Introduction
Type 1 diabetes is an autoimmune disease often diagnosed in childhood that is characterised by the loss of insulin producing beta cells, which leads to hyperglycaemia [
1]. Even with strict glycaemic control using rigorous insulin management, microvascular complications including diabetic nephropathy [
2] and diabetic cardiomyopathy [
3] develop over time. The microvascular complications associated with diabetes are key drivers of morbidity (and ultimately mortality) rates, and therefore put a significant economic burden on healthcare providers. Most notably, there are no specific therapeutic interventions that prevent, delay or reduce the microvascular complications associated with diabetes. Diabetic nephropathy is the primary cause of death in 21% of people with type 1 diabetes [
4], and cardiovascular disease, which includes diabetic cardiomyopathy, accounts for 44% of all fatalities in type 1 diabetes [
5]. Both pathologies are characterised by an impairment in function (kidney [proteinuria [
6]], heart [impairment in systolic contractility [
7]]) caused by local inflammation [
8], endothelial dysfunction [
9] and loss of survival pathways, the latter of which predisposes tissues to injury.
Resolution of inflammation and modulation of survival pathways are critical to normal physiology and are often altered in disease states. Annexin A1 (ANXA1) is a 37 kDa member of the multigene annexin family with anti-inflammatory and pro-resolving activities [
10]. The N-terminus of each family member varies giving each its unique functionality [
11]. ANXA1 binds formyl peptide receptor 2 (FPR2/ALX), downregulating the production of proinflammatory processes but activating pro-survival pathways via protein kinase C [
12]. ANXA1 levels are modulated in many disease states including cancer [
13], multiple sclerosis [
14], cystic fibrosis [
15] and obesity/the metabolic syndrome [
16]. Human recombinant ANXA1 (hrANXA1) or its N-terminal peptide (Ac2-26) have therapeutic benefits in many experimental models of disease, including rheumatoid arthritis [
17], atherosclerosis [
18] and nonalcoholic steatohepatitis [
19].
The role of ANXA1 as a pro-resolving-like molecule in type 1 diabetes is unknown. Therefore, this study was designed to investigate: (1) the plasma levels of ANXA1 in individuals with diabetes with/without nephropathy; (2) the role of endogenous ANXA1 in an animal model of type 1 diabetes and in the subsequent development of microvascular complications (diabetic cardiomyopathy and diabetic nephropathy); (3) if prophylactic daily administration with hrANXA1 prevents diabetic cardiomyopathy and/or nephropathy; and (4) if therapeutic intervention with hrANXA1 (after microvascular complications have developed) can halt the progression of diabetic cardiomyopathy and/or nephropathy.
Discussion
ANXA1 levels are elevated in individuals with type 1 diabetes. The main conceptually novel findings of this study are: (1) Anxa1
−/− mice challenged with STZ developed a more severe diabetic phenotype and more severe systolic cardiac and renal dysfunction compared with diabetic WT mice; (2) prophylactic treatment of diabetic mice with hrANXA1 did not prevent the development of a diabetic phenotype but attenuated the development of both cardiac and renal dysfunction; and (3) therapeutic administration of hrANXA1 (after both cardiac and renal dysfunction had developed) halted the progression of both cardiac and renal dysfunction seen in diabetic WT mice. Thus, endogenous ANXA1 prevents the development of end-organ injury in a murine model of type 1 diabetes and therapeutic hrANXA1 prevents (prophylactic administration) or halts (late therapeutic administration) the progression of systolic cardiac dysfunction and proteinuria caused by diabetes in these animals without altering the underlying diabetic phenotype.
Type 1 diabetes is characterised by elevated blood glucose levels, which drives low-grade chronic inflammation and thus contribute to tissue/organ damage. Healthy donors have low levels of ANXA1 in plasma [
26]. Here we report that ANXA1 is elevated in individuals with type 1 diabetes with and without nephropathy (Fig.
1a). Additionally, we show that diabetic individuals have elevated systemic inflammation levels, which are further elevated by nephropathy. Elevated plasma CRP is strongly associated with adverse outcomes in myocardial infarction, stroke and coronary heart disease, which are common causes of death in people with type 1 diabetes. We hypothesise that the elevation in circulating ANXA1 is a compensatory mechanism to protect tissues from the deleterious effects of hyperglycaemia, independent of the presence of diabetic nephropathy and not as a consequence of a rise in systemic inflammation (ESM Fig.
