Characterization of a Trpc6 Transgenic Mouse Associated with Early Onset FSGS.

RATIONALE: Mutations in Transient Receptor Potential Channel 6 (TRPC6) gene are associated with autosomal dominant focal and segmental glomerulosclerosis (FSGS). The majority of the identified mutations affect the ion channel function. Since calcium channels are promising candidate drug targets, there is an an urgent need for a mouse model to assess new therapeutic drugs and to help delineate the pathogenic process leading to FSGS. We have previously reported the generation of three independent transgenic mouse lines carrying different Trpc6 mutations that display a glomerular disease comparable to the phenotype presented by individuals with FSGS. However, the utility of these models for drug testing is dampened by the late-onset of the presentation and the mild phenotypic manifestations. METHODOLOGY: In order to obtain a time-effective mouse model for Trpc6-associated FSGS we generated a new transgenic mutant Trpc6 mouse model emulating the amino acid change carried by the first pediatric patient of FSGS associated with a TRPC6 mutation: M132T. RESULTS: Mice carrying the orthologous Trpc6 M131T transgene showed early onset proteinuria and early signs of FSGS. When exploring molecular consequences of the overexpression of this mutated form of Trpc6 in podocytes, differences in expression levels of Axin2 and ß-catenin were found in glomeruli from transgenic Trpc6 M131T mice. These data supports the proposed molecular mechanisms related to the activation of calcineurin-NFAT/Wnt signaling, as outcome of the increased calcium influx caused by the mutated form of Trpc6. CONCLUSION: Given that the Trpc6 M131T mouse develops an early onset of FSGS-like phenotypes it represents a promising model for studying the pathogenesis of FSGS caused by TRpC6, facilitating the assessment of new drugs as treatments and allowing further studies to understand underlying molecular pathways involved in the development of the TRPC6 mediated disease.

Active trafficking of alpha 1 antitrypsin across the lung endothelium.

The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency.

The Wnt/ß-catenin pathway cross-talks with STAT3 signaling to regulate survival of retinal pigment epithelium cells.

Wnt/ß-catenin signaling is an essential pathway that regulates numerous cellular processes, including cell survival. The molecular mechanisms contributing to pro-survival Wnt signaling are mostly unknown. Signal transducer and activator of transcription proteins (STATs) are a well-described family of transcription factors. STAT3 induces expression of anti-apoptotic genes in many tissues and is a downstream mediator of protective growth factors and cytokines. In this study, we investigated whether pro-survival Wnt signaling is mediated by STAT3. The Wnt3a ligand activated Wnt signaling in the retinal pigment epithelium ARPE-19 cell line and significantly increased the viability of cells exposed to oxidative stress. Furthermore, Wnt3a increased STAT3 activation and nuclear translocation, as measured by an antibody against phosphorylated STAT3. Reducing STAT3 levels with siRNA eliminated Wnt3a-dependent protection from oxidative stress. Together, these data demonstrate a previously unknown link between Wnt3a-mediated activation of STAT3 and cell survival, and indicate cross-talk between two important pro-survival signaling pathways.

The Wnt signaling pathway protects retinal ganglion cell 5 (RGC-5) cells from elevated pressure.

The Wnt pathway is an essential signaling cascade that regulates survival and differentiation in the retina. We recently demonstrated that retinal ganglion cells (RGCs) have constitutively active Wnt signaling in vivo. However, the role of Wnt in RGC viability or function is unknown. In this study, we investigated whether Wnt protects the retinal ganglion cell line RGC-5 from elevated pressure, oxidative stress, and hypoxia injuries. Expression of RGC marker genes in the RGC-5 cultures was confirmed by immunocytochemistry and PCR. We demonstrated that the Wnt3a ligand significantly reduced pressure-induced caspase activity in RGC-5 cells (n = 5, P = 0.03) and decreased the number of TUNEL-positive cells (n = 5, P = 0.0014). Notably, Wnt3a-dependent protection was reversed by the Wnt signaling inhibitor Dkk1. In contrast, Wnt3a did not protect RGC-5 cells from oxidative stress or hypoxia. Furthermore, Wnt3a significantly increased growth factor expression in the presence of elevated pressure but not in the presence of oxidative stress and hypoxia. These results indicate that Wnt3a induces injury-specific survival pathways in RGC-5 cells, potentially by upregulating neuroprotective growth factors. Therefore, activation of the Wnt pathway by Wnt3a could be investigated further as a tool to develop novel molecular therapeutic strategies for the prevention of RGC death in retinal disease.

