Different effects of CsA and FK506 on aquaporin-2 abundance in rat primary cultured collecting duct cells.

Calcineurin (Cn) inhibitors (CnI) such as cyclosporine A (CsA) and FK506 are nephrotoxic immunosuppressant drugs, which decrease tubular function. Here, we examined the direct effect of CnI on aquaporin-2 (AQP2) expression in rat primary cultured inner medullary collecting duct cells. CsA (0.5-5 µM) but not FK 506 (0.01-1 µM) decreased expression of AQP2 protein and messenger RNA (mRNA) in a concentration and time dependent manner, without affecting mRNA stability. This effect was observed despite similar inhibition of Cn activity by both CnI, thereby suggesting that the CsA-dependent decrease in AQP2 expression was Cn independent. Another inhibitor of cyclophilin A, the primary intracellular target of CsA, had no effect on AQP2 expression. In order to investigate the mechanism of decreased AQP2 transcription, we studied activation status of two suggested transcriptional regulators of AQP2, cAMP-responsive element binding protein (CREB), and tonicity enhancer binding protein (TonEBP). Localization of TonEBP, as well as TonEBP-mediated gene transcription, was not affected by CsA. Phosphorylation of CREB at an activating phosphorylation site (S133) was decreased by CsA, but not by FK506. However, both CnI did not affect cellular cAMP levels. We show that CsA decreases transcription of AQP2, a process that is in part independent of Cn or cyclophilin A and suggests dependence on decreased activity of CREB.

Clinical efficacy of dissolvable microneedles armed with anti-melanogenic compounds to counter hyperpigmentation.

BACKGROUND: Hyperpigmentation is a complex physiological process associated with alterations of skin color due to melanin overproduction and distribution. Both intrinsic and extrinsic factors influence discoloration with the involvement of multiple pathways in cooperative manners. Restoring natural skin color requires a multi-targeted approach. Latest advances in anti-melanogenic compounds and microneedle delivery system have presented opportunities for tackling hyperpigmentationsuccessfully. AIMS: This work was aimed at assessing the dermal tolerability and efficacy of hyaluronic acid-based microneedle patches loaded with anti-melanogenic actives for improvements of skin discoloration. PATIENTS/METHODS: The test products consist of hyaluronic acid with niacinamide, ascorbic acid 2-glucoside, tranexamic acid, resveratrol, 4-n-butyl-resorcinol, and Halidrys siliquosa extract. In a monocentric 12 weeks clinical trial, the HA-MNs patches were applied to the affected areas of the faces on subjects with hyperpigmented skin. The color properties of the skin were analyzed spectrophotometrically. RESULTS: The products were tolerated remarkably; none of the subjects informed any primary or cumulative skin responses. Evaluation of color related measurable skin properties provided insight into the general effectiveness: The individual topology measurements showed 51.4% noticeable improvements in hyperpigmented zones, which were also in good agreement with the personal impressions of the subjects determined by questionnaires before and after the treatments. CONCLUSIONS: The concentrated blends of actives in microneedle patches act in a multi-targeted manner, and they could be worked in a complementary fashion for the improvement of skin color and appearance. The study has established the overall convenience of the HA-MNs with the formulation of anti-melogenic compounds against skin discoloration.

Cyclosporin-A induced toxicity in rat renal collecting duct cells: interference with enhanced hypertonicity induced apoptosis.

BACKGROUND/AIMS: Rat renal inner medullary collecting duct (IMCD) cells are physiologically exposed to a wide range of ambient tonicity. To maintain their function upon changes in osmolality, IMCD cells induce expression of osmoprotective and antiapoptotic genes, mainly mediated by the transcription factor Tonicity Enhancer Binding Protein (TonEBP). Some drugs like Cyclosporin-A (CsA) are discussed to interfere with the activity of TonEBP and thereby mediate their nephrotoxic effects. The aim of our study was to further understand CsA toxicity during elevation of ambient osmolality. METHODS: First we examined cytotoxicity of CsA in IMCD exposed to elevated tonicity. Employing microarray analysis of gene expression, real-time PCR and immunoassays, we scrutinized pathways contributing to this effect. RESULTS: We show that in IMCD cells CsA but not FK506 increases apoptosis upon an increase in tonicity. This effect is independent of cellular TonEBP localization or activity and reactive oxygen species. Microarray studies revealed marked quantitative differences in gene expression. Functional analysis showed overrepresentation of genes associated with cell death in presence of CsA. This correlated with increased mRNA expression of genes associated with the death receptor pathway and detection of TNFα in culture medium of cells treated with CsA. CONCLUSION: Our results show that CsA cytotoxicity is induced under elevated ambient osmolality and that death receptor signaling probably contributes to CsA cytotoxicity.

