ELUCIDATING THE ROLE OF MICRORNA (MIR)-299A-5P IN ENHANCING FIBROSIS AND PRO-APOPTOTIC SIGNALLING PATHWAYS, THEREBY PROMOTING DIABETIC KIDNEY DISEASE.

Abstract

LAY ABSTRACT: Diabetic kidney disease (DKD) is a major complication of diabetes mellitus, developing in up to 40% of patients with diabetes. DKD is the leading cause of kidney diseases globally, associated with reduced quality of life and increased mortality. A key early problem is the buildup of extracellular matrix proteins, mainly due to mesangial cell activation and proximal tubular cell damage. The major goal of my research is to show the role that microRNA-299a-5p plays in promoting kidney damage over time in DKD. My research showed that in a diabetic setting microRNA-299a-5p is increased, and this in turn reduces the antifibrotic proteins follistatin and cripto-1 thereby increasing the expression of the proteins known to promote kidney scarring, activin A and transforming growth factor β1 (TGFβ1). Since follistatin and cripto-1 naturally inhibit these fibrosis-promoting proteins, their suppression by microRNA-299a-5p worsens kidney damage. My research also showed that microRNA-299a-5p is elevated in diabetic kidneys from both mice and humans, as well as in mesangial cells exposed to high glucose. When we increased this microRNA, mesangial cells produced more extracellular matrix proteins, mimicking the effects of high glucose. Blocking microRNA-299a-5p, however, prevented these harmful effects. In a diabetic mouse model, inhibiting microRNA-299a-5p reduced kidney damage, preventing protein loss in urine (albuminuria), kidney enlargement, loss of important kidney cells (podocytes), and excessive extracellular matrix buildup. This was linked to higher levels of follistatin and cripto-1, suggesting that microRNA-299a-5p plays a key role in kidney fibrosis and may be a promising target for new treatments in DKD. Additionally, I showed that in the kidney proximal tubules, this microRNA causes damage and death to the tubules, a hallmark of DKD progression. The effect of the microRNA on the kidney proximal tubules is seen through the reduction of the anti-apoptotic mitogen activated protein 3 kinase 2 expression which eventually leads to the disruption of its signalling pathway.

The studies in this thesis collectively emphasize the importance of microRNA-299a-5p inhibition in preventing the debilitating effects of DKD. Additionally, it provides a non-invasive method of detecting kidney disease progression by assessing the level of microRNA-299a-5p. As specific microRNAs are dysregulated early in the course of DKD even before significant kidney damage occurs, this makes them serve as both early biomarkers and therapeutic targets. Current treatments for DKD such as blood pressure control, glucose-lowering drugs, and medications that block the renin-angiotensin system can slow disease progression but often do not fully prevent kidney damage. These therapies mainly target symptoms or single pathways and are not tailored to the molecular drivers of the disease. However, microRNAs act as master regulators of gene expression and can control entire networks of genes involved in key disease processes such as inflammation, fibrosis (scarring), oxidative stress, and cell death. This means that targeting a single microRNA such as microRNA-299a-5p has the potential to modify multiple disease mechanisms at once, offering a broader and more upstream therapeutic approach.

Lastly, this thesis provides a broader view of the targets of this microRNA, which if synthetically increased in DKD patients, has the potential of conferring protection against kidney decline. By correcting microRNA imbalances, we could intervene earlier and more precisely, moving from a “one-size-fits-all” model towards personalized, mechanism-based treatment that addresses the root causes of DKD rather than just its consequences.

SCIENTIFIC ABSTRACT: Diabetic kidney disease (DKD) is a major complication of diabetes mellitus, affecting up to 40 percent of individuals with the condition. As the leading cause of end stage kidney disease, DKD significantly diminishes quality of life and contributes to increased mortality. In Canada, approximately one in ten people, equivalent to four million individuals, are affected by some form of kidney disease. This imposes an estimated annual cost of forty billion dollars on the healthcare system for treatment and management of the disease. A hallmark of early DKD progression is the accumulation of extracellular matrix proteins within the glomerulus, primarily driven by mesangial cell activation. Despite extensive efforts to target the underlying molecular mechanisms of DKD, the search for optimal biomarkers and effective therapeutic targets remains ongoing. There is a critical need to identify novel molecular targets and therapeutic strategies to improve clinical outcomes for patients with this debilitating condition. Recent studies have highlighted microRNAs (miRs) as promising candidates for both diagnostic and therapeutic applications. Given that miR expression profiles are often altered in disease states such as DKD, they offer a compelling avenue for the development of innovative interventions.

The first study in this thesis investigates the role of the novel miR-299a-5p in promoting fibrosis and the accumulation of extracellular matrix proteins in the kidneys of type 1 diabetic Akita mice. This miR was found to be significantly upregulated in human diabetic kidney biopsies and in kidneys across multiple mouse models of type 1 diabetes. Bioinformatic analyses identified cripto-1 and follistatin as direct targets of miR-299a-5p through binding to their respective 3' untranslated regions. Follistatin is a well-established anti-fibrotic protein that inhibits activin A, while cripto-1 suppresses transforming growth factor beta 1 (TGFβ1), both of which are key pro-fibrotic cytokines. In vitro studies using primary mesangial cells showed that miR-299a-5p was expressed in these cell lines and validated our previous findings, which demonstrated that overexpression of miR-299a-5p significantly reduced the basal expression of cripto-1 and follistatin while enhancing the expression of fibrotic proteins. Conversely, inhibition of miR-299a-5p prevented high glucose-induced fibrotic protein expression and restored basal levels of cripto-1 and follistatin. Interestingly, co-administration of cripto-1 and follistatin produced an additive anti-fibrotic effect in the presence of miR-299a-5p overexpression.

Building upon these findings, the therapeutic relevance of miR-299a-5p inhibition was assessed in vivo using type 1 diabetic Akita mice overexpressing TGFβ1, a model characterized by kidney fibrosis. Inhibition of miR-299a-5p using locked nucleic acid technology led to significant reductions in albuminuria, kidney hypertrophy, and fibrosis. Notably, circulating miR-299a-5p which we suspect were packaged as extracellular vesicles (exosomes) was detected in the serum of these diabetic mice, supporting its potential utility as a non-invasive biomarker in diabetic kidney disease. The second study focuses on the role of miR-299a-5p in proximal tubular apoptosis, an increasingly recognized contributor to DKD progression beyond glomerular injury. We identified mitogen-activated protein kinase kinase kinase 2 (MAP3K2), which is highly expressed in proximal tubules, as a direct target of miR-299a-5p. MAP3K2 activates extracellular signal-regulated kinase 5 (Erk5), a kinase involved in anti-inflammatory and anti-apoptotic pathways via activation of zinc finger transcription factors such as the Kruppel-like factor family and nuclear factor erythroid 2-related factor 2. Using human proximal tubular cells which expressed this miR, we showed that inhibition of miR-299a-5p attenuated high glucose-induced tubular apoptosis and preserved the protein expression of both MAP3K2 and Erk5.

Recognizing the importance of sex as a biological variable in disease pathogenesis, the final study of this thesis describes the development of a robust mouse model to study sex differences in DKD progression. A high dose treatment with streptozotocin protocol was optimized to induce comparable type 1 diabetes in both male and female CD1 mice. Using this model, we demonstrate that DKD develops similarly in both sexes, thereby enabling future studies to assess sex-specific expression patterns of miR-299a-5p and to determine whether its regulation is modulated by diabetic pathology.