1b). ANXA1 is found in many tissues, including the heart and kidney, but is also highly expressed in macrophages and other immune cells [
26]. Here we report that ANXA1 levels were lower in both the heart and kidney, suggesting that hyperglycaemia causes ANXA1 to be released from tissue stores. ANXA1 secretion is needed so it can signal to the FRP2 in both an autocrine and paracrine manner to activate tissue/organ protective pathways [
12,
27].
We therefore wanted to understand the role of endogenous ANXA1 in the development of type 1 diabetes and in tissue/organ protection in the mouse. Mice challenged with STZ developed a diabetic phenotype with elevated non-fasted blood glucose levels and impairment in OGTT secondary to a very large (>75%) reduction in serum insulin levels. In individuals with type 1 diabetes, beta cell mass is irreversibly reduced by 70–80% at the time of diagnosis [
28]; similar loss of beta cells is recorded in mice administered STZ [
29]. We report here that the diabetic phenotype (OGTT) caused by STZ is more severe in diabetic
Anxa1
−/− mice, although the serum insulin levels are reduced to a similar degree (Table
1). This finding suggests that endogenous ANXA1 limits the development of hyperglycaemia in an insulin-independent manner.
Even individuals who undergo rigorous insulin management develop microvascular complications over time [
30]. Here we report that diabetic
Anxa1
−/− mice have more severe cardiac and renal dysfunction compared with diabetic WT mice, suggesting that endogenous ANXA1 limits the development of end-organ injury/dysfunction in mice with diabetes (Figs
2,
3). As hypertrophy is an important contributor in diabetes-associated cardiac and renal dysfunction we evaluated both intra-ventricular septum diameter [
31] and glomerular size; and report that both are significantly increased in diabetic mice (Figs
2,
3). Notably, both glomerular hypertrophy and interstitial fibrosis were further significantly augmented in diabetic
Anxa1
−/− mice when compared with diabetic WT mice. Cardiac hypertrophy is a common response to external stressors, including hyperglycaemia and oxidative stress [
32]; this compensatory process evolves into a decompensated state with profound changes in contractile dysfunction and extracellular remodelling [
33]. Similarly, renal hypertrophy is also a compensatory mechanism [
34]; in the case of type 1 diabetes, excessive renal hyperfiltration due to activation of the renin–angiotensin pathways [
35] puts stress on glomeruli and proximal tubules [
35], manifesting in functional decline.
Having found that endogenous ANXA1 limits the development of microvascular complications associated with diabetes, we wanted to investigate whether pharmacological intervention with hrANXA1 can attenuate the development of diabetic cardiomyopathy and nephropathy. Previously, both full-length AXNA1 and the Ac2-26 peptide (the N-terminal functional fragment of ANXA1) have been used in vivo and both elicit biological function [
17‐
19]). In this study, we chose to use full-length hrANXA1, as the dose of full-length ANXA1 needed to induce biological function is up to 20 times less than that of the Ac2-26 peptide [
36] and 14 times less than that needed to induce changes in gene expression in terms of molarity [
37].
Treatment of mice challenged with STZ with hrANXA1 (weeks 1–13) did not alter the diabetic phenotype (reduced insulin levels, elevated blood glucose and impaired OGTT), but attenuated the cardiac dysfunction and the proteinuria caused by diabetes, suggesting that hrANXA1 reduces the cardiac and renal injury caused by hyperglycaemia and excessive oxidative stress (organ protection rather than reduction of diabetes). Most notably, we also report that a late therapeutic intervention (weeks 8–13), when a diabetic phenotype and a moderate degree of cardiac and renal dysfunction had already occurred, halted the further decline in both cardiac and renal function seen in diabetic WT mice. It should be noted that administration of ANXA1 during the administration of STZ (days 1–5) was also not sufficient to reduce the diabetic phenotype or give long-term protection to organs.
