the kidneys of patients with diabetic nephropathy. However, the mechanism by which the diabetic conditions may affect cell-ECM interactions is unknown. Existing hypotheses suggest a role of glucose in direct modification of ECM. Here, we have demonstrated that carbonyl compound methylglyoxal (MGO) completely inhibited endothelial cell adhesion to recombinant alpha3 noncollagenous 1 domain of type IV collagen mediated via a short collagenous region containing RGD (Arg-Gly-Asp) sequence as well as binding of purified alpha(v)beta(3) integrin to this protein. Specific MGO adducts of the arginine residue were detected within RGD sequence using mass spectrometry. Modification by carbonyl compounds glyoxal or glycolaldehyde had similar but smaller effects. MGO strongly inhibited adhesion of renal glomerular cells, podocytes, and mesangial cells to native collagen IV and laminin-1 as well as binding of collagen IV to its major receptor in glomerular cells, alpha(1)beta(1) integrin. In contrast, modification of these proteins by glucose had no effect on cell adhesion. Pyridoxamine, a promising drug for treatment of diabetic nephropathy, protected cell adhesion and integrin binding from inhibition by MGO. We suggest that in diabetes, perturbation of integrin-mediated cell-matrix interactions occurs via the modification of critical arginine residues in renal ECM by reactive carbonyl compounds. This mechanism may contribute to the development of diabetic nephropathy.

Aldehydes↗
PubMed2005

The human glomerular podocyte is a novel target for insulin action.

Microalbuminuria is significant both as the earliest stage of diabetic nephropathy and as an independent cardiovascular risk factor in nondiabetic subjects, in whom it is associated with insulin resistance. The link between disorders of cellular insulin metabolism and albuminuria has been elusive. Here, we report using novel conditionally immortalized human podocytes in vitro and human glomeruli ex vivo that the podocyte, the principal cell responsible for prevention of urinary protein loss, is insulin responsive and able to approximately double its glucose uptake within 15 min of insulin stimulation. Conditionally immortalized human glomerular endothelial cells do not respond to insulin, suggesting that insulin has a specific effect on the podocyte in the glomerular filtration barrier. The insulin response of the podocyte occurs via the facilitative glucose transporters GLUT1 and GLUT4, and this process is dependent on the filamentous actin cytoskeleton. Insulin responsiveness in this key structural component of the glomerular filtration barrier may have central relevance for understanding of diabetic nephropathy and for the association of albuminuria with states of insulin resistance.

Actins↗
PubMed2004

Vascular endothelial growth factor and nephrin interact and reduce apoptosis in human podocytes.

Vascular endothelial growth factor (VEGF) is anti-cytotoxic in podocytes. Moreover, it has been suggested that nephrin, a cell adhesion molecule of the podocyte slit diaphragm, can contribute to antiapoptotic mechanisms in these cells. We therefore investigated whether VEGF signals to reduce apoptosis and the role of nephrin in this survival mechanism. Flow cytometry showed that podocytes with nephrin mutations had a significantly greater proportion of apoptosis. Although VEGF reduced apoptosis in human conditionally immortalized podocytes [wild-type (WT)] by 18.1% of control (P < 0.001), it was unable to do so in nephrin-deficient human conditionally immortalized podocytes. Moreover, Western blotting and immunodetection with an anti-nephrin antibody showed that the phosphorylation of nephrin, compared with serum-starved WTs, was significantly increased (ratio of 3.36 +/- 1.2 to control, P < 0.05) by VEGF treatment and significantly reduced by treatment with a neutralizing VEGF monoclonal antibody (mAb) (ratio of 0.2 +/- 0.09 to control, P < 0.05). The AKT signaling pathway has been implicated in nephrin-mediated inhibition of apoptosis in transfected cells, but the role of this pathway has not previously been shown in podocytes. Surprisingly, exogenous VEGF decreased AKT/PKB phosphorylation in normal podocytes but increased it in nephrin-deficient podocytes. We suggest therefore that both exogenous and endogenous (podocyte derived) VEGF can stimulate the phosphorylation of nephrin and through this action may prevent podocyte apoptosis. However, the involvement of AKT in this survival response in normal human podocytes is not clear.

Apoptosis↗
PubMed2004

Role of CD4+ and CD8+ T cells in murine skin and heart allograft rejection across different antigenic desparities.

