Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Two warm Neptunes transiting HIP 9618 revealed by TESS and Cheops

peer reviewedHIP 9618 (HD 12572, TOI-1471, TIC 306263608) is a bright (G = 9.0 mag) solar analogue. TESS photometry revealed the star to have two candidate planets with radii of 3.9 ± 0.044 R (HIP 9618 b) and 3.343 ± 0.039 R (HIP 9618 c). While the 20.77291 d period of HIP 9618 b was measured unambiguously, HIP 9618 c showed only two transits separated by a 680-d gap in the time series, leaving many possibilities for the period. To solve this issue, CHEOPS performed targeted photometry of period aliases to attempt to recover the true period of planet c, and successfully determined the true period to be 52.56349 d. High-resolution spectroscopy with HARPS-N, SOPHIE, and CAFE revealed a mass of 10.0 ± 3.1M for HIP 9618 b, which, according to our interior structure models, corresponds to a 6.8 ± 1.4 per cent gas fraction. HIP 9618 c appears to have a lower mass than HIP 9618 b, with a 3-sigma upper limit of 50 d, opening the door for the atmospheric characterization of warm (Teq < 750 K) sub-Neptunes

Regulatory Effects of Sestrin 3 (SESN3) in BCR-ABL Expressing Cells

<div><p>Chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) are characterized by the presence of the BCR-ABL oncoprotein, which leads to activation of a plethora of pro-mitogenic and pro-survival pathways, including the mTOR signaling cascade. We provide evidence that in BCR-ABL expressing cells, treatment with tyrosine kinase inhibitors (TKIs) results in upregulation of mRNA levels and protein expression of sestrin3 (SESN3), a unique cellular inhibitor of mTOR complex 1 (mTORC1). Such upregulation appears to be mediated by regulatory effects on mTOR, as catalytic inhibition of the mTOR kinase also induces SESN3. Catalytic mTOR inhibition also results in upregulation of SESN3 expression in cells harboring the TKI-insensitive T315I-BCR-ABL mutant, which is resistant to imatinib mesylate. Overexpression of SESN3 results in inhibitory effects on different Ph+ leukemic cell lines including KT-1-derived leukemic precursors, indicating that SESN3 mediates anti-leukemic responses in Ph+ cells. Altogether, our findings suggest the existence of a novel mechanism for the generation of antileukemic responses in CML cells, involving upregulation of SESN3 expression.</p></div

Differential effects of SESN3 and SESN2 overexpression on mTOR and MAPK signaling effectors.

<p><b>A</b>. KT-1 cells were transiently nucleofected with either empty vector (E.V.) or SESN3 expressing plasmid and were analyzed for the presence of ROS by flow cytometry, following 30 minutes of staining with DCFDA at the time-points indicated. Data are as percent control empty vector for each time-point and represent means ± SE of 3 independent experiments. <b>B</b>. KT1 cells were transiently nucleofected with either empty vector (E.V.) or SESN2 expressing plasmid. Cells were lysed 48 hours post-nucleofection Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN2 or GAPDH as indicated. <b>C</b>. KT1 cells were transiently nucleofected with either empty vector (E.V.) or SESN3 expressing plasmid. Cells were lysed 24 hours post-nucleofection Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. <b>D</b>. KT-1 cells were transiently nucleofected with either empty vector (E.V.) or SESN3 expressing plasmid. Expression of mTOR was quantified at 24 hours post-nucleofection either by quantitative RT-PCR. Data are expressed as fold increase in the Sesn3-nucleofected samples over E.V.-nucleofected samples normalized to GAPDH and represent means ± S.E. of 3 independent experiments. <b>E</b>. KT1 cells were transiently nucleofected with either empty vector (E.V.), SESN2 or SESN3 expressing plasmids. Cells were lysed 48 hours post-nucleofection and equal amounts of protein from cell lysates from the same experiment for each panel were resolved separately by SDS-PAGE and immunoblotted with the indicated antibodies. <b>F–G</b>. KT1 cells were transiently nucleofected with either empty vector (E.V.), SESN2 or SESN3 expressing plasmids. Cells were lysed 48 hours post-nucleofection and equal amounts of protein were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Blots were subsequently stripped and reprobed with the respective antibodies against the total form of the protein. Densitometry analysis of the representative blots is shown.</p

Inhibitory effects of SESN3 but not SESN2 on primitive BCR-ABL expressing leukemic progenitors.

