Fig 1.
Study protocols and validation of rat urinary exosomes.
(A) Schematic of the experimental protocols, (B) Representative electron microscope images of exosomes vesicles isolated from 18 hours rat urine at X10,000 magnification. The images shows small, round vesicles of size between 30-120nm. Below is the representative immunoblots for TSG101 proteins in urinary exosome samples from, untreated diabetic (DM), non-diabetic control (CTRL) and insulin treated diabetic rats (DM + INS). Exosome pellet from urine samples from all rats were positive for TSG101 protein, an exosomal marker protein.
Fig 2.
Validation of diabetes induction in rats.
(A) Line diagram showing mean blood glucose, and (B-C) 18-hour urine volume and water intake in untreated diabetic (DM, n = 10), non-diabetic control (CTRL, n = 6) and insulin treated diabetic rats (DM + INS, n = 6) at following weeks during the course of the study 3rd, 6th and 9th weeks post-injections *p≤0.05 versus DM+ INS and δ p≤0.05 versus CTRL by One-Way ANOVA followed by pair wise multiple comparison (at each time point) testing (n = 6-10/group).
Fig 3.
Urine albumin excretion in rats.
Line plot showing average urine albumin excretion (mg of albumin in 18 hours urine collected from untreated diabetic (DM, n = 10), non-diabetic control (CTRL, n = 6) and insulin treated diabetic rats (DM + INS, n = 6) at following weeks during the course of the study (3rd, 6th and 9th weeks post-injections. #p ≤ 0.05 as compared to 3rd week by unpaired t-test (n = 10/time point). *p≤0.05 versus DM+ INS and δ p≤0.05 versus CTRL by One-Way ANOVA followed by pair wise multiple comparison (at each time point) testing (n = 6-10/group).
Fig 4.
Kidney tissue pathology in rats.
Representative (A) PAS and (C) MT stained images of kidney tissue sections from untreated diabetic (DM), non-diabetic control (CTRL) and insulin treated diabetic rats (DM + INS) after 3rd, 6th and 9th weeks of diabetes induction (n = 3/group/time point), Bar graph showing a semi-quantitative analysis for (B) glomerulosclerotic index (GI) and (D) tubulointerstitial fibrosis index (TFI) kidneys tissues from these (n = 3/group/time point). #p≤0.05 as compared to 3rd week within the group by unpaired t-test, (n = 3/time point). *p≤0.05 versus DM+ INS and δ p≤0.05 versus CTRL by One-Way ANOVA followed by pair wise multiple comparison (at each time point) testing (n = 3/group).
Fig 5.
Change in Urinary exosomal miRNA-451-5p and miR-16 levels during the course of the study in rats.
(A) Line diagram showing average fold expression of (A) miR-451-5p and (B) miR-16 in urinary exosomes from untreated diabetic (DM, n = 10), non-diabetic control (CTRL, n = 6) and insulin treated diabetic rats (DM + INS, n = 6) at 3rd, 6th and 9th weeks post-injection. Fold expression was calculated using the 2- ΔCT method, where ΔCT = CTmiRNA—CTU6snRNA. The values were log transformed for analysis. #p≤0.05 versus week 3 by paired t-test, (n = 6-10/time point). (C) Scatter plot with regression fit line showing the relationship between miR-451-5p at 6th week and urine albumin at 9th week.
Fig 6.
Kidney tissue levels of miR-451-5p and miR-16 and their association with renal pathology at the end of the study.
Bar graphs showing (A) average fold expression of miR-451-5p and miR-16expression, fold expression was calculated using the 2- ΔCT method, where ΔCT = CTmiRNA—CTU6snRNA; and (B) semi-quantitative analysis of pathology for glomerulosclerotic index (GI) and tubulointerstitial fibrosis index (TFI) in kidney-tissue from untreated diabetic (DM), non-diabetic control (CTRL) and insulin treated diabetic rats (DM + INS) at 10th week of the study 1 (n = 6-10/group). (C) Scatter plot with regression fit line showing the relationship between miR-451-5p and miR-16 expression with TFI and GI scores. *p≤0.05 versus DM+ INS and δ p≤0.05 versus CTRL by One-Way ANOVA followed by pair wise multiple comparison testing (n = 6-10/time point).
Fig 7.
Kidney tissue levels of IL-6 and MMP-9 in rats.
Bar graphs showing average fold expression of (A) IL-6 and (B) MMP-9 in kidney-tissue from untreated diabetic (DM), non-diabetic control (CTRL) and insulin treated diabetic rats (DM + INS) at 10th week of the study 1 (n = 6-10/group). *p≤0.05 versus DM+ INS and δ p≤0.05 versus CTRL by One-Way ANOVA followed by pair wise multiple comparison testing (n = 6-10/time point).