Peter W. Mathieson

Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters

Reabsorption of filtered solutes from the glomerular filtrate and excretion of waste products and xenobiotics are the main functions of the renal proximal tubular (PT) epithelium. A human PT cell line expressing a range of functional transporters would help to augment current knowledge in renal physiology and pharmacology. We have established and characterized a conditionally immortalized PT epithelial cell line (ciPTEC) obtained by transfecting and subcloning cells exfoliated in the urine of a healthy volunteer. The PT origin of this line has been confirmed morphologically and by the expression of aminopeptidase N, zona occludens 1, aquaporin 1, dipeptidyl peptidase IV and multidrug resistance protein 4 together with alkaline phosphatase activity. ciPTEC assembles in a tight monolayer with limited diffusion of inulin-fluorescein-isothiocyanate. Concentration and time-dependent reabsorption of albumin via endocytosis has been demonstrated, together with sodium-dependent phosphate uptake. The expression and activity of apical efflux transporter p-glycoprotein and of baso-lateral influx transporter organic cation transporter 2 have been shown in ciPTEC. This established human ciPTEC expressing multiple endogenous organic ion transporters mimicking renal reabsorption and excretion represents a powerful tool for future in vitro transport studies in pharmacology and physiology.

THE INTERLEUKIN-10-1082 G/A POLYMORPHISM: ALLELE FREQUENCY IN DIFFERENT POPULATIONS AND FUNCTIONAL SIGNIFICANCE

Abstract. Genotypic variation in the human interleukin-10 (IL-10) promoter may account for marked inter-individual variation in IL-10 production and may influence susceptibility to autoimmune diseases. The G/A polymorphism at position -1082 has been linked to high/low IL-10 producer status. We directly tested the functional significance of this polymorphism using DNA-binding assays and reporter gene assays, examined allele frequencies in two geographically distinct populations and assessed intra- and inter-individual variation in IL-10 production in vitro according to genotype. Functional analyses showed that the -1082 region contains a putative ETS-like transcription factor-binding site, and nuclear factors from a monocyte cell line bind to this region. Transient transfection studies in an Epstein-Barr virus-transformed B cell line indicated that the -1082 A allele confers a two fold increase in transcriptional activity of the IL-10 promoter compared to the G allele. There was marked inter-individual variation in IL-10 production by peripheral blood mononuclear cells in vitro, with no consistent effect of genotype.

Morphine Induces Albuminuria by Compromising Podocyte Integrity

Morphine has been reported to accelerate the progression of chronic kidney disease. However, whether morphine affects slit diaphragm (SD), the major constituent of glomerular filtration barrier, is still unclear. In the present study, we examined the effect of morphine on glomerular filtration barrier in general and podocyte integrity in particular. Mice were administered either normal saline or morphine for 72 h, then urine samples were collected and kidneys were subsequently isolated for immunohistochemical studies and Western blot. For in vitro studies, human podocytes were treated with morphine and then probed for the molecular markers of slit diaphragm. Morphine-receiving mice displayed a significant increase in albuminuria and showed effacement of podocyte foot processes. In both in vivo and in vitro studies, the expression of synaptopodin, a molecular marker for podocyte integrity, and the slit diaphragm constituting molecules (SDCM), such as nephrin, podocin, and CD2-associated protein (CD2AP), were decreased in morphine-treated podocytes. In vitro studies indicated that morphine modulated podocyte expression of SDCM through opiate mu (MOR) and kappa (KOR) receptors. Since morphine also enhanced podocyte oxidative stress, the latter seems to contribute to decreased SDCM expression. In addition, AKT, p38, and JNK pathways were involved in morphine-induced down regulation of SDCM in human podocytes. These findings demonstrate that morphine has the potential to alter the glomerular filtration barrier by compromising the integrity of podocytes.