Oxidative epithelial host defense is regulated by infectious and inflammatory stimuli.

Epithelia express oxidative antimicrobial protection that uses lactoperoxidase (LPO), hydrogen peroxide (H(2)O(2)), and thiocyanate to generate the reactive hypothiocyanite. Duox1 and Duox2, found in epithelia, are hypothesized to provide H(2)O(2) for use by LPO. To investigate the regulation of oxidative LPO-mediated host defense by bacterial and inflammatory stimuli, LPO and Duox mRNA were followed in differentiated primary human airway epithelial cells challenged with Pseudomonas aeruginosa flagellin or IFN-gamma. Flagellin upregulated Duox2 mRNA 20-fold, but upregulated LPO mRNA only 2.5-fold. IFN-gamma increased Duox2 mRNA 127-fold and upregulated LPO mRNA 10-fold. DuoxA2, needed for Duox2 activity, was also upregulated by flagellin and IFN-gamma. Both stimuli increased H(2)O(2) synthesis and LPO-dependent killing of P. aeruginosa. Reduction of Duox1 by siRNA showed little effect on basal H(2)O(2) production, whereas Duox2 siRNA markedly reduced basal H(2)O(2) production and resulted in an 8-fold increase in Nox4 mRNA. In conclusion, large increases in Duox2-mediated H(2)O(2) production seem to be coordinated with increases in LPO mRNA and, without increased LPO, H(2)O(2) levels in airway secretion are expected to increase substantially. The data suggest that Duox2 is the major contributor to basal H(2)O(2) synthesis despite the presence of greater amounts of Duox1.

Molecular heterogeneity and alternative splicing of human lactoperoxidase.

Human lactoperoxidase (LPO) exists as two distinct molecules independent of glycosylation. The N-terminus of one form is blocked and has not been identified while the other is proteolytically processed at the N-terminus similar to myeloperoxidase. Our analysis identified alternatively spliced human LPO mRNAs that may explain the observed molecular heterogeneity of LPO. Two mRNAs omit propeptide encoding exons while retaining the 5' exon encoding the secretion signal, consistent with the heterogeneity and suggesting a possible functional role for the propeptide. Two LPO forms were expressed using baculovirus and both showed similar enzyme activity. LC/MS/MS analysis of trypsin digested, partially purified, salivary LPO confirmed the larger unprocessed LPO is present in saliva. To compare variant expression patterns, antisera were raised against purified recombinant (rhLPO) as well as against an antigenic peptide sequence within the exons encoding the propeptide region. Immunohistochemistry demonstrated proLPO was differently localized within gland cells compared to other forms of LPO. The data suggested splice variants may contribute to LPO molecular heterogeneity and its regulation by intracellular compartmental localization.

Epithelial organic cation transporters ensure pH-dependent drug absorption in the airway.

Most inhaled beta(2)-adrenergic agonist and anticholinergic bronchodilators have low lipid solubility because of their transient or permanent positive net charge at physiologic pH. Airway absorption of these cationic drugs is incompletely understood. We examined carrier-mediated mechanisms of cationic drug uptake by human airway epithelia. Airway tissues and epithelial cells, obtained from lung donors without preexisting lung disease, were evaluated for organic cation transporter expression by quantitative RT-PCR and immunofluorescence. For in vitro functional studies on primary airway epithelial cells, uptake of the cationic fluorophore 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP+) was characterized. Quantitative RT-PCR analysis demonstrated high mRNA levels for two polyspecific organic cation/carnitine transporters, OCTN1 and OCTN2, in human airway epithelia. Immunofluorescence of human airway sections confirmed OCTN1/2 protein expression, with a predominant localization to the apical portion of epithelial cells. Primary airway epithelial cells showed a carrier-mediated, temperature-sensitive and saturable uptake of ASP(+). Seventy-five to eighty percent of ASP(+) uptake was inhibited by L-carnitine, an OCTN2-carried zwitterion. The uptake was pH dependent, with approximately 3-fold lower rates at acidic (pH 5.7) than at alkaline (pH 8.2) extracellular pH. Albuterol and formoterol inhibited ASP(+) uptake, suggesting that all these molecules are carried by the same transport mechanism. These findings demonstrate the existence and functional role of a pH-dependent organic cation uptake machinery, namely OCTN1 and OCTN2, in human airway epithelia. We suggest that epithelial OCTN1/2 are involved in the delivery of inhaled cationic bronchodilators to the airway tissue.

