Effects of Tolvaptan on Oxidative Stress in ADPKD: A Molecular Biological Approach

Autosomal dominant polycystic disease (ADPKD) is the most frequent monogenic kidney disease. It causes progressive renal failure, endothelial dysfunction, and hypertension, all of which are strictly linked to oxidative stress (OxSt). Treatment with tolvaptan is known to slow the renal deterioration rate, but not all the molecular mechanisms involved in this effect are well-established. We evaluated the OxSt state in untreated ADPKD patients compared to that in tolvaptan-treated ADPKD patients and healthy subjects. OxSt was assessed in nine patients for each group in terms of mononuclear cell p22phox protein expression, NADPH oxidase key subunit, MYPT-1 phosphorylation state, marker of Rho kinase activity (Western blot) and heme oxygenase (HO)-1, induced and protective against OxSt (ELISA). p22phox protein expression was higher in untreated ADPKD patients compared to treated patients and controls: 1.42 ± 0.11 vs. 0.86 ± 0.15 d.u., p = 0.015, vs. 0.53 ± 0.11 d.u., p < 0.001, respectively. The same was observed for phosphorylated MYPT-1: 0.96 ± 0.28 vs. 0.68 ± 0.09 d.u., p = 0.013 and vs. 0.47 ± 0.13 d.u., p < 0.001, respectively, while the HO-1 expression of untreated patients was significantly lower compared to that of treated patients and controls: 5.33 ± 3.34 vs. 2.08 ± 0.79 ng/mL, p = 0.012, vs. 1.97 ± 1.22 ng/mL, p = 0.012, respectively. Tolvaptan-treated ADPKD patients have reduced OxSt levels compared to untreated patients. This effect may contribute to the slowing of renal function loss observed with tolvaptan treatment.

Functional Validation of cas9/guideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8′-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.

Functional Validation of cas9/guideRNA Constructs for Site-directed Mutagenesis of Triticale ABA8’OH1 loci

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like Triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in Triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the Triticale ABA 8&rsquo;-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in Triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in Triticale genes.