Podocyte actin in health, disease and treatment

Focal segmental glomerulosclerosis (FSGS) is a common and important cause of nephrotic syndrome. It is now generally accepted that the predominant glomerular lesion in FSGS is injury to podocytes (visceral glomerular epithelial cells). Podocytes have a complex cellular architecture with interdigitating processes maintained by a precise organization of actin filaments in the cellular cytoplasm. Electron microscopic analysis of the glomerular capillary wall shows that, on the urinary side, the foot processes of the podocytes are organized in a precise manner with slits between them through which filtration occurs (Figure 1A). It has been known for decades that a cardinal feature of the glomerular capillary wall in proteinuric states is flattening or effacement of the foot processes so that this precise organization has been disrupted: it is clear from examination of the podocyte cytoplasm that this effacement is associated with flattening of the actin filaments (Figure 1B). Doubts have been expressed about the specificity of this finding, including by this author [1] citing, for example, that similar changes are seen in children with severe protein malnutrition in kwashiorkor [2]. This observation is still unexplained, but in recent years, it has become ever clearer that the precise organization and regulation of the actin cytoskeleton in the podocyte is essential for the maintenance of its normal structure and function, that disruption thereof is a feature of podocyte diseases and is associated with proteinuria, and perhaps most importantly of all that therapeutic agents which have beneficial effects in nephrotic syndrome are capable of restoring the podocyte actin cytoskeleton. Early work that supports this line of thought came from the same group, led by Martin Pollak, responsible for the paper under discussion here. Firstly, they reported that mutations in the gene encoding alpha-actinin IV, which plays an important role in actin polymerization, were associated with autosomal dominant late-onset familial FSGS [3]. Next, they showed that the mutant form of the protein binds more avidly to actin and affects the mechanical properties of actin gels, providing an explanation for its effect on podocyte structure [4]. Another group then showed that podocyte-specific transgenic expression of the mutant alpha-actinin IV gene in mice leads to FSGS [5], demonstrating that it is the effects of the gene in the podocyte rather than any other cell that is responsible for the disease. The final proof of the causative role of the mutation came in experiments in which the group of Pollak showed that, when the mutant gene was ‘knocked-in’ in mice, the animals developed FSGS, showing that this gene defect alone is capable of causing the disease [6]. The mechanisms underlying the effects of the mutant protein have now been demonstrated in detail [7]. Most recently, the group of Pollak provided further evidence that the physicochemical characteristics of actin fibres formed with the mutant alpha-actinin IV show altered flexibility that can explain the effects on the podocyte [8]. Other groups have reported different gene mutations which affect the actin cytoskeleton and also cause FSGS: for instance, in the gene encoding CD2-associated protein which encodes a protein that is important in linking to actin fibres [9]. However, nephrologists have inevitably asked whether these rare familial forms of FSGS are analogous to the much more common sporadic forms of the disease. Shared mechanisms are likely to exist, but does the actin cytoskeleton play a similar role in idiopathic FSGS? We do not yet know for sure, but recent observations on the mechanisms of action of drugs which are effective in FSGS have caused real excitement and shown the way to more specific forms of treatment. Calcineurin inhibitors, especially cyclosporin, are widely used in the treatment of proteinuric diseases including FSGS; their use originally being based on the assumption that the diseases are immune-mediated and the immunosuppressive effects of such drugs are likely to be helpful. Faul et al. [10] made the novel observation that the anti-proteinuric effects of cyclosporin can be explained by direct effects on the podocyte actin cytoskeleton (and therefore the cells shape) and are independent of its effects on T lymphocytes. The mechanism involves synaptopodin, a key stabilizer of the actin cytoskeleton in podocytes. When synaptopodin is phosphorylated, it is protected from degradation. Calcineurin (which is blocked by cyclosporin) dephosphorylates synaptopodin and allows its degradation. Thus, cyclosporin prevents degradation of

The podocyte cytoskeleton in health and in disease

The podocyte is a key cell in the selective filtering action of the glomerular capillary wall. Podocyte injury is of pathogenetic and prognostic significance in human glomerular disease; podocyte repair and regeneration are important therapeutic targets. In particular, podocyte function is dependent on the cells actin cytoskeleton: this maintains their complex structure. Alterations in the actin cytoskeleton arise from a variety of genetic and acquired causes. Therapeutic agents that are beneficial in proteinuric disease may act at least partly by restoring the cell shape via effects on the actin cytoskeleton. Recent studies of podocytes in vivo and in vitro are described, highlighting clinically relevant observations and those that help us understand the ways in which we may harness natures own mechanisms to repair and/or renew these specialized glomerular cells, with a particular focus on their actin cytoskeleton. Drugs that have beneficial effects on podocytes can improve our ability to treat important renal diseases including diabetic nephropathy. Currently available agents can be applied in this way and the rapid progress in the study of podocytes is highlighting new therapeutic targets that can bring even more specificity.

A String of Beads

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