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

TRPM4 promotes cellular contractility and wound healing.

<p>A) Three-dimensional (3D) invasion assay of TREx293-TRPM4 cells. TRPM4 expression was induced by adding 1 μg/mL Tetracycline to the media (n = 3, p<0.05 compared to control). *, significant difference (p<0.05) versus non-stimulated cell control. Statistical analysis was performed using a Mann-Whitney test. B) Fibroblasts migration from skin grafts (E) treated with DMSO or 20 μM 9-phenanthrol. Grafts were fixed 5 days after explant and labeled with Hoechst (blue). Scale bar: 1 mm. C) Quantification of the experiment shown in (B). The data correspond to cell counts from 3 independent experiments (13 and 12 explants for DMSO and 9-phenanthrol treatments, respectively). *, significant difference (p<0.05) versus DMSO controls. Statistical analysis was performed using a Mann-Whitney test. D) Three dimensional contraction assay of MEFs transfected with shRNA^Scramble and shRNA^TRPM4. Contraction was induced by incubating the immersed cells with 10% v/v serum for 48 h. The upper graph represents the collected data for 4 independent assays. *, significant difference (p<0.05) versus shRNA^Scramble controls. **, significant difference (p<0.05) versus shRNA^TRPM4. Statistical analysis was performed using a two-way ANOVA test. E) Frames (t = 0 and 14 min) from time lapse of untreated, DMSO and 9-phenanthrol treated wounds in zebrafish tails. Scale bar: 1 mm. F) Quantification of the wound closure experiments from (E) (n = 10 larvae per condition). Statistical analysis was performed using a two-way ANOVA test. G) Excisional cutaneous wounds were created using a 3 mm biopsy punch. Images from the time course of wound closure in the presence of DMSO (control) and 9-phenanthrol (n = 5 mice). Scale bar: 1.5 mm. H) Wound closure was monitored measuring the area of the wound on the indicated days post-wounding. Statistical analysis was performed using a two-way ANOVA test. I) Images of skin wounds at 3 days post-wounding. Bottom panels show magnifications of the areas marked in the upper panels. Arrowheads mark the epithelial tissue. Scale bar: 50 μm. J) Images of wounds at 5 days post-wounding. The dashed lines indicate the limit of the wound area. Scale bar: 500 μm.</p

TRPM4 regulates the number and size of focal adhesions.

<p>A) Representative immunoblot from MEFs subjected to shRNA-mediated TRPM4 knock down. Membranes were incubated with mouse anti-TRPM4 mAb, and anti-Grp75 mAb as a loading control. B) Graph of the densitometric analyses of three independent immunoblot experiments. Statistical analysis was performed using a Mann Whitney test. C) Immunofluorescence labeling of MEFs transfected with shRNA^Scramble, and shRNA^TRPM4. Cells were labeled with Hoechst (blue), and mouse anti-TRPM4 mAb (green); tRFP (red) was used as transfection marker. Scale bar corresponds to 10 μm. D) Immunofluorescence labeling of MEFs transfected with shRNA^Scramble and shRNA^TRPM4. Cells were labeled with Hoechst (blue), and mouse anti-vinculin mAb (green); tRFP (red) was used as transfection marker. Scale bar corresponds to 10 μm. Quantification of FA number (E) and areas (F) from the shRNA-transfected cells (n = 15 cells for shRNA^Scramble and n = 15 cells for shRNA^TRPM4 from 7 independent experiments). G) Immunofluorescence labeling of MEFs treated with DMSO (0.1% v/v) and 10 μM 9-phenanthrol. Cells were labeled with Hoechst (blue) and mouse anti-vinculin mAb (green). Scale bar corresponds to 10 μm. Graphs of FA number (H) and areas (I) are shown (20 cells per condition, n = 3, p<0.05). The number and areas of the FAs were analyzed using NIH/ImageJ software. The graphs correspond to mean ± standard deviation. Statistical analysis was performed using a Mann-Whitney test.</p

TRPM4 controls cellular migration <i>via</i> Rac1 GTPase activity.

<p>A) MEFs were incubated with DMSO (0.1% v/v final) vehicle or 10 μM 9-phenanthrol for 16 h. F-actin was labeled with the F-actin stain Alexa 555 phalloidin (red) and Hoechst (blue). B) Quantified results from A. The percentage of wound closure is expressed as mean ± standard deviation; s.d. (n = 3, p<0.05). *, significant difference (p<0.05) versus DMSO controls. Statistical analysis was performed using a Mann-Whitney test. C) Graph from Transwell Boyden chamber migration assays of MEFs transfected with shRNA^Scramble and shRNA^TRPM4. Cells were stimulated with 10% v/v serum for 16 h. The bars represent the mean ± s.d. (n = 3; p<0.05 compared to control). *, significant difference (p<0.05) versus shRNA^Scramble controls. Statistical analysis was performed using a two-way ANOVA test. D) Graph from Transwell Boyden chamber migration assays of MEFs transfected with shRNA^Scramble and shRNA^TRPM4 and coexpressing Rac1(Q61L). Cells were stimulated with 10% v/v serum for 16 h. The bars represent the mean ± s.d. (n = 3; p<0.05 compared to control). *, significant difference (p<0.05) versus shRNA^Scramble/Mock controls. **, significant difference (p<0.05) versus shRNA^TRPM4/Mock controls. Statistical analysis was performed using a two-way ANOVA test.</p

TRPM4 localizes to focal adhesions.

<p>A) Classification of putative TRPM4-associated proteins identified by LC-MS/MS. B) FA-related proteins identified as putative TRPM4 interacting proteins. C) TRPM4 localizes to FAs in MEFs. Cells were labeled with Hoechst (blue), mouse anti-TRPM4 mAb (green), and tRFP (red). D) Magnification of the section from C. E) TRPM4 (green) colocalizes with Focal Adhesion Kinase (FAK, magenta) in MEFs. F) Amplification of the region marked in E. G) Biochemical isolation of FA complexes (FA) from MEFs. The cell body fraction is labeled as CB. TRPM4 localizes to FAs in mouse pancreatic (H) and skin fibroblasts (I); Human Umbilical Vein Endothelial Cells (HUVEC) (J) and astrocytes (K). Scale bar: 5 μm.</p

TRPM4 regulates FA turnover.

<p>(A) Representative time-lapse imaging from MEFs coexpressing shRNA^Scramble or shRNA^TRPM4 with EGFP-Paxillin. Assembling FAs are marked with arrows. Arrowheads mark FAs undergoing turnover. Scale bar: 5 μm. Quantification of assembly (B) and disassembly rates (C) of FAs from live-cell time-lapse recordings of EGFP-Paxillin transfected MEFs. Statistical analysis was performed using a Mann-Whitney test from 7 independent experiments.</p