<p><b>A</b>. KT-1 cells were transiently nucleofected with either empty vector (E.V.) or a SESN3 expressing plasmid. Levels of SESN3 were quantified at 24 hours post-nucleofection by immunoblotting. <b>B</b>. KT-1 cells transiently nucleofected with either empty vector (E.V.) or SESN3 were plated in methylcellulose 24 hours post-nucleofection. Leukemic CFU-L colonies were allowed to develop in clonogenic assays in methylcellulose and scored on day 6. Data are expressed as percentage of control untreated colonies and represent means ± S.E. of 5 independent experiments. <b>C</b>. KT-1 cells were transiently nucleofected with either empty vector (E.V.) or SESN2 expressing plasmid. Levels of SESN2 were quantified at 24 hours post-nucleofection by immunoblotting. <b>D</b>. KT-1 cells transiently nucleofected with either empty vector (E.V.) or SESN2 expressing plasmid were incubated in clonogenic assays in methylcellulose. Leukemic CFU-L colonies were scored on day 6 and data are expressed as percentage of control untreated colonies and represent means ± S.E. of 4 independent experiments. <b>E</b>. BV173R cells were transiently nucleofected with either empty vector (E.V.) or SESN2 or SESN3 expressing plasmid. 48 hours post-transfection, equal number of cells were plated and allowed to proliferate for 120 hours. Proliferation was measured by WST-1 assay at the indicated times. Data are expressed as the absorbance at 450 nm and represent means ± S.E. from 3 independent experiments, * p = 0.0018 comparing SESN3 nucleofected cells vs. E.V. nucleofected cells on day 4, ** p = 0.0068 comparing SESN3 nucleofected cells vs. E.V. nucleofected on day 5.</p

Induction of SESN3 mRNA expression by ponatinib in T315I-BCR-ABL expressing cells.

<p><b>A</b>. Ba/F3 cells stably transfected with T315I-BCR-ABL were treated with ponatinib (10 nM) or imatinib (1 µM) for 12 hours as indicated. RNA was extracted and expression of SESN3 mRNA was determined by quantitative RT-PCR, using β-actin for normalization. Data are expressed as fold increase in the treated samples over untreated samples and represent means ± S.E. of 3 independent experiments. <b>B</b>. BV173R cells were treated with ponatinib (100 nM) for 12 hours. RNA was extracted and expression of SESN3 mRNA was determined by quantitative RT-PCR, using GAPDH for normalization. Data are expressed as fold increase in the treated samples over untreated samples and represent means ± S.E. of 4 independent experiments.</p

mTOR inhibition but not BCR-ABL inhibition upregulates SESN3 in T315I-BCR-ABL expressing cells.

<p><b>A</b>. BV173 or BV173R cells were treated with imatinib mesylate (5 µM) for 12 hours. RNA was extracted and expression of SESN3 mRNA was determined by quantitative RT-PCR, using GAPDH for normalization. Data are expressed as fold increase in the treated samples over untreated samples and represent means ± S.E. of 3 independent experiments. <b>B</b>. BV173 or BV173R cells were treated with either imatinib (5 µM) or nilotinib (100 nM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. <b>C</b>. Ba/F3 cells stably transfected with WT-BCR-ABL or T315I-BCR-ABL were treated with either imatinib (1 µM) or nilotinib (100 nM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. <b>D</b>. BV173 and BV173R cells were treated with OSI-027 (5 µM) for 12 hours. RNA was extracted and expression of SESN3 mRNA was determined by quantitative RT-PCR, using GAPDH for normalization. Data are expressed as fold increase in the treated samples over untreated samples and represent means ± S.E. of 3 independent experiments. <b>E</b>. BV173 or BV173R cells were treated with OSI-027 (5 µM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. <b>F</b>. Ba/F3 cells stably transfected with WT-BCR-ABL or T315I-BCR-ABL were treated with OSI-027 (5 µM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated.</p