The factors that influence the relative contribution of the T cell subsets to allograft rejection remain unclear. We compared skin and heart rejection in CD4 Knockout (KO), and CD8 KO mice across full-, minor-, and class II histocompatibility antigen (HA) mismatches. Skin allografts were rejected by either CD4+ or CD8+ T cells alone at any degree of antigenic mismatch. However, either the absence of CD4+ cells or a lesser degree of HA mismatch resulted in prolongation of graft survival. In contrast, fully allogeneic heart grafts were accepted in CD4 KO recipients, and minor HA mismatched heart grafts were accepted by both CD4 KO and CD8 KO mice. Thus, the T cell subsets required for allograft rejection are determined by the immunogenicity of the tissue transplanted. In the absence of CD8+ T cells, perforin and Fas ligand (FasL) but not granzyme B mRNA were detected in rejecting grafts. Thus, granzyme B is a CD8+ cytotoxic T lymphocyte (CTL)-specific effector molecule.

Animals↗
PubMed2004

The cellular basis of albuminuria.

The appearance of albumin in the urine has long been recognized as a cardinal feature of kidney disease and more recently has been shown to also be an independent cardiovascular risk factor associated with insulin resistance. Recent studies on rare human genetic variants, targeted gene disruption in mouse models and cultured glomerular cells in vitro have dramatically improved our understanding of the cellular and molecular basis of albuminuria. This review aims to summarize the current state of knowledge, to illustrate known mechanisms of proteinuria in disease states and to suggest a possible explanation for the link between albuminuria and insulin resistance.

Albuminuria↗
PubMed2004

Angiopoietin 1 and vascular endothelial growth factor modulate human glomerular endothelial cell barrier properties.

Normal glomerular filtration depends on the combined properties of the three layers of glomerular capillary wall: glomerular endothelial cells (GEnC), basement membrane, and podocytes. Podocytes produce endothelial factors, including angiopoietin 1 (ang1), and vascular endothelial growth factor (VEGF), whereas GEnC express their respective receptors Tie2 and VEGFR2 in vivo. As ang1 acts to maintain the endothelium in other vascular beds, regulating some actions of VEGF, these observations suggest a mechanism whereby podocytes may direct the unique properties of the glomerular endothelium. This interaction was investigated by studies on the barrier properties of human GEnC in vitro. GEnC were examined for expression of endothelium-specific markers by immunofluorescence and Western blotting and for typical responses to TNF-alpha by a cell-based immunoassay. Expression of angiopoietin and VEGF receptors was examined similarly. Barrier properties of GEnC monolayers cultured on porous supports were investigated by measurement of transendothelial electrical resistance (TEER) and passage of labeled albumin. Responses to a cAMP analogue and thrombin were examined before those to ang1 and VEGF. Results confirmed the endothelial origin of GEnC and their expression of Tie2 and VEGFR2. GEnC formed monolayers with a mean TEER of 30 to 40 Omega/cm(2). The cAMP analogue and thrombin increased and decreased TEER by 34.4 and 14.8 Omega/cm(2), respectively, with corresponding effects on protein passage. Ang1 increased TEER by 11.4 Omega/cm(2) and reduced protein passage by 45.2%, whereas VEGF reduced TEER by 12.5 Omega/cm(2) but had no effect on protein passage. Both ang1 and VEGF modulate GEnC barrier properties, consistent with potential in vivo roles; ang1 stabilizing the endothelium and resisting angiogenesis while VEGF induces the high permeability to water, characteristic of the glomerular endothelium.

Angiopoietin-1↗
PubMed2004

Human podocytes possess a stretch-sensitive, Ca2+-activated K+ channel: potential implications for the control of glomerular filtration.

Podocytes express many proteins characteristic of smooth muscle, such as actin and myosin. They also express receptors to several vasoactive agents, including acetylcholine and angiotensin II; these phenotypic properties suggest that podocytes are not static entities but may respond to physiologic stimuli. The electrophysiologic properties of a conditionally immortalized human podocyte cell line that expresses the specific podocyte proteins nephrin, podocin, and synaptopodin were examined by patch clamp. Channels that were highly K(+)-selective and had a conductance of 224 +/- 11.5 pS in symmetrical 150 mM K(+) solutions were identified. Channel activity was Ca(2+)- and voltage-dependent, being increased with an increase in Ca(2+) or depolarization, and inhibited by penitrem A. The conductance and voltage- and Ca(2+)-dependence suggest that this is the large-conductance calcium-activated K(+) channel, BK (KCNMA1)-this was supported by reverse transcription-PCR experiments that showed the presence of the BK encoding mRNA, along with expression of KCNMB subunit types 3 and 4. In sections of human glomeruli, immunocytochemistry revealed that BK co-localizes with the podocyte-specific protein nephrin, indicating that these channels are present in native human podocytes. In whole-cell experiments, penitrem A inhibited outward currents to the same extent as tetra-ethyl ammonium (TEA) but did not affect the membrane potential. Channel activity was also increased by applying suction to the patch pipette or by dilution of the bathing medium, indicating that these channels are stretch sensitive. Thus, these channels do not contribute to the resting membrane potential but are activated by a rise in intracellular Ca(2+), membrane depolarization, cell swelling, or membrane stretch. By implication, these results suggest that podocytes may be able to respond to changes in the glomerular capillary pressure and modulate the GFR.

