Comparison of renal function parameters between WT1 positive and WT1 negative diabetic subjects.

<p>Box plots comparing; A) Estimated GFR; B) Urine protein-to- creatinine ratio; C) Urine albumin-to-creatinine ratio; and D) serum Creatinine levels between WT1 positive and WT1 negative diabetic patients. The boxes indicate median and 25th and 75th percentiles; Outliers are indicated by closed dots. Data were compared by the Mann-Whitney U test. p<0.05 was considered significant.</p

Comparison of WT-1 expression and presence of proteinuria (ACR) in diabetic patients at various eGFR cutoffs.

<p>Bar graph showing percentage of patents detected with proteinuria or WT1 expression in urinary exosomes at various cutoff values of eGFR between 60–90 ml. min⁻¹/1.73 m²). WT-1 expression was detected in higher percentage of patients at earlier fall in GFR (eGFR<70/80/90 ml. min⁻¹/1.73 m²).</p

Detection of WT1 protein in urinary exosomes of diabetic patients with or without proteinuria.

<p>A) Representative immunoblots for WT1 and TSG101 proteins in urinary exosome samples from, type 1 diabetic patients with or without proteinuria and healthy controls. Exosomal protein obtained from same urine volume was loaded for all the samples. B) Frequency of WT1 expression in urinary exosomes from diabetic patients with or without proteinuria and healthy controls. All subjects were positive for TSG101 protein, an exosomal marker (data not shown). Densitometry analysis of WT1 bands in: C) Type-1 diabetic patients, using Mann-Whitney U test, and D) Proteinuria and Non-Proteinuria groups, using ANOVA rank test. The boxes indicate median and 25th and 75th percentiles; Outliers are indicated by closed dots. p<0.05 was considered significant.</p

Change in Urinary exosomal miRNA-451-5p and miR-16 levels during the course of the study in rats.

<p>(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 3^rd, 6^th and 9^th weeks post-injection. Fold expression was calculated using the 2^{- ΔCT} method, where ΔCT = CT_miRNA—CT_U6snRNA. 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 6^th week and urine albumin at 9^th week.</p

Validation of diabetes induction in rats.

<p>(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).</p

Study protocols and validation of rat urinary exosomes.

<p>(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.</p

Kidney tissue pathology in rats.

<p>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 3^rd, 6^th and 9^th 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 3^rd 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).</p

Kidney tissue levels of miR-451-5p and miR-16 and their association with renal pathology at the end of the study.

<p>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 = CT_miRNA—CT_U6snRNA; 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 10^th 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).</p

Kidney tissue levels of IL-6 and MMP-9 in rats.

<p>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 10^th 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).</p