Dysregulated expression but redundant function of the long non-coding RNA HOTAIR in diabetic kidney disease
Long non-coding RNAs (lncRNAs) are garnering increasing attention for their putative roles in the pathogenesis of chronic diseases, including diabetic kidney disease (DKD). However, much about in vivo lncRNA functionality in the adult organism remains unclear. To better understand lncRNA regulation and function in DKD, we explored the effects of the modular scaffold lncRNA HOTAIR (HOX antisense intergenic RNA), which approximates chromatin modifying complexes to their target sites on the genome.
Experiments were performed in human kidney tissue, in mice with streptozotocin-induced diabetes, the db/db mouse model of type 2 diabetes, podocyte-specific Hotair knockout mice and conditionally immortalised mouse podocytes.
HOTAIR was observed to be expressed by several kidney cell-types, including glomerular podocytes, in both human and mouse kidneys. However, knockout of Hotair from podocytes had almost no effect on kidney structure, function or ultrastructure. Glomerular HOTAIR expression was found to be increased in human DKD, in the kidneys of mice with streptozotocin-induced diabetes and in the kidneys of db/db mice. Likewise, exposure of cultured mouse podocytes to high glucose caused upregulation of Hotair expression, which occurred in a p65-dependent manner. Although HOTAIR expression was upregulated in DKD and in high glucose-exposed podocytes, its knockout did not alter the development of kidney damage in diabetic mice. Rather, in a bioinformatic analysis of human kidney tissue, HOTAIR expression closely paralleled the expression of its genic neighbour, HOXC11, which is important to developmental patterning but which has an uncertain role in the adult kidney.
Many lncRNAs have been found to bind to the same chromatin modifying complexes. Thus, there is likely to exist sufficient redundancy in the system that the biological effects of dysregulated lncRNAs in kidney disease may often be inconsequential. The example of the archetypal scaffold lncRNA, HOTAIR, illustrates how lncRNA dysregulation may be a bystander in DKD without necessarily contributing to the pathogenesis of the condition. In the absence of in vivo validation, caution should be taken before ascribing major functional roles to single lncRNAs in the pathogenesis of chronic diseases.
KeywordsDiabetic kidney disease Diabetic nephropathy Epigenetics Histone HOTAIR Kidney lncRNA Podocyte
Corepressor for element-1-silencing transcription factor
Chromatin immunoprecipitation by RNA purification
Chronic kidney disease
Diabetic kidney disease
Enhancer of zeste homologue 2
Gene Expression Omnibus
HOX antisense intergenic RNA
Cre positive wild-type mice
Podocyte-specific Hotair knockout mice
Long non-coding RNA
Lysine-specific histone demethylase 1A
Polycomb repressive complex 2
Quantitative reverse transcription-PCR
Repressor element-1 silencing transcription factor
Short interference RNA
α-Smooth muscle actin
The authors thank MD Golam Kabir (Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, ON, Canada) for technical assistance. Parts of this work were presented at the Keystone Symposium Unraveling the Secrets of Kidney Disease, Whistler, BC, Canada (3–7 March 2019).
SM conceived the idea, designed and performed the experiments, analysed the data and revised and edited the manuscript. MJH performed experiments and analysed data. KT, SNB, VGY, HV and TAA designed and performed the experiments and analysed the data. DN and SC performed the bioinformatic analyses. SLA performed the histological studies. BBB and YL performed the in vivo studies. KEW performed the electron microscopy. LG and FSS contributed to the design of the human studies, sourced archived human tissue and provided the clinical data. AA conceived the idea, designed the experiments, analysed the data and wrote the manuscript. All authors reviewed, edited and approved the final version of the manuscript. AA is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
This work was supported by a project grant from the Canadian Institutes of Health Research to AA (PJT 153284). The study sponsor was not involved in the design of the study; the collection, analysis, and interpretation of data; writing the report; or the decision to submit the report for publication. SM was supported by a Diabetes Canada Post-doctoral Fellowship. MJH is a recipient of a Scholarship from the Research Training Centre of St Michael’s Hospital and a Banting and Best Diabetes Centre - Novo Nordisk Studentship. KT was supported by a Research Internship Abroad from the São Paulo Research Foundation (Fapesp 2016/04591-1). SNB was supported by a Keenan Family Foundation KRESCENT Post-doctoral Fellowship through the Kidney Foundation of Canada, by a Heart and Stroke/Richard Lewar Center of Excellence Fellowship Award and by a Banting and Best Diabetes Centre Hugh Sellers Post-doctoral Fellowship. VGY is supported by a Diabetes Canada Post-doctoral Fellowship. TAA was supported by a King Abdullah Foreign Scholarship. AA is a recipient of a Diabetes Investigator Award from Diabetes Canada.
Duality of interest
AA has received research support from Boehringer Ingelheim and AstraZeneca, is listed as an inventor on a patent application (WO 2015/128453) from Boehringer Ingelheim, has received an unrestricted educational grant from Eli Lilly and has received honoraria from Novo Nordisk, Eli Lilly and Boehringer Ingelheim, Abbott and Dexcom.
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