Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Mismatch dependent uracil/thymine-DNA glycosylases excise exocyclic hydroxyethano and hydroxypropano cytosine adducts

Exocyclic adducts of DNA bases, such as etheno- and hydroxyalkano- ones, are generated by a variety of bifunctional agents, including endogenously formed products of lipid peroxidation. In this work we selectively modified cytosines in the 5'-d(TTT TTT CTT TTT CTT TTT CTT TTT T)-3' oligonucleotide using: chloroacetaldehyde to obtain 3,N(4)-alpha-hydroxyethano- (HEC) and 3,N(4)-etheno- (epsilonC), acrolein to obtain 3,N(4)-alpha-hydroxypropano- (HPC) and crotonaldehyde to obtain 3,N(4)-alpha-hydroxy-gamma-methylpropano- (mHPC) adducts of cytosine. The studied adducts are alkali-labile which results in oligonucleotide strain breaks at the sites of modification upon strong base treatment. The oligonucleotides carrying adducted cytosines were studied as substrates of Escherichia coli Mug, human TDG and fission yeast Thp1p glycosylases. All the adducts studied are excised by bacterial Mug although with various efficiency: epsilonC <HEC <HPC <mHPC. The yeast enzyme excises efficiently epsilonC<HEC<HPC, whereas the human enzyme excises only epsilonC. The pH-dependence curves of excision of eC, HEC and HPC by Mug are bell shaped and the most efficient excision of adducts occurs within the pH range of 8.6-9.6. The observed increase of excision of HEC and HPC above pH 7.2 can be explained by deprotonation of these adducts, which are high pK(a) compounds and exist in a protonated form at neutrality. On the other hand, since epsilonC is in a neutral form in the pH range studied, we postulate an involvement of an additional catalytic factor. We hypothesize that the enzyme structure undergoes a pH-induced rearrangement allowing the participation of Lys68 of Mug in catalysis via a hydrogen bond interaction of its epsilon-amino group with N(4) of the cytosine exocyclic adducts

VSX2 and ASCL1 Are Indicators of Neurogenic Competence in Human Retinal Progenitor Cultures

<div><p>Three dimensional (3D) culture techniques are frequently used for CNS tissue modeling and organoid production, including generation of retina-like tissues. A proposed advantage of these 3D systems is their potential to more closely approximate <i>in vivo</i> cellular microenvironments, which could translate into improved manufacture and/or maintenance of neuronal populations. Visual System Homeobox 2 (VSX2) labels all multipotent retinal progenitor cells (RPCs) and is known to play important roles in retinal development. In contrast, the proneural transcription factor Acheate scute-like 1 (ASCL1) is expressed transiently in a subset of RPCs, but is required for the production of most retinal neurons. Therefore, we asked whether the presence of VSX2 and ASCL1 could gauge neurogenic potential in 3D retinal cultures derived from human prenatal tissue or ES cells (hESCs). Short term prenatal 3D retinal cultures displayed multiple characteristics of human RPCs (hRPCs) found <i>in situ</i>, including robust expression of VSX2. Upon initiation of hRPC differentiation, there was a small increase in co-labeling of VSX2+ cells with ASCL1, along with a modest increase in the number of PKCα+ neurons. However, 3D prenatal retinal cultures lost expression of VSX2 and ASCL1 over time while concurrently becoming refractory to neuronal differentiation. Conversely, 3D optic vesicles derived from hESCs (hESC-OVs) maintained a robust VSX2+ hRPC population that could spontaneously co-express ASCL1 and generate photoreceptors and other retinal neurons for an extended period of time. These results show that VSX2 and ASCL1 can serve as markers for neurogenic potential in cultured hRPCs. Furthermore, unlike hESC-OVs, maintenance of 3D structure does not independently convey an advantage in the culture of prenatal hRPCs, further illustrating differences in the survival and differentiation requirements of hRPCs extracted from native tissue vs. those generated entirely <i>in vitro</i>.</p></div

VSX2+ hRPCs are abundant within short and long term cultures of 3D optic vesicles derived from hESCs.

<p>20 days after initiation of retinal differentiation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135830#pone.0135830.ref020" target="_blank">20</a>], WA09 hESCs formed optic vesicle structures (OVs) comprised of VSX2+ hRPCs co-expressing (<b>A</b>) KI67, (<b>B</b>) NESTIN, and (<b>C</b>) SOX2. (<b>D-F</b>) At day 50, the majority of VSX2+ hRPCs remained KI67+. <b>(G)</b> VSX2+/KI67+ progenitors were also present at day 50 in hESC-OVs derived from the WA01 line. 50 day VSX2+ hRPCs continued to express (<b>H</b>) NESTIN and (<b>I</b>) SOX2. (<b>J</b>) At 20, 50, and 90 days of differentiation, the percentages of VSX2+ or KI67+ cells and (<b>K</b>) VSX2+ cells co-labeled with other progenitor markers were quantified. Nuclei were visualized with DAPI and cell count data is expressed as % immunopositive cells. Scale bar: 50 μm (panels A-D,H,I); 20 μm (panels E,F,G).</p

NOTCH inhibition augments production of PKCα+ neurons in short term prenatal human retinal neurospheres.

