Urine MicroRNA as Potential Biomarkers of Autosomal Dominant Polycystic Kidney Disease Progression: Description of miRNA Profiles at Baseline

<div><p>Background</p><p>Autosomal dominant polycystic kidney disease (ADPKD) is clinically heterogenic. Biomarkers are needed to predict prognosis and guide management. We aimed to profile microRNA (miRNA) in ADPKD to gain molecular insight and evaluate biomarker potential.</p><p>Methods</p><p>Small-RNA libraries were generated from urine specimens of ADPKD patients (N = 20) and patients with chronic kidney disease of other etiologies (CKD, N = 20). In this report, we describe the miRNA profiles and baseline characteristics. For reference, we also examined the miRNA transcriptome in primary cultures of ADPKD cyst epithelia (N = 10), normal adult tubule (N = 8) and fetal tubule (N = 7) epithelia.</p><p>Results</p><p>In primary cultures of ADPKD kidney cells, miRNA cistrons mir-143(2) (9.2-fold), let-7i(1) (2.3-fold) and mir-3619(1) (12.1-fold) were significantly elevated compared to normal tubule epithelia, whereas mir-1(4) members (19.7-fold), mir-133b(2) (21.1-fold) and mir-205(1) (3.0-fold) were downregulated (P<0.01). Expression of the dysregulated miRNA in fetal tubule epithelia resembled ADPKD better than normal adult cells, except let-7i, which was lower in fetal cells. In patient biofluid specimens, mir-143(2) members were 2.9-fold higher in urine cells from ADPKD compared to other CKD patients, while expression levels of mir-133b(2) (4.9-fold) and mir-1(4) (4.4-fold) were lower in ADPKD. We also noted increased abundance mir-223(1) (5.6-fold), mir-199a(3) (1.4-fold) and mir-199b(1) (1.8-fold) (P<0.01) in ADPKD urine cells. In ADPKD urine microvesicles, miR-1(2) (7.2-fold) and miR-133a(2) (11.8-fold) were less abundant compared to other CKD patients (P<0.01).</p><p>Conclusions</p><p>We found that in ADPKD urine specimens, miRNA previously implicated as kidney tumor suppressors (miR-1 and miR-133), as well as miRNA of presumed inflammatory and fibroblast cell origin (miR-223/miR-199), are dysregulated when compared to other CKD patients. Concordant with findings in the primary tubule epithelial cell model, this suggests roles for dysregulated miRNA in ADPKD pathogenesis and potential use as biomarkers. We intend to assess prognostic potential of miRNA in a followup analysis.</p></div

miRNA cistron clusters differentially expressed between ADPKD-derived and normal kidney-derived primary tubular epithelial cultures^*.

*<p>, Cutoff for presentation in this table is p-value<0.05 and FDR<0.2 in the ADPKD vs. normal comparison.</p

miRNA sequence variants that were found to be differentially expressed between ADPKD and non-ADPKD specimens.

<p>(<b>A</b>) miRNA sequence variants that were found to be differentially expressed between ADPKD cyst-derived primary cultures and normal adult kidney derived cultures (see complete list in Table S15 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086856#pone.0086856.s005" target="_blank">File S1</a>). (<b>B</b>) Box plot showing a composite score derived from the information in (A). The median frequency of each miRNA variant across the samples was used to generate the score; 1 point was added (or reduced) for each miRNA variant with frequency above (or below) the median frequency. (<b>C</b>) Bar plot showing a composite score derived in a manner similar to (B) from patient' urine sediment cell sequence data (contributing miRNA variants are depicted in Table S16 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086856#pone.0086856.s005" target="_blank">File S1</a>).</p

Mature miRNA differentially expressed between ADPKD (N = 20) and other CKD (N = 20) patient urine exosomal preparations^*.

*<p>, Cutoff for presentation in this table is p-value<0.05.</p

Hierarchically clustered study samples grouped by type and clinical characteristics.

<p>including additional non-study specimens for reference (columns) and miRNA cistrons (rows). High relative read frequency (log₂-transformed) is indicated by bright yellow shades; low frequencies in dark blue. Corresponding numerical data is presented in the supplementary tables. Abbreviations: ADPKD, autosomal dominant polycystic kidney disease; CKD, chronic kidney disease; immort, immortalized; CPM, counts per million; F, female; M, male.</p

miRNA cistron clusters differentially expressed between ADPKD (N = 20) and other CKD (N = 20) patient urine sediment cells^*.

*<p>, Cutoff for presentation in this table is p-value<0.05 and FDR<0.2.</p

Validation of top 20 most down-regulated genes.

<p>Shown here are means of gene expression levels relative to vehicle control group ± standard errors (SE); the vehicle control group was normalized as 1. Taking Dhrs3 expression as an example, 0.01 and 2.88 in the AGN193109 group and the DEAB+tRA group indicate 100-fold reduction and 2.88-fold induction relative to the vehicle group, respectively.</p>*<p>p<0.05 vs vehicle,</p>**<p>p<0.01 vs vehicle,</p>***<p>p<0.001 vs vehicle;</p>∧<p>p<0.05 vs AGN193109,</p>∧∧<p>p<0.01 vs AGN193109,</p>∧∧∧<p>p<0.001 vs AGN193109;</p>+<p>p<0.05 vs DEAB,</p>++<p>p<0.01 vs DEAB,</p>+++<p>p<0.001 vs DEAB; NA: Not amplified.</p

Top 20 most up-regulated genes.

<p>Shown here are mean fold-changes of gene expression compared to vehicle control from three experimental groups. Genes were sorted by fold-changes of AGN193109 group compared to vehicle group. Minus and plus numbers indicate folds of suppression and induction, respectively, in comparison to the vehicle control group, which was normalized as 1.</p

Regulation of Bmp7 and Foxa1 by AGN193109 and 4-(diethylamino)benzaldehyde (DEAB) in pilot study.

<p>mRNA expression of Bmp7 (<b>Ai</b>) and Foxa1 (<b>Aii</b>) was suppressed by AGN193109; the suppression was at least partially abolished in the presence of 0.2 µM tRA. <b>Bi.</b> Bmp7 mRNA was suppressed by DEAB; the suppression was reversed to a level slightly higher than basal level in the presence of 0.01 µM tRA. <b>Bii.</b> Expression of Foxa1 mRNA was suppressed by DEAB; the suppression was partially abolished in the presence of 0.01 µM tRA. Each dot represents mean value of three technical replicates from a single biological experiment. *, **, and ***: p<0.05, p<0.01, and p<0.001, respectively.</p

Candidate target genes of endogenous tRA/RARs: number of induced and suppressed genes.

<p>A total of 31 and 94 genes were up-(red) and down-(green) regulated, respectively, by AGN193109 and by DEAB; regulation of these genes were at least partially abolished in the presence of tRA. Of the 31 up-regulated genes, 24 were up-regulated by AGN193109 by less than 1.5-fold, 7 by more than 1.5-fold but less than 2-fold, and none by 2-fold and more; none of the genes were up-regulated by DEAB by 1.5-fold and more. Of the 94 down-regulated genes, 38 were down-regulated by AGN193109 by less than 1.5-fold, 37 by more than 1.5-fold but less than 2-fold, and 19 by 2-fold and more; 68 were down-regulated by DEAB by less than 1.5-fold, 14 by more than 1.5-fold but less than 2-fold, and 12 by 2-fold and more.</p