Efficacy of bioactive peptides loaded on hyaluronic acid microneedle patches: A monocentric clinical study.

BACKGROUND: Aging skin is a gradual physiological process associated with functional and structural changes of the skin. Both intrinsic and extrinsic factors influence skin aging by the involvement of multiple pathways. Restoring natural skin conditions requires a multi-targeted approach. Recent developments in both bioactive peptides and microneedle delivery system have presented an opportunity for tackling premature skin aging. AIMS: This study was aimed at evaluating the dermal tolerability and efficacy of hyaluronic acid-based microneedle patches loaded with bioactives for restoration of the skin properties including hydration, wrinkle reduction, density, and thickness. PATIENTS/METHODS: The test product of HA-MNs comprises arginine/lysine polypeptide, acetyl octapeptide-3, palmitoyl tripeptide-5, adenosine, and seaweed extracts. In the monocentric 12-week clinical trial, the HA-MNs patches were applied to the outer corner of the right and left eye and a defined area on the volar forearm on healthy subjects with aged skin. Instrumental analysis of skin properties was determined. RESULTS: The product was tolerated excellently; none of the subjects reported any primary or cumulative skin reactions. Assessment of measurable skin properties provides insight into the general effectiveness: The fine lines/wrinkles showed 25.8% noticeable decrease; the skin hydration measurements demonstrated 15.4% improvement; the skin density and thickness in the dermis increased 14.2% and 12.9%, respectively. CONCLUSIONS: The composition of the microneedle patches works in a multi-targeted manner and all ingredients might possibly be acted synergistically for the improvement of skin structure, function, and appearance. The study has demonstrated the overall usefulness of the HA-MNs with careful formulation for skin care applications.

Atrial natriuretic peptide and nitric oxide signaling antagonizes vasopressin-mediated water permeability in inner medullary collecting duct cells.

AVP and atrial natriuretic peptide (ANP) have opposite effects in the kidney. AVP induces antidiuresis by insertion of aquaporin-2 (AQP2) water channels into the plasma membrane of collecting duct principal cells. ANP acts as a diuretic factor. An ANP- and nitric oxide (NO)/soluble guanylate cyclase (sGC)-induced insertion of AQP2 into the plasma membrane is reported from different models. However, functional data on the insertion of AQP2 is missing. We used primary cultured inner medullary collecting duct (IMCD) cells and digital holographic microscopy, calcein-quenching measurements, and immunofluorescence and Western blotting to analyze the effects of ANP and NO donors on AQP2 phosphorylation, membrane expression, and water permeability. While AVP led to acceleration in osmotically induced swelling, ANP had no effect. However, in AVP-pretreated cells ANP significantly decreased the kinetics of cell swelling. This effect was mimicked by 8-bromo-cGMP and blunted by PKG inhibition. Stimulation of the NO/sGC pathway or direct activation of sGC with BAY 58-2667 had similar effects to ANP. In cells treated with AVP, AQP2 was predominantly localized in the plasma membrane, and after additional incubation with ANP AQP2 was mostly localized in the cytosol, indicating an increased retrieval of AQP2 from the plasma membrane by ANP. Western blot analysis showed that ANP was able to reduce AVP-induced phosphorylation of AQP2 at position S256. In conclusion, we show that the diuretic action of ANP or NO in the IMCD involves a decreased localization of AQP2 in the plasma membrane which is mediated by cGMP and PKG.

Translocation of aquaporin-containing vesicles to the plasma membrane is facilitated by actomyosin relaxation.

Docking and fusion of vesicles to the plasma membrane is a fundamental process in living cells. An established model for the trafficking of vesicles is based on primary epithelial cells from the collecting duct of the nephron. Upon stimulation with the signaling peptide arginine-vasopressin (AVP), aquaporin-containing vesicles are directed to the plasma membrane. Since aquaporin selectively enhances the water permeability of plasma membranes, this process helps to balance the water content of the organism. A mechanism has been suggested involving local depolymerization of F-actin to facilitate the movement of vesicles to the membrane. Since F-actin is the major component of cytoskeletal restoring forces, AVP-stimulated cells can be expected to lose rigidity. Here, we used atomic force microscopy force mapping to test whether AVP alters cell stiffness. The Young's modulus of living epithelial cells at 37 degrees C was continuously monitored, yielding a 51% decrease of Young's modulus after the addition of AVP. The data demonstrate that not the depolymerization of actin but a relaxation of actomyosin interaction facilitates vesicle translocation.