Role of Iron in Collagen-expressing Proangiogenic Macrophages in Diabetic Nephropathy

ABSTRACT

Diabetic nephropathy is the leading cause of end-stage renal disease (ESRD) worldwide. The number of patients requiring hemodialysis for diabetes-related kidney disease has increased dramatically over the past two decades. Diabetes mellitus remains the leading cause of end-stage kidney disease (ESKD) in the United States, accounting for approximately 40% of patients requiring renal replacement therapy. Therefore, it is important to identify novel interventions to halt the progression of diabetic nephropathy.

Inflammatory mechanisms, including macrophage (MΦ) recruitment into the glomerulus and tubulointerstitial region, play an important role in diabetic nephropathy. In diabetic kidneys, macrophage development and accumulation depend on angiotensin II. Iron accumulation occurs in diabetic nephropathy. Iron dysregulation and heme oxygenase-1 (HO-1), an inducible regulator of heme and iron metabolism, have anti-inflammatory effect, so we need to assess their pro-fibrotic role and oxidative stress; they may promote macrophage profibrotic responses. These macrophages also play a central role in recycling iron from senescent erythrocytes. They can contain substantial intracellular and extracellular pools of catalytic iron, which promote free radical formation and oxidative stress.

We investigated the role of iron in collagen-secreting macrophages under hyperglycemic conditions. Human peripheral blood mononuclear cells (PBMCs) were cultured with normal (5 mM) or high (25 mM) glucose (GLUC) for 2–8 days in the presence of angiotensin II (Ang II). Ang II (0.1 or 1.0 µM) was added for 24 hours to 4 days. We assessed macrophage phenotype, procollagen-1 (PC-1), ferroportin (FPN), heme oxygenase-1 (HO-1), and angiogenic markers using immunofluorescence, Western blotting, and flow cytometry. Catalytic iron levels were measured, and the effect of iron chelation was evaluated using deferiprone (DFP; 25 µM). We examined human kidney tissue for CD163 and procollagen-1 expression. Our results showed that high glucose increased the number of CD163+ and CD68+ macrophages expressing procollagen-1. Ang II further enhanced these responses. Hyperglycemia and Ang II increased catalytic iron levels (P < 0.0001) and altered HO-1 and FPN expression, consistent with activation of iron- and oxidative stress-related pathways. Collagen-expressing macrophages displayed an angiogenic phenotype, with increased lectin and acetylated low-density lipoprotein (LDL) expression. Deferiprone significantly reduced macrophage adhesion, PC-1 expression, and angiogenic markers. In human kidney tissue, CD163+ macrophages and procollagen-1+ cells were detected in the interstitium and sclerotic glomeruli, with procollagen-1+ cells predominantly localized to the interstitium.

Hyperglycemia and Ang II promote iron accumulation and the development of collagen-secreting, fibrotic, and proangiogenic macrophages. These cells may play an important pathophysiological role in the progression of diabetic nephropathy. The association with FPN and HO-1 suggests that iron recycling pathways may be involved in this response. Iron chelation attenuated these macrophage phenotypes, supporting the iron–FPN–HO-1 axis as a potential therapeutic target in diabetic nephropathy.

Our findings identify catalytic iron accumulation as a potential driver of a collagen-secreting, proangiogenic macrophage phenotype induced by the combined dysmetabolic and renin–angiotensin stress associated with diabetes.

Keywords: Angiogenic markers, Angiotensin II, Catalytic iron, diabetic nephropathy, Deferiprone, iron chelation, Glucose, pro-collagen and profibrotic positive macrophages, peripheral blood mononuclear cells