<p>The percentages of cells immunopositive for (<b>A</b>) ASCL1 and (<b>B</b>) other selected hRPC and neuronal markers following DAPT treatment was determined by immunocytochemistry. (<b>C</b>) RT-PCR analysis was used to evaluate expression of VSX2 and ASCL1 in prenatal human retinal tissue and short (1 week) and long (2 month) term prenatal retinal neurosphere cultures. (<b>D</b>) Phase photomicrographs of dissociated prenatal retinal neurospheres reveal profound cell morphology differences between short term and long term cultures. Cell counts are expressed as % immunopositive cells. *p<0.05; **p<0.01. Scale bars in panel E: 50 μm.</p

Neurogenesis decreases over time in human prenatal retinal neurosphere cultures.

<p>VSX2 immunoreactivity is not associated with (<b>A</b>) RECOVERIN+ cells in prenatal retinal tissue but does co-label a small subset of (<b>B</b>) βIII TUBULIN+ neurons. A similar pattern of VSX2 co-expression with (<b>C</b>) RECOVERIN and (<b>D</b>) βIII TUBULIN is observed in short term prenatal retinal neurosphere cultures; <i>arrow</i> in panels B and D indicate VSX+/βIII TUBULIN+ cells. (<b>E</b>) A subpopulation of PKCα+ cells also co-labels with VXS2 (<i>arrow</i> and <i>arrowhead</i> in panel E indicate VSX+/PKCα+ and VSX2-/PKCα+ cells, respectively). (<b>F</b>) The percentage of cells expressing VSX2 and selected neuronal markers were quantified in short term cultures. (<b>G</b>) Human prenatal retinal neurosphere cultures (n = 5) from day 79–108 gestation tissue were sampled at 1 week, 1 month, and 2 months, and the number of cells immunolabeled with selected neuronal markers was quantified. Nuclei were visualized with DAPI and cell count data is expressed as % immunopositive cells. ND: nondetectable. Scale bars: 50 μm (panels A,B); 20 μm (panels C-E).</p

Prenatal retinal neurospheres lose VSX2 expression over time in culture.

<p>KI67+ hRPCs in the outer neuroblastic layer of 96 day human prenatal retina co-express the neural stem cell markers (<b>A</b>) NESTIN and (<b>B</b>) SOX2. Short term prenatal retinal neurosphere cultures also contain abundant (<b>C</b>) KI67+/NESTIN+ and (<b>D</b>) KI67+/SOX2+ hRPCs. Nearly all VSX2+ hRPCs in short term prenatal retinal neurosphere cultures co-label with (<b>E</b>) NESTIN and (<b>F</b>) SOX2. Prenatal retinal neurosphere cultures (n = 5) from 79–108 day gestation tissue were sampled at 1 week, 1 month, and 2 months. After 2 months, very little VSX2 immunostaining is detected, although (<b>G</b>) NESTIN and (<b>H</b>) SOX2 remain highly expressed. The percentage of VSX2, KI67, NESTIN, and SOX2 immunopositive cells were quantified (<b>I</b>) in short term cultures and (<b>J</b>) over a 2 month period. Nuclei were visualized with DAPI and cell count data is expressed as % immunopositive cells. Scale bars: 50 μm (panels A,B); 20 μm (panels C-H).</p

Short term cultures of human retinal neurospheres retain a robust population of VSX2+ proliferating progenitor cells from source prenatal retinal tissue.

<p>VSX2+/KI67+ proliferating hRPCs were observed in the outer neuroblastic layer of the developing retina at (<b>A</b>) 59 days, (<b>B</b>) 73 days, (<b>C</b>) 85 days, (<b>D</b>) 93 days, and (<b>E</b>) 108 days of gestation. (<b>F-I</b>) VSX2+/KI67+ co-labeled cells were also present in dissociated cells from short term prenatal retinal neurospheres established from retinal tissue of similar gestational ages. (<b>J</b>) Short term prenatal retinal neurospheres were dissociated and immunostained to determine the percentage of cells expressing VSX2 and/or KI67. Nuclei were visualized with DAPI. The insert is a 4X magnification of the indicated area in panel A. Scale bars: 100 μm (panel A); 50 μm (panels B-E); 20 μm (panels F-I).</p