MicroRNAs 1915–3p, 2861, and 4532 Are Associated with Long-Term Renal Function Decline in Type 1 Diabetes

To the Editor:

Diabetic kidney disease (DKD)¹ is the leading cause of end-stage renal disease worldwide (1). Microalbuminuria is the earliest indicator of DKD in diabetes mellitus (DM). Microalbuminuria is clinically sensitive but not specific to this disease, which in some patients progresses with rapid glomerular filtration rate (GFR) decline (2). Research on new urinary biomarkers had demonstrated the ability of microRNAs (miRNAs) to discriminate between individuals with DKD and either healthy or non-DKD individuals (3).

In an article published in Clinical Chemistry, Cardenas-Gonzalez et al. identified miRNAs miR-1915-3p, miR-2861, and miR-4532 as novel urinary miRNA biomarkers in DKD (3). Despite validating the results in different DM cohorts, the study's cross-sectional design was unable to address causality or prediction of subsequent outcomes.

To further evaluate the prognostic potential of these new biomarkers, we tested the correlation between the expression of the 3 miRNAs and long-term estimated GFR (eGFR) decline in a cohort of 113 individuals with type 1 diabetes (T1D) who were recruited from 2013 to 2017 at the Diabetes Outpatient Clinic, Hospital das Clinicas. At the time of recruitment, spot urine samples were collected in sterile RNase-free flasks, centrifuged for 10 min, 3000g at 4 °C, and frozen at −80 °C without additives or protease inhibitors within 4 h from collection.

Clinical and demographic data [sex, age, T1D duration, use of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, and glycohemoglobin (HbA_1c) values] were collected starting from the date each individual was enrolled at the clinic until 2018 (mean ± SD follow-up, 11 ± 2.9 years). Renal function evolution was assessed by all measurements of serum creatinine performed during the entire follow-up period (at least 1 per year of follow-up, median of 3 measurements per year). Individuals with eGFR decline of ≥5 mL/min/1.73 m²/year of follow-up were classified as decliners, and those with stable eGFR (change <5 mL/min/1.73 m²/year of follow-up) were classified as nondecliners (4).

Participants were mostly female (61%), diagnosed with DM at the age of 11 years (6–18) [median (25th to 75th percentiles)] with an HbA_1c of 8.3% (7.5–9.4), 23 years (17–29) of DM duration, and 76% had eGFR ≥60 mL/min/1.73 m² at the time of urine collection. There was no difference between groups regarding age at diagnosis, DM duration, and HbA_1c at the time of urine collection.

Total miRNA was isolated from 200 μL of urine using QIAzol Lysis Reagent and Serum Plasma miRNeasy Mini Kit; reverse transcription and preamplification were performed using miScript II RT Kit and miScript PreAMP PCR Kit, respectively. Real-time quantitative PCR was performed using Custom miScript miRNA PCR Array (Qiagen) in a QuantStudio7 (Life Technologies). Urinary creatinine (UCr) was measured using a creatinine colorimetric assay kit (Cayman Chemicals).

Caenorhabditis elegans miR-39, miRNA reverse transcription control, and positive PCR control were used to assess RNA isolation efficiency and reverse transcription efficiency and to test for the presence of PCR inhibitors, respectively. Raw Ct values were log2-transformed and normalized by subtracting the mean of the reverse transcription control and positive PCR control. UCr was used to normalize urine dilution (2^-dCt/UCr).

The Wilcoxon test was used in the cross-sectional analysis. Linear mixed effects models (random effects models) were used to test the association between each miRNA and the longitudinal change in eGFR during the follow-up period. Sex, DM duration, use of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, HbA_1c, and follow-up time and its interaction with the normalized expression of each miRNA were used as fixed effects. Because measurements of the same individuals were taken repeatedly through time, we used each participant's identification number as a random effect, which accounted for differences on the initial eGFR, intraindividual changes of eGFR over time, and correlation among repeated measurements on the same individual. Each miRNA was evaluated separately. P values <0.05 were considered statistically significant.

Internal correlation of the urinary contents of the 3 miRNAs was 0.85 (miR-2861 vs miR1915-3p), 0.87 (miR-4532 vs miR-1915-3p), and 0.93 (miR-4532 vs miR-2861); P < 0.0001. In the cross-sectional analysis, expression of miR-1915-3p, miR-2861, and miR-4532 was down-regulated (P < 0.0001) in individuals classified as decliners, yielding an area under the ROC curve of 0.84 [−3.84 (94.7%; 67.7%)] for miR-1915-3p [cutoff (clinical sensitivity; specificity)], 0.84 [−7.8 (89.4%; 75.2%)] for miR-2861, and 0.82 [−3.6; 94.7%; 66.6%)] for miR-4532, corroborating the findings from Cardenas-Gonzalez (3). In the longitudinal analysis, expression levels of miR-1915-3p, miR-2861, and miR-4532 significantly modified the slope of eGFR across time (P < 0.0001), therefore associating with long-term eGFR decline (Fig. 1).

Fig. 1. miRNAs miR-1915–3p, miR-2861, and miR-4532 associate with long-term eGFR decline in individuals with T1D.

Cross-sectional analysis of miRNA expression in T1D classified as nondecliners and decliners. The horizontal lines indicate mean ± SE (standard error). Linear mixed models estimate ± SE for the expression of each miRNA and its contribution on modifying the slope of eGFR in individuals with T1D (Type 1 diabetes) across time.

In summary, we have confirmed that the miRNAs previously described as novel biomarkers for DKD are associated with long-term eGFR decline in T1D, adding more evidence for the participation of these miRNAs in DKD. Additional mechanistic studies are necessary to better understand the role of these miRNAs in the pathophysiology of DKD.

1 Nonstandard abbreviations

DKD

diabetic kidney disease

DM

diabetes mellitus

GFR

glomerular filtration rate

miRNA

microRNA

eGFR

estimated glomerular filtration rate

T1D

type 1 diabetes

HbA_1c

glycohemoglobin

UCr

urinary creatinine.