The molecular and functional phenotype of glomerular podocytes reveals key features of contractile smooth muscle cells.

The glomerular podocyte is a highly specialized cell, with the ability to ultrafilter blood and support glomerular capillary pressures. However, little is known about either the genetic programs leading to this functionality or the final phenotype. We approached this question utilizing a human conditionally immortalized cell line, which differentiates from a proliferating epithelial phenotype to a differentiated form. We profiled gene expression during several time points during differentiation and grouped the regulated genes into major functional categories. A novel category of genes that was upregulated during differentiation was of smooth muscle-related molecules. We further examined the smooth muscle phenotype and showed that podocytes consistently express the differentiated smooth muscle markers smoothelin and calponin and the specific transcription factor myocardin, both in vitro and in vivo. The contractile contribution of the podocyte to the glomerular capillary is controversial. We demonstrated using two novel techniques that podocytes contract vigorously in vitro when differentiated and in real time were able to demonstrate that angiotensin II treatment decreases monolayer resistance, morphologically correlating with enhanced contractility. We conclude that the mature podocyte in vitro possesses functional apparatus of contractile smooth muscle cells, with potential implications for its in vivo ability to regulate glomerular dynamic and permeability characteristics.

Increased EP4 receptor expression in colorectal cancer progression promotes cell growth and anchorage independence.

Cyclooxygenase-2 and prostaglandin E(2) (PGE(2)) levels are increased in colorectal cancers and a subset of adenomas. PGE(2) signaling through the EP4 receptor has previously been associated with colorectal tumorigenesis. However, changes in EP4 expression during adenoma to carcinoma progression have not been investigated, neither has whether levels of EP4 influence important markers of malignant potential, such as anchorage-independent growth or the tumors growth response to PGE(2). We report using immunohistochemistry that in vivo EP4 receptor protein expression was increased in colorectal cancers (100%) as well as adenomas (36%) when compared with normal colonic epithelium. EP4 expression was also higher in colorectal carcinoma compared with adenoma cell lines and increased with in vitro models of tumor progression. Adenoma (PC/AA/C1 and RG/C2) and carcinoma cell lines (HT29) were growth stimulated by PGE(2) up to 0.5 micromol/L. However, although carcinoma and transformed adenoma (PC/AA/C1SB10C, a transformed derivative of PC/AA/C1) cells remain stimulated by higher doses of PGE(2) (10 micromol/L), the adenoma cell lines were inhibited. Interestingly, enforced expression of EP4 in the adenoma cell line, RG/C2, resulted in stimulation of growth by 10 micromol/L PGE(2) and promoted anchorage-independent growth. Both in vivo and in vitro data from this study suggest that increased EP4 receptor expression is important during colorectal carcinogenesis. We propose that high levels of PGE(2) in a tumor microenvironment would select for cells with increased EP4 expression, and that the EP4 receptor may therefore represent an important target for colorectal cancer prevention and treatment.

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.

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.

Expression of functional toll-like receptor-2 and -4 on alveolar epithelial cells.

The recognition of potentially harmful microorganisms involves the specific recognition of pathogen-associated molecular patterns (PAMPs) and the family of Toll-like receptors (TLRs) is known to play a central role in this process. TLR-4 is the major recognition receptor for lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, whereas TLR-2 responds to bacterial products from gram-positive organisms. Although resident alveolar macrophages are the first line of defense against microbial attack, it is now understood that the alveolar epithelium also plays a pivotal role in the innate immunity of the lung. The purpose of the current study was to determine whether human primary type II alveolar epithelial cells (ATII) express functional TLR-2 and TLR-4 and how they may be regulated by inflammatory mediators. We have used reverse transcriptase-polymerase chain reaction and flow cytometry to determine basal and inducible expression on ATII. We have used highly purified preparations of the gram-positive bacterial product lipoteichoic acid (LTA) and LPS to look at the functional consequences of TLR-2 and TLR-4 ligation, respectively, in terms of interleukin-8 release. We have shown that human primary ATII cells express mRNA and protein for both TLR-2 and TLR-4, which can be modulated by incubation with LPS and tumor necrosis factor. Furthermore, we have demonstrated that these receptors are functional. This suggests that ATII have the potential to contribute significantly to the host defense of the human alveolus against bacteria.

Primary human alveolar type II epithelial cell chemokine release: effects of cigarette smoke and neutrophil elastase.

An early response to cigarette smoke is an influx of leukocytes into the lung. Alveolar epithelial type II (ATII) cells may contribute by releasing chemokines in response to cigarette smoke and neutrophil elastase (NE). Human ATII cells were purified from normal regions of lungs resected for carcinoma (n = 14). In vitro, these cells exhibited ATII cell characteristics: lamellar bodies, apical microvilli, tight junctions, and expressed surfactant apoprotein C. Basal ATII cell release of five chemokines ranked as follows: monocyte chemotactic protein (MCP)-1 > interleukin (IL)-8 > growth-related oncogene (GRO)-alpha > macrophage inflammatory protein (MIP)-1alpha > regulated on activation, normal T cell expressed and secreted (RANTES). MIP-1alpha and RANTES were often not detectable. After stimulation with a mixture of lipopolysaccharide/endotoxin (LPS), tumor necrosis factor-alpha, IL-1beta, and IFN-gamma, MCP-1 and IL-8 secretion rose 4-6-fold, whereas GRO-alpha rose 25-fold. NE stimulated IL-8 mRNA expression, and 10nM NE stimulated IL-8 secretion; however, 100 nM NE caused a decrease in extracellular IL-8, MCP-1, and GRO-alpha, attributed to proteolysis. Cigarette smoke extract (CSE) inhibited IL-8 mRNA expression and release of all chemokines. Glutathione protected against the effects of CSE, suggesting oxidative mechanisms. GRO-alpha, important in growth and repair, was sensitive to both stimulation, by LPS:cytokines, and inhibition, by CSE. Thus, contrary to the original hypothesis, high concentrations of NE and CSE resulted in reduced extracellular chemokine levels. We hypothesize that reduced ATII cell-derived chemokine levels compromise alveolar repair, contributing to cigarette smoke-induced alveolar damage and emphysema.

