Serum circulating microRNA profiling for identification of potential markers of diabetic nephropathy in black African South Africans with type 2 diabetes mellitus
Date
2020
Authors
Valentin, Stefan Drikus
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Abstract
Introduction: Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterised by
insulin resistance. The prevalence of T2DM is increasing with an estimated 14.2 million adults
with T2DM expected in Africa by the year 2040. Poor glycaemic control, over a prolonged period
of time, leads to diabetic micro- and macrovascular complications. Of the microvascular
complications, diabetic nephropathy (DN) has been implicated in causing the majority of deaths
associated with T2DM. DN is characterised by progressive decline of renal function, resulting in
end-stage renal disease. With early intervention, kidney damage can be prevented or even
reversed, however no test currently exists for early detection of DN. Currently, urine albumin-to creatinine ratios (UACR) are used to monitor kidney function and diagnose DN. UACR as a marker
is unspecific and unreliable, and there is a need for a more sensitive and specific marker. Serum
circulating microRNA (miRNA) have shown promise as potential novel biomarkers as they are
stable and often differentially expressed in different pathological states. This study, therefore,
aimed to detect differentially expressed serum miRNAs in black South African participants with
T2DM with and without microalbuminuria (MA) to identify potential biomarkers for early
detection of DN. In addition, the prevalence of polymorphisms in the HMGA2 and TGF-β1 gene
and their associations with markers of kidney function were investigated. Further, the use of
serum cystatin C (SCC) as an alternative to serum creatinine for estimation of glomerular filtration
rate (GFR; used to measure kidney function and to classify the stage of kidney disease) was
assessed.
Methods: Black South African participants with T2DM (n = 238) were recruited from
diabetes clinics at Charlotte Maxeke Johannesburg Academic Hospital and Chris Hani
Baragwanath Hospital. A subset of participants (n = 14) were classified into two groups based on
the absence (UACR < 2 mg/mmol; controls, n = 7) or presence (UACR 2-20 mg/mmol; cases, n =
7) of MA. The participants were matched for age, medication use, and anthropometry. miRNA
expression profiles were analysed using the Affymetrix® GeneChip® 4.0 assay and transcriptome
analysis console (TAC) software. Two differentially expressed miRNAs were selected for
validation through real-time quantification polymerase chain reaction (PCR) in a subset of 40
participants (20 participants with the lowest UACR, and 20 participants with the highest UACR).
Two genes that were targeted by the differentially expressed miRNA were selected for
polymorphism analysis. The total cohort was genotyped for HMGA2 rs1114167319, and TGF-β1
V
rs1800417 polymorphism by amplification refractory mutation systems (ARMS) PCR. In addition,
participants were genotyped for the HMGA2 rs1114167320 polymorphism by PCR restriction
fragment length polymorphism (RFLP).
SCC concentrations were measured using a R&D enzyme-linked immunosorbent assay
(ELISA). Estimated GFR (eGFR) were calculated through the use of two creatinine-based
equations (modification of diet in renal disease study (MDRD) equation without ethnic factor
correction and the chronic kidney disease epidemiology collaboration (CKD-EPI) equation), and
three SCC-based equations. Passing-Bablok linear regression and Band-Altman plots were
generated to evaluate correlation and method agreement between the eGFR equations, using
the MDRD equation as the reference method.
Results: The study population consisted of a 238 black South Africans with T2DM. The
cohort had a mean age of 56.6 ± 9.88 years and a mean duration of disease of 11.5 ± 8.82 years.
The majority of the participants were female (61.8 %), and they had poor glycaemic control
(HbA1c of 8.68 ± 2.73 %).
Differential miRNA expression profiling (> 2-fold difference between cases and controls)
identified nine miRNAs. miR-455-3p and let-7b-5p were upregulated, and miR-4740-3p, miR 7704, miR-101-5p, miR-5189-3p, miR-1273g-3p, miR-16-5p and miR-6880-5p were
downregulated in cases relative to controls. Real-time quantitative PCR validation of expression
levels of miR-455-3p and let-7b-5p found that expression levels of both miRNAs were higher in
the group with the lowest UACR compared to the group with the highest UACR (miR-455-3p: 40.5
[39.1; 42.8] vs 42.5 [41.3; 45.3] CT value, p = 0.043; and let-7b-5p: 26.3 ± 1.50 vs 27.6 ± 2.03 CT
value, p = 0.022).
The HMGA2 rs111416319 and rs111416320 major alleles were the C (0.51) and A allele
(1.0), respectively. The major allele for the TGF-β1 rs1800471 polymorphism was the G allele
(0.64). None of the polymorphisms were in Hardy-Weinberg equilibrium (HWE). Despite not
being in HWE, we did perform additional analyses with rs111416319 and rs1800471.
rs111416319 was not associated with any markers of kidney function or DM. Similarly, rs1800471
was not associated with markers of kidney function, however, participants with the GG genotype
had significantly lower glucose concentrations compared to participants with the GC/CC
genotype (6.60 [4.80; 8.80] vs. 8.10 [5.70; 11.3] mmol/l; p = 0.011).
The classification of stages of kidney disease using the five different eGFR equations
resulted in significantly different clinical classifications of kidney function (p < 0.001). The MDRD
VI
and CKD-EPI equation correlated strongly (r = 0.945; p < 0.001) however there was poor
agreement between the equations with a bias of -39.7 %. There was good agreement between
SCC equation 2 and equation 3 with the MDRD equation with a bias of -7.71 % and -7.10 %,
respectively.
Conclusion: Let-7b-5p and miR-455-3p protect against the development of DN. Let-7b-5p
binds to the TGF-β1 receptor 1 inhibiting the TGF-β1 signalling pathway. This pathway has been
shown to play a role in DN by enhancing glucose-induced cell hypertrophy and increased
expression of extracellular matrix (ECM) genes (e.g. collagen and fibronectin). In addition, let-7b 5p targets the 3’ untranslated region of the HMGA2 gene, that has been implicated in activation
of the TGF-β1 signalling pathway. Thus, increased expression of let-7b-5p leads to decreased
renal fibrosis via inhibiting HMGA2 and TGF-β1 receptor expression. miR-455-3p overexpression
decreases ECM synthesis and inflammatory cytokines resulting in protection from renal fibrosis.
Furthermore, TGF-β1 inhibits the expression of miR-455-3p, thus activating renal fibrotic
pathways, resulting in reduced kidney function and DN development.
Further studies are required in the South African black population to confirm the role of
these miRNAs in DN. Longitudinal studies need to be conducted to confirm the role of let-7b-5p
and miR-455-3p as possible early markers of DN. The eGFR equations showed significant variation
in terms of classification of kidney disease and cannot be used interchangeably
Description
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Medicine to the Faculty of Health Sciences, School of Pathology, University of the Witwatersrand, Johannesburg, 2020