Efficacy of various drugs in the prevention of posterior capsule opacification: experimental study of rabbit eyes.

PURPOSE: To assess the efficacy of various drugs in the prevention of posterior capsule opacification (PCO) in a closed capsular bag technique. SETTING: Ophthalmic Biophysics Center, Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida, USA. METHODS: Lens material was removed using phacoaspiration or phacoemulsification through a microcapsulorhexis according to the hardness of the crystalline lens correlated with the weight and age of the rabbits. A mixture of an ophthalmic viscosurgical device (sodium hyaluronate 1.4% [SHA]) and a drug was injected into the empty capsular bag, allowed to remain inside for 3 minutes, and removed. The capsular bag was rinsed with balanced salt solution (BSS) and refilled with SHA. In a group of rabbits, the capsulorhexis was sealed with a minicapsulorhexis valve (MCV). Rabbits were treated with 1 of the following: SHA (control), BSS, mitomycin-C (MMC, 0.2 mg/mL), ethylenediaminetetraacetic acid (EDTA) (10 mM and 15 mM), 5-fluorouracil (5-FU, 33 mg/mL), acetic acid (3%, 0.3%, and 0.003%), and distilled water. RESULTS: Upon completion of the study, the control and treated eyes had PCO and new lens material (not residual). Anterior capsule proliferation was observed in eyes treated with 5-FU. The order of PCO appearance (earliest to latest) was as follows: 15 mM EDTA, SHA, MMC, acetic acid 0.3%, acetic acid 3%, BSS, distilled water (small animals; no MCV), acetic acid 0.003%, 5-FU, 10 mM EDTA, and distilled water (large animals; MCV). The earliest appearance was day 1 postoperatively and the latest, day 47. CONCLUSIONS: Distilled water and 10 mM EDTA treatments were the most efficient in retarding the appearance of PCO.

Transcellular thiocyanate transport by human airway epithelia.

Human airway mucosa synthesizes and secretes lactoperoxidase (LPO). As H(2)O(2) and thiocyanate (SCN(-)) are also present, a functional LPO antibacterial defence system exists in the airways. SCN(-) concentrations in several epithelial secretions are higher than in serum, although the mechanisms of transepithelial transport and accumulation in these secretions are unknown. To examine SCN(-) accumulation in secretions, human airway epithelial cells, re-differentiated at the air-liquid interface, were used in open-circuit conditions. [(14)C]SCN(-), in the basolateral medium, was transported across the epithelium and concentrated tenfold at the apical surface. Measurement of the transepithelial potential showed that the basolateral compartment was positive relative to the apical surface (13.7 +/- 1.8 mV) and therefore unfavourable for passive movement of SCN(-). Transport was dependent on basolateral [SCN(-)] and saturable (K(m,app) = 69 +/- 25 microM); was inhibited by increased apical [SCN(-)]; and was dependent on the presence of basolateral Na(+). Perchlorate (K(i,app) = 0.6 +/- 0.05 microM) and iodide (K(i,app) = 9 +/- 8 microM) in the basolateral medium reversibly inhibited transport, but furosemide did not. Iodide was also transported (K(m,app) = 111 +/- 69 microM). RT-PCR and immunohistochemistry confirmed expression of Na(+)-I(-) symporter (NIS) in the airways. SCN(-) transport was insensitive to apical disulphonic acid Cl(-) channel blockers, but sensitive to apical glibenclamide and arylaminobenzoates. Forskolin and dibutyryl cAMP increased transport. These data suggest SCN(-) transport may occur through basolateral NIS-mediated SCN(-) concentration inside cells, followed by release through an apical channel, perhaps cystic fibrosis transmembrane conductance regulator.