Interstitial microRNA miR-214 attenuates inflammation and polycystic kidney disease progression
Ronak Lakhia, Matanel Yheskel, Andrea Flaten, Harini Ramalingam, Karam Aboudehen, Silvia Ferrè, Laurence Biggers, Abheepsa Mishra, Christopher Chaney, Darren P. Wallace, Thomas Carroll, Peter Igarashi, Vishal Patel
Ronak Lakhia, Matanel Yheskel, Andrea Flaten, Harini Ramalingam, Karam Aboudehen, Silvia Ferrè, Laurence Biggers, Abheepsa Mishra, Christopher Chaney, Darren P. Wallace, Thomas Carroll, Peter Igarashi, Vishal Patel
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Research Article Nephrology

Interstitial microRNA miR-214 attenuates inflammation and polycystic kidney disease progression

Abstract

Renal cysts are the defining feature of autosomal dominant polycystic kidney disease (ADPKD); however, the substantial interstitial inflammation is an often-overlooked aspect of this disorder. Recent studies suggest that immune cells in the cyst microenvironment affect ADPKD progression. Here we report that microRNAs (miRNAs) are new molecular signals in this crosstalk. We found that miR-214 and its host long noncoding RNA Dnm3os are upregulated in orthologous ADPKD mouse models and cystic kidneys from humans with ADPKD. In situ hybridization revealed that interstitial cells in the cyst microenvironment are the primary source of miR-214. While genetic deletion of miR-214 does not affect kidney development or homeostasis, surprisingly, its inhibition in Pkd2- and Pkd1-mutant mice aggravates cyst growth. Mechanistically, the proinflammatory TLR4/IFN-γ/STAT1 pathways transactivate the miR-214 host gene. miR-214, in turn as a negative feedback loop, directly inhibits Tlr4. Accordingly, miR-214 deletion is associated with increased Tlr4 expression and enhanced pericystic macrophage accumulation. Thus, miR-214 upregulation is a compensatory protective response in the cyst microenvironment that restrains inflammation and cyst growth.

Authors

Ronak Lakhia, Matanel Yheskel, Andrea Flaten, Harini Ramalingam, Karam Aboudehen, Silvia Ferrè, Laurence Biggers, Abheepsa Mishra, Christopher Chaney, Darren P. Wallace, Thomas Carroll, Peter Igarashi, Vishal Patel

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Figure 5

miR-214 directly inhibits TLR4.

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miR-214 directly inhibits TLR4. (A) The approach used to identify direct...
(A) The approach used to identify direct miR-214 targets is shown. There were 2735 evolutionarily conserved miR-214 mRNA targets based on the prediction by TargetScan software. Intersecting this list with RNA-Seq data identified 88 mRNAs that were differentially expressed in Pkd2-KO miR-214–/– kidneys compared with Pkd2-KO kidneys. Among these, 56 mRNAs were upregulated after miR-214 deletion in Pkd2-KO mice. (B) Heatmap showing upregulation of the 56 miR-214 targets in Pkd2-KO miR-214–/– (n = 3) compared with Pkd2-KO kidneys (n = 3). Tlr4 was identified as one of the putative miR-214 targets (red arrow). (C) Analysis of the 56 upregulated mRNAs demonstrated that inflammation-associated pathways are the predominant signature of miR-214 deletion. (D) Q-PCR analysis confirmed that Tlr4 expression was upregulated in Pkd2-KO miR-214–/– (red circles, n = 6) compared with Pkd2-KO kidneys (green circles, n = 6). (E) Q-PCR showing that, compared with scramble control, treatment with miR-214 mimic repressed Tlr4 expression in mIMCD3 (n = 6), MEF (n = 4), and RAW 264.7 cells (n = 5). (F) To further validate Tlr4 as a direct miR-214 target, we used CRISPR/Cas9 to generate a monoclonal mIMCD3 cell line with targeted deletion of the miR-214 binding site in Tlr4 3′ UTRs (TlrΔmiR-214). Q-PCR analysis showed that miR-214–mediated Tlr4 repression was abolished in TlrΔmiR-214 (n = 5) but not in control mIMCD3 cells (n = 4–5). *P < 0.05; Student’s unpaired t test (D–F); error bars indicate SEM.