Correlation between enterococcal biofilm formation in vitro and medical-device-related infection potential in vivo.

Hospital-acquired infections caused by enterococci have increased dramatically since the 1970s. Many nosocomial enterococcal bloodstream infections are associated with medical devices such as central venous catheters. The ability to form biofilm on medical devices is a potential virulence trait that may allow enterococci to cause infections in the expanding population of patients managed with such devices. In this study, the hypothesis that increased ability to form biofilm in vitro is associated with medical-device-related infection in vivo was tested. A microplate assay was employed to assess biofilm-forming characteristics of enterococci in 0.9 % (w/v) sodium chloride, an oligotrophic environment, and BHI, a nutrient-rich environment. Results were compared in isolates from different sources of infection. One hundred and nine enterococcal bloodstream isolates were assayed. Biofilm formation on microplates was demonstrated by all Enterococcus faecalis isolates and 16/38 (42 %) Enterococcus faecium isolates. E. faecalis isolates produced significantly more biofilm than E. faecium isolates in both media (P < 0.0001, Mann-Whitney U test). E. faecalis isolates from intravascular-catheter-related bloodstream infections (CRBSIs) produced significantly more biofilm than non-CRBSI isolates (P < 0.0001), or isolates of uncertain clinical significance (P < 0.0001). Biofilm formed by E. faecium isolates was not significantly affected by culture medium and did not differ between isolates from the different clinical categories. In conclusion, there was significantly more biofilm formed by E. faecalis isolates causing CRBSI compared with isolates from other types of infection or from isolates of uncertain clinical significance. The ability of E. faecalis isolates to form biofilm in vitro appears to be a marker of a virulence trait that enhances the ability of isolates to cause CRBSI.

Co-localization of nephrin, podocin, and the actin cytoskeleton: evidence for a role in podocyte foot process formation.

The discovery of the genes for nephrin and podocin, which are mutated in two types of congenital nephrotic syndrome, was pivotal in establishing the podocyte as the central component of the glomerular filtration barrier. In vivo the proteins have been localized to the podocyte slit diaphragm, and there is recent evidence for interaction between the two via the adapter molecule CD2AP. We describe in a human podocyte cell line, the subcellular distribution of nephrin, podocins, and CD2AP and their functional interaction with the cytoskeleton. In addition to membrane expression, nephrin and podocin were detected intracellularly in a filamentous pattern. Double immunolabeling and depolymerization studies showed that nephrin and podocin partially co-localize with actin, most strikingly seen protruding from the tips of actin filaments, and are dependent on intact actin polymers for their intracellular distribution. Treatment of differentiated podocytes with puromycin aminonucleoside, an agent that causes foot process effacement in vivo, disrupted actin and nephrin simultaneously, with loss of cell surface localization. We demonstrate an intimate relationship between nephrin podocin and filamentous actin, and reason that disruption of nephrin/podocin could be a final common pathway leading to foot process effacement in proteinuric diseases.

Enterococcal intravascular catheter-related bloodstream infection: management and outcome of 61 consecutive cases.

Enterococci are an increasingly important cause of intravascular catheter-related bloodstream infection (CRBSI), but the evidence base for treating such cases is limited. Successful antimicrobial treatment of CRBSI while leaving the central venous catheter (CVC) in situ has been reported for some bacteria, such as coagulase-negative staphylococci, but the effectiveness of this approach for treating enterococcal CRBSI is unknown. We aimed to determine the effectiveness of treatment options for enterococcal CRBSI and whether CVC removal is mandatory. Treatment and outcome was determined in a 3 year cohort of patients with enterococcal CRBSI from a university teaching hospital. All episodes of enterococcal bacteraemia during the study (n = 268) were examined to identify the cohort of 61 CRBSIs. Outcomes were determined for various antimicrobial regimens with or without CVC removal. Forty-eight episodes were managed with CVC removal and 13 were managed with the CVC in situ. Forty of 48 (83%) and five of 13 (38%) episodes were cured with the CVC removed or left in situ, respectively. All five episodes cured with the CVC in situ were treated with a cell wall-acting antimicrobial plus an aminoglycoside. This antimicrobial combination was significantly more effective than either ampicillin or vancomycin monotherapy (P < 0.05), or antimicrobials to which isolates were not susceptible (P < 0.01) when the CVC remained in situ. We conclude that enterococcal CRBSI can be treated successfully without CVC removal. The combination of a cell wall-acting antimicrobial with an aminoglycoside was the most effective regimen when the CVC remained in situ in this small group of patients. Although CVC removal was associated with a high cure rate, it did not guarantee treatment success.