What are the mechanisms by which ANXA1 elicits protection against cardiac and renal dysfunction associated with diabetes? We show here for the first time that the development of type 1 diabetes (caused by STZ) in mice is associated with (1) a reduction in endogenous ANXA1 levels in both the heart and the kidney (increased secretion) and (2) a decline in cardiac and renal dysfunction. Thus, we speculated that endogenous ANXA1 protects both the heart and the kidney (and hence reduces organ injury/dysfunction) by acting on pro-survival and anti-inflammatory pathways.
Hyperglycaemia leads to activation (phosphorylation) of mitogen-activated protein kinases (MAPKs) p38, JNK and ERK1/2, hypertrophy and fibrosis in the heart [
32‐
38] and kidney [
39,
40]. While little evidence is available to suggest strong tissue specific activation of proinflammatory pathways in STZ-induced diabetes, pharmacological inhibition or genetic deletion of any of these three MAPKs reduces microvascular complications (diabetic nephropathy, cardiomyopathy and retinopathy) caused by type 1 diabetes in rodents [
41,
42]. Indeed we found no activation of NF-κB or the inflammasome in our model of STZ-induced diabetes (ESM Fig.
4). However, we observed a significant increase in activated (phosphorylated) p38, JNK and ERK1/2 in diabetic WT mice. Interestingly,
Anxa1
−/− mice, even in the absence of diabetes (no STZ challenge), had constitutive activation (phosphorylation) of p38, JNK and ERK1/2 in the heart and kidney. The degree of activation of p38, JNK and ERK1/2 was further exacerbated when
Anxa1
−/− mice were challenged with STZ. As diabetic
Anxa1
−/− mice also had excessive renal hypertrophy and fibrosis, we speculate that the excessive activation of these known proinflammatory and profibrotic signalling pathways are key drivers of the excessive pathology seen in
Anxa1
−/− mice. This hypothesis is supported by the following two findings: (1) treatment of diabetic WT mice with hrANXA1 attenuated the activation of p38, JNK and ERK1/2 and the cardiac and renal dysfunction caused by diabetes; and (2) even when hrANXA1 was given therapeutically (weeks 8–13) this resulted in attenuation of the activation of p38, JNK and ERK1/2 as well as the organ dysfunction caused by diabetes.
In addition, we also investigated the effect of diabetes with or without hrANXA1 treatment on the degree of activation of Akt survival pathways, which is in part regulated by IRS-1 [
43]. Diabetic mice demonstrated a significant decrease in the phosphorylation of Ser
473 on Akt (indicating a reduction in activity of the kinase) in the heart and kidney. One effect of this inhibition is that organs are less resistant to stressor stimuli and subsequent organ injury. In contrast, treatment with hrANXA1 attenuated the decline in Akt activation caused by diabetes. Akt is a member of the phosphoinositide-3 kinase (PI3K) signal transduction pathway. Insulin signalling through IRS-1 regulates PI3K activity and PI3K can activate Akt (phosphorylation on Ser
473). Activated Akt controls inflammatory and pro-survival responses [
44]. Most notably, activation of the Akt survival pathway reduces organ injury in many conditions associated with inflammation including sepsis-induced organ dysfunction [
20,
45], haemorrhagic shock-induced organ dysfunction [
25,
46], myocardial infarction [
47] and diabetes [
48]. Specifically, in cardiac and renal ischaemia/reperfusion injury treatment with the Ac2-16 peptide tissue necrosis was reduced by activation of Akt [
49,
50].
In conclusion, we report for the first time that ANXA1 levels are elevated in the plasma of individuals with type 1 diabetes. We have also clearly demonstrated that endogenous ANXA1 plays a key role in protecting the heart and kidney from functional decline in an animal model of type 1 diabetes. Specifically, we have shown that key mediators of the MAPK pathway (p38, JNK and ERK1/2) are constitutively activated in Anxa1
−/− mice. Administration of hrANXA1 attenuated both cardiac and renal dysfunction caused by STZ induction of diabetes. While late treatment can halt both cardiac and renal dysfunction. We propose that attenuation of MAPK pathway signalling and restoration/activation of Akt survival pathways mediates these effects. Thus, we propose that treatment with hrANXA1 may represent a novel new intervention of microvascular complications caused by type 1 diabetes.