Role of Canagliflozin on function of CD34+ve endothelial progenitor cells (EPC) in patients with type 2 diabetes

Abstract

Background: Endothelial progenitor cells (EPCs) has been shown to be dysfunctional in both type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD) leading to poor regeneration of endothelium and renal perfusion. EPCs have been shown to be a robust cardiovascular disease (CVD) risk indicator. Effect of sodium glucose channel inhibitors (SGLT2i) such as Canagliflozin (CG) on a cellular biomarker such as CD34+ve progenitor cells, which may help predict CVD risk, in patients with T2DM with established CKD has not been explored.

Methods: This is a pilot study where 29 subjects taking metformin and/or Insulin were enrolled in a 16 week, double blind, randomized placebo matched trial, with a low dose 100 mg CG as the intervention group compared to matched placebo. Type 2 diabetes subjects (30-70 years old), with hemoglobin A1c (HbA1c) of 7-10%, were enrolled. CD34+ve cell number, migratory function, gene expression along with vascular parameters such as arterial stiffness, serum biochemistry pertaining to cardio-metabolic health, resting energy expenditure and body composition were measured. Data were collected at week 0, 8 and 16. A mixed model regression analysis was done and p value less than 0.05 was considered statistically significant.

Results: A significant expression of CXCR4 receptor with a concomittant increase in migratory function of CD34+ve cells was observed in CG treated group as compared to placebo group. Gene expression analysis of CD34+ve cells showed an increase in expression of antioxidants (superoxide dismutase 2 or SOD2, Catalase and Glutathione Peroxidase or GPX) and notable endothelial markers (PECAM1, VEGF-A, and NOS3). A significant reduction in glucose and HbA1c levels were observed along with improved systolic and diastolic blood pressure in the CG group. A significant increase in adiponectin (p = 0.006) was also noted in treatment group. Urinary exosomal protein leak in urine, examining podocyte health (podocalyxin, Wilm's tumor and nephrin) showed reduction with CG CONCLUSION: Low dose Canagliflozin has a beneficial effect on CD34+ cell function, serum biochemistry and urinary podocyte specific exosomes in type 2 diabetes.

Keywords: Canagliflozin; Endothelial function; Progenitor cells; Type 2 diabetes; Urinary exosomes.

Fig. 1

A significant difference in mean systolic blood pressure (a) and diastolic blood pressure (b) is observed between Canagliflozin and placebo groups. In Canagliflozin group both decreased from visit 1 to 2 and increased by visit 3. Whereas in placebo group both increased from visit 1 to 2 and decreased by visit 3. c HbA1c went down substantially in Canagliflozin group whereas it increased in Placebo group. The sharp decrease in HbA1c is seen in visit 1to visit 2 (p = 0.0919) and is stabilized from visit 2 to visit 3 in Canagliflozin group. d Glucose levels also followed the same trend as HbA1c in Canagliflozin group by sharp decrease from visit 1 to 2 and then stabilized till visit 3. Whereas, in placebo group glucose levels increased from visit 1 to 3. e A significant rise in adiponectin levels is noted in Canagliflozin group where as a sharp decrease is observed in placebo group from visit 2 to 3. f Inflammatory marker serum IL6 levels decreased. The difference was significant from visit 2 to 3 (p = 0.0259) in Canagliflozin group as compared to placebo group

Fig. 2

Migration Assay in response to SDF1alpha at 10 ng/ml concentration and gene expression of SDF1a receptor, CXCR-4 on CD34+ve cells. a Mean migration of CD34+ve cells towards SDF1-α (at 10 ng/ml) increased from visit 1 to 3 in Canagliflozin group as compared to placebo group (for visit 1 to 3 p = 0.03 and for visit 2–3 p = 0.05). b CXCR4 gene expression on CD34 positive cells is increased (p = 0.06) in Canagliflozin group as compared to placebo group at visit 3

Fig. 3

The effect of Canagliflozin on CD34+ve cell antioxidants gene expression. Fold change in gene expression of antioxidants SOD2 (a) in CD34+ve cells are increased in Canagliflozin group from visit 1 to 3. However, the same for the placebo group is decreased. Whereas the antioxidants, b Catalase (CAT) and c GPX3 gene expression increased in CD34+ve cells in Canagliflozin group from visit 2 to 3. However, the same for the placebo group is decreased. Overall, all the antioxidant genes showed increased expression going from visit 2 to visit 3, whereas the placebo there was a downward trend with catalase expression reaching statistical difference, overall

Fig. 4

The effect of Canagliflozin on CD34+ve cell Endothelial markers gene expression. Fold change in gene expression of Endothelial markers a VEGF-A, b KDR and c PECAM and endothelial function marker d NOS3 on CD34+ve cells increased significantly in Canagliflozin group from visit 1 to 3. However, the same for the placebo group is decreased. Gene expression is normalized to 18S

Fig. 5

Urinary exosome markers a Wilm’s tumor-1 (WT-1), b Nephrin and c Podocalyxin (PODXL) were identified by Western blot and followed by quantification. A trend in reduced mean band intensities for Urinary exosome markers are observed in Canagliflozin group from visit 1 to visit 3 as compared to placebo group