Background: Sodium-glucose cotransporter-2 inhibitors (SGLT-2i's) improve heart failure (HF) related outcomes. The mechanisms underlying these benefits are not well understood, but diuretic properties may contribute. Traditional diuretics, such as furosemide, induce substantial neurohormonal activation contributing to the limited improvement in intravascular volume often seen with these agents. However, the proximal tubular site of action of the SGLT-2i's may help circumvent these limitations. Methods: 20 patients with type-2 diabetes and chronic, stable HF completed a randomized placebo-controlled crossover study of empagliflozin 10mg daily vs. placebo. Patients underwent an intensive 6-hour biospecimen collection and cardio-renal phenotyping at baseline and again after 14 days of study drug. After a 2-week washout, patients crossed over to the alternate therapy with repeat of the above protocol. Results: Oral empagliflozin was rapidly absorbed as evidenced by a 27-fold increase in urinary glucose excretion by 3 hours (p<0.0001). Fractional excretion of sodium (FENa) increased significantly with empagliflozin monotherapy vs. placebo (FENa 1.2 ± 0.7% vs. 0.7 ± 0.4% p=0.001) and there was a synergistic effect in combination with bumetanide (FENa 5.8 ± 2.5% vs. 3.9 ± 1.9%, p=0.001). At 14 days, the natriuretic effect of empagliflozin persisted, resulting in a reduction in blood volume (-208mL, IQR -536 to 153 mL vs -14mL, IQR -282 to 335 mL, p=0.035), and plasma volume (-138mL, IQR -379 to 154mL ± 453 mL, p=0.04). This natriuresis was not, however, associated with evidence of neurohormonal activation as change in norepinephrine was superior (p = 0.02) and all other neurohormones similar (p<0.34) during the empagliflozin vs. placebo period. Furthermore, there was no evidence of potassium wasting (p=0.20), or renal dysfunction (p>0.11 for all biomarkers), whereas both serum magnesium (p<0.001) and uric acid levels (p=0.008) improved. Conclusions: Empagliflozin causes significant natriuresis, particularly when combined with loop diuretics, resulting in an improvement in blood volume. However, off-target electrolyte wasting, renal dysfunction, and neurohormonal activation were not observed. This favorable diuretic profile may offer significant advantage in the management of volume status in HF patients and may represent a mechanism contributing to the superior long-term HF outcomes observed with these agents. Clinical Trial Registration: URL: https://clinicaltrials.gov Unique Identifier: NCT03029760

DNA nonhomologous end-joining (NHEJ) and homologous recombination are two distinct pathways of DNA double-strand break repair in mammalian cells. Biochemical and genetic studies showed that DNA ends can also be joined via microhomology-mediated end joining (MHEJ), especially when proteins responsible for NHEJ, such as Ku, are reduced or absent. While it has been known that Ku-dependent NHEJ requires DNA ligase IV, it is unclear which DNA ligase(s) is required for Ku-independent MHEJ. In this study, we used a cell-free assay to determine the roles of DNA ligases I, III and IV in MHEJ and NHEJ. We found that siRNA mediated down-regulation of DNA ligase I or ligase III in human HTD114 cells led to impaired end joining that was mediated by 2-, 3- or 10-bp microhomology. In addition, nuclear extract from human fibroblasts harboring a mutation in DNA ligase I displayed reduced MHEJ activity. Furthermore, treatment of HTD114 nuclear extracts with an antibody against DNA ligase I or III also significantly reduced MHEJ. These data indicate that DNA ligases I and III are required in MHEJ. DNA ligase IV, on the contrary, is not required in MHEJ but facilitates Ku-dependent NHEJ. Therefore, MHEJ and NHEJ require different DNA ligases.

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