Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease

Background: Tubulointerstitial fibrosis represents the key underlying pathology of Chronic Kidney Disease (CKD), yet treatment options remain limited. In this study, we investigated the role of connexin43 (Cx43) hemichannel-mediated adenosine triphosphate (ATP) release in purinergic-mediated disassembly of adherens and tight junction complexes in early tubular injury. Methods: Human primary proximal tubule epithelial cells (hPTECs) and clonal tubular epithelial cells (HK2) were treated with Transforming Growth Factor Beta1 (TGFβ1) ± apyrase, or ATPγS for 48h. For inhibitor studies, cells were co-incubated with Cx43 mimetic Peptide 5, or purinergic receptor antagonists Suramin, A438079 or A804598. Immunoblotting, single-cell force spectroscopy and trans-epithelial electrical resistance assessed protein expression, cell-cell adhesion and paracellular permeability. Carboxyfluorescein uptake and biosensing measured hemichannel activity and real-time ATP release, whilst a heterozygous Cx43+/- mouse model with unilateral ureteral obstruction (UUO) assessed the role of Cx43 in vivo. Results: Immunohistochemistry of biopsy material from patients with diabetic nephropathy confirmed increased expression of purinergic receptor P2X7. TGFβ1 increased Cx43 mediated hemichannel activity and ATP release in hPTECs and HK2 cells. The cytokine reduced maximum unbinding forces and reduced cell-cell adhesion, which translated to increased paracellular permeability. Changes were reversed when cells were co-incubated with either Peptide 5 or P2-purinoceptor inhibitors. Cx43+/- mice did not exhibit protein changes associated with early tubular injury in a UUO model of fibrosis. Conclusion: Data suggest that Cx43 mediated ATP release represents an initial trigger in early tubular injury via its actions on the adherens and tight junction complex. Since Cx43 is highly expressed in nephropathy, it represents a novel target for intervention of tubulointerstitial fibrosis in CKD

In vitro propagation of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen] as affected by carbon sources

This study aimed to establish a protocol for in vitro propagation of two accessions (Ac) of Pfaffia glomerata (Ac 4 and Ac 13) and to evaluate the effect of different carbon sources on the production of 20-hydroxyecdysone (20E) in leaves and roots. For the assessment of axillary shoot proliferation in vitro, nodal segments were inoculated onto Murashige and Skoog (MS) medium supplemented with 2.22 μM 6-benzyladenine and 2.68 μM α-naphthaleneacetic acid and carbon sources (glucose or sucrose) at varying concentrations (0.1, 0.2, or 0.3 M). To assess the in vitro production of 20E, nodal segments were inoculated into Magenta® containers containing MS medium with different carbon sources (glucose, sucrose, or glucose + sucrose at 0.1 or 0.2 M) and placed in plastic bags with bacterial filters. Both experiments were composed of five repetitions for each treatment and analyzed after 30 d of culture. Multiple shoot formations were genotype-dependent when segments were cultivated on a medium supplemented with glucose or sucrose at 0.1 M, yielding 35 and 43 shoots per explant for Ac 4 and 4.4 and 2.8 shoots per explant for Ac 13, respectively. For the 20E content, significant effects were also observed among accessions and carbon sources. Ac 13 had the highest average 20E levels for both roots and leaves. Under the experimental conditions, Ac 4 had more favorable characteristics for large-scale multiplication than Ac 13, and glucose at 0.2 M was the best carbon source for the cultivation of Pfaffia, both for producing multiple shoots and for in vitro 20E production. This is the first report using a combination of auxin and cytokinin to enable effective Pfaffia in vitro axillary shoot proliferation from nodal explants