| Abstract |
Systemic lupus erythematosus (SLE) is a challenging autoimmune disease with manifestations
derived from the involvement of multiple organs including the kidneys, the joints, the nervous
system as well as hematopoietic organs. Lupus nephritis (LN) is one of the most severe
manifestations of SLE and a major risk factor for morbidity and mortality. The frequency is
particularly high in juvenile-onset SLE and in patients of African ancestry. Ten percent of
patients with LN will develop end stage renal disease (ESRD). The main pathological feature
in LN is immune complex formation and deposition in the kidney, which results in
intraglomerular inflammation with recruitment of leucocytes and activation and proliferation
of resident renal cells. Gene expression studies in LN have revealed gene networks that involve
not only immune cell infiltration but also extracellular matrix formation and renal tissue repair
and fibrosis.
Micro-RNAs (miRNAs) come from a large family of small non-coding RNA sequences, which
are about 22 nucleotides in length. They constitute critical posttranscriptional regulators of
gene expression and act either as translational inhibitors of gene expression or by degrading
mRNA transcripts since they bind to complementary sequences in the 3’ untranslated region
(3’UTR) of target mRNAs. Multiple studies have shown that a cluster of key miRs are highly
expressed in the kidney and play a critical role in renal development and function. They also
affect the diabetic or polycystic kidney, the kidney with IgA, acute injury or renal cell
carcinoma.
The role of miRNAs as transcriptional modulators in the pathogenesis of SLE has recently
emerged and their regulatory effects on DNA methylation pathway, type I IFN pathway,
estrogen and regulatory T-cells are becoming clear. MiRNA expression has been studied in
peripheral blood mononuclear cells (PBMCs), CD4 T cells, kidney biopsy tissue and Epstein-
Barr virus transformed B-cell lines from patients with SLE.
In this study, we investigated the role of miRNAs in human LN. To this end, we analysed the
miRNA profile in kidney biopsy samples of patients with LN by TaqMan based array aiming
to identify novel genes involved in disease pathogenesis. We identified important miRNAgene
pairs and constructed gene networks that are potentially involved in LN pathogenesis.
We focused on miR-422a, which is the most upregulated miRNA that correlates with active histological lesions and its target Kallikrein-4 (KLK-4), a serine esterase with putative
renoprotective properties.
Compared to normal tissue a 24-miRNA signature defines human LN with 9 miRNAs upregulated
and 15 miRNAs down-regulated, and miR-422a exhibits the highest up-regulation
(17.2-fold). These mi-RNAs with altered expression are predicted to target genes involved in
inflammatory signaling pathways, membrane transporters and cell homeostasis such as TGF-
β, protein kinase A, ERK/MAPK, NF-κB, HNF4A, Wnt/β-catenin signaling. Bioinformatic
analysis predicted that miR-422a has a binding site in the 3’UTR of KLK4 gene, a member of
the kallikrein family of serine proteases. This was validated by overexpression of miR-422a,
which suppressed by 65% KLK4 luciferase activity and by 82% KLK4 mRNA levels in
Human embryonic kidney - 293 cells (HEK-293).
In order to monitor miR-422a/KLK4 expression during LN progression, we used NZB/W F1
lupus mice. We found that at early stages miR-422a was 4.1-fold up-regulated, while KLK4
mRNA levels were 3.4-fold down-regulated while at later stages miR-422a was 9.4-fold upregulated
and KLK4 mRNA levels were 7.6-fold down-regulated. In unaffected NZW kidney
and human control specimens, KLK4 protein was strongly expressed, mainly in the cytoplasm
of renal tubular as well as mesangial cells. In contrast, KLK4 showed absent or faint or
moderate expression in renal parenchymal cells of proteinuric NZB/W F1 and most patients
with active LN. Together, these data implicate miRNAs and KLK4, a secreted serine esterase
with angiogenic and extracellular matrix remodeling properties, in the pathogenesis of
immune-mediated LN.
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