Calcium↗
PubMed2003

Differentiated human podocytes endogenously express an inhibitory isoform of vascular endothelial growth factor (VEGF165b) mRNA and protein.

Despite production by podocytes of the proangiogenic molecule vascular endothelial growth factor-A (VEGF), the glomeruli are not sites of angiogenesis. We recently described mRNA expression of an inhibitory splice variant of VEGF (VEGF165b) in normal kidney (Bates DO, Cui TG, Doughty JM, Winkler M, Sugiono M, Shields JD, Peat D, Gillatt D, and Harper SJ. Cancer Res 62: 4123-4131, 2002). Available anti-VEGF antibodies do not distinguish stimulatory from inhibitory VEGF families. To assess the production of VEGF165 (stimulatory) and VEGF165b (inhibitory) isoforms by human podocytes, we examined both primary cultured and conditionally immortalized human podocytes using family- and isoform-specific RT-PCR. In addition, VEGF protein production was analyzed in podocytes, using isoform-specific double-strand small-interference RNAs (siRNA). RT-PCR demonstrated the production of VEGF189 mRNA by podocytes of both phenotypes. In contrast, on differentiation there was a splicing change from VEGF165 to VEGF165b mRNA. In addition, VEGF protein in the supernatant of conditionally immortalized, differentiated podocytes was reduced by VEGF165b siRNA to 20+/-11% of the level of mock-transfected cells (P < 0.01). No reduction was seen with mismatch siRNA. Moreover, there was no reduction in VEGF protein concentration in the supernatant of primary cultured, dedifferentiated human podocytes (109+/-8% of mismatch siRNA, P > 0.1). In conclusion, differentiated but not dedifferentiated human podocytes secrete significant amounts of VEGF165b protein. It is possible that this may explain the paradox of high VEGF production in the glomerulus but no angiogenesis. Furthermore, the existence of this splicing switch in relation to podocyte phenotype suggests that alternative splicing of the VEGF pre-RNA is a regulated process that is open to manipulation and therefore could be a target for novel cancer therapies.

Cell Differentiation↗
PubMed2003

Functional evidence that vascular endothelial growth factor may act as an autocrine factor on human podocytes.

Vascular endothelial growth factor (VEGF) is expressed by renal glomerular epithelial cells (podocytes) and is thought to be protective against nephrotoxic agents. VEGF has been shown to be an autocrine survival factor in neuropilin-1-positive, VEGF receptor-negative breast carcinoma cells. Normal human podocytes are also known to express neuropilin-1, VEGF, and are VEGF-R2 negative. Here, we investigated whether a similar VEGF autocrine loop may exist in podocytes. Podocyte cytosolic calcium concentration ([Ca(2+)](i)) was analyzed in primary cultured and conditionally immortalized podocytes using ratiometric fluorescence measurement. Cytotoxicity was determined by lactate dehydrogenase assay, proliferation by [(3)H]-thymidine incorporation, and cell counts by hemocytometric assay. VEGF decreased [Ca(2+)](i) in primary podocytes (from 179 +/- 36 to 121 +/- 25 nM, P < 0.05) and conditionally immortalized podocytes (from 95 +/- 10 to 66 +/- 8 nM, P < 0.02) in the absence of extracellular calcium. The type III receptor tyrosine-kinase inhibitor PTK787/ZK222584 abolished this reduction. VEGF increased podocyte [(3)H]-thymidine incorporation (3,349 +/- 283 cpm, control 2,364 +/- 301 cpm, P < 0.05) and cell number (4.5 +/- 0.7 x 10(4)/ml, control 2.6 +/- 0.5 x 10(4)/ml, P < 0.05) and decreased cytotoxicity (5.9 +/- 0.7%, control 12 +/- 3%, P < 0.05), whereas a monoclonal antibody to VEGF increased cytotoxicity. Electron microscopy of normal human glomeruli demonstrated that the glomerular VEGF is mostly podocyte cell membrane associated. These results indicate that one of the functions of VEGF secreted from podocytes may be to act as an autocrine factor on calcium homeostasis and cell survival.

Adult↗