Iron is suggested to play a detrimental role in the progression of chronic kidney disease (CKD). The kidney recycles iron back into the circulation. However, the localization of proteins relevant for physiological tubular iron handling and their potential role in CKD remain unclear. We examined associations between iron deposition, expression of iron handling proteins and tubular injury in kidney biopsies from CKD patients and healthy controls using immunohistochemistry. Iron was deposited in proximal (PT) and distal tubules (DT) in 33% of CKD biopsies, predominantly in pathologies with glomerular dysfunction, but absent in controls. In healthy kidney, PT contained proteins required for iron recycling including putative iron importers ZIP8, ZIP14, DMT1, iron storage proteins L- and H-ferritin and iron exporter ferroportin, while DT only contained ZIP8, ZIP14, and DMT1. In CKD, iron deposition associated with increased intensity of iron importers (ZIP14, ZIP8), storage proteins (L-, H-ferritin), and/or decreased ferroportin abundance. This demonstrates that tubular iron accumulation may result from increased iron uptake and/or inadequate iron export. Iron deposition associated with oxidative injury as indicated by heme oxygenase-1 abundance. In conclusion, iron deposition is relatively common in CKD, and may result from altered molecular iron handling and may contribute to renal injury.

In patients with iron overload disorders, increasing number of reports of renal dysfunction and renal iron deposition support an association between increased iron exposure and renal injury. In systemic iron overload, elevated circulating levels of transferrin-bound (TBI) and non-transferrin-bound iron (NTBI) are filtered to the renal proximal tubules, where they may cause injury. However, the mechanisms of tubular iron handling remain elusive. To unravel molecular renal proximal tubular NTBI and TBI handling, human conditionally immortalized proximal tubular epithelial cells (ciPTECs) were incubated with 55 Fe as NTBI and fluorescently labeled holo-transferrin as TBI. Ferrous iron importers ZIP8 and ZIP14 were localized in the ciPTEC plasma membrane. Whereas silencing of either ZIP8 or ZIP14 alone did not affect 55 Fe uptake, combined silencing significantly reduced 55 Fe uptake compared to control (p < 0.05). Furthermore, transferrin receptor 1 (TfR1) and ZIP14, but not ZIP8, colocalized with early endosome antigen 1 (EEA1). TfR1 and ZIP14 also colocalized with uptake of fluorescently labeled transferrin. Furthermore, ZIP14 silencing decreased 55 Fe uptake after 55 Fe-Transferrin exposure (p < 0.05), suggesting ZIP14 could be involved in early endosomal transport of TBI-derived iron into the cytosol. Our data suggest that human proximal tubular epithelial cells take up TBI and NTBI, where ZIP8 and ZIP14 are both involved in NTBI uptake, but ZIP14, not ZIP8, mediates TBI-derived iron uptake. This knowledge provides more insights in the mechanisms of renal iron handling and suggests that ZIP8 and ZIP14 could be potential targets for limiting renal iron reabsorption and enhancing urinary iron excretion in systemic iron overload disorders. Electronic supplementary material The online version of this article (10.1007/s10534-019-00183-7) contains supplementary material, which is available to authorized users.

In physiological conditions, circulating iron can be filtered by the glomerulus and is almost completely reabsorbed by the tubular epithelium to prevent urinary iron wasting. Increased urinary iron concentrations have been associated with renal injury. However, it is not clear whether increased urinary iron concentrations in patients are the result of increased glomerular iron filtration and/or insufficient tubular iron reabsorption and if these processes contribute to renal injury. We measured plasma and urine iron parameters and urinary tubular injury markers in healthy human subjects ( n = 20), patients with systemic iron overload ( n = 20), and patients with renal tubular dysfunction ( n = 18). Urinary iron excretion parameters were increased in both patients with systemic iron overload and tubular dysfunction, whereas plasma iron parameters were only increased in patients with systemic iron overload. In patients with systemic iron overload, increased urinary iron levels were associated with elevated circulating iron, as indicated by transferrin saturation (TSAT), and increased body iron, as suggested by plasma ferritin concentrations. In patients with tubular dysfunction, enhanced urinary iron and transferrin excretion were associated with distal tubular injury as indicated by increased urinary glutathione S-transferase pi 1-1 (GSTP1-1) excretion. In systemic iron overload, elevated urinary iron and transferrin levels were associated with increased injury to proximal tubules, indicated by increased urinary kidney injury marker 1 (KIM-1) excretion. Our explorative study demonstrates that both glomerular filtration of elevated plasma iron levels and insufficient tubular iron reabsorption could increase urinary iron excretion and cause renal injury.

Iron can catalyze reactive oxygen species (ROS) formation, causing cellular injury. In systemic iron overload, renal tubular epithelial cells are luminally exposed to high iron levels due to glomerular filtration of increased circulating iron. Reports of tubular dysfunction and iron deposition in β-thalassemia major support an association between increased chronic iron exposure and renal tubular injury. In acute iron exposure, Nuclear factor-erythroid 2-related factor 2 (Nrf2) may protect from iron-induced injury, whereas chronic renal stress may lead to Nrf2 exhaustion. We studied the cytotoxic mechanisms of chronic iron exposure using human conditionally immortalized proximal tubular epithelial cells (ciPTECs). Long-term iron exposure resulted in iron accumulation, cytosolic ROS formation and increased heme oxygenase 1 (HMOX-1) mRNA expression (all p < 0.001). This was accompanied by nuclear translocation of Nrf2 and induction of its target protein NQO1, which both could be blocked by the Nrf2 inhibitor trigonelline. Interestingly, iron and trigonelline incubation reduced ROS production, but did not affect HMOX-1 mRNA levels. Moreover, ferritin protein and CHOP mRNA expression were induced in combined iron and trigonelline incubated cells (p < 0.05). Together, these findings suggest that chronic iron exposure induces oxidative stress and that exhaustion of the antioxidant Nrf2 pathway may lead to renal injury.

In the original version of this Article, the name of author Marius C. van den Heuvel was incorrectly given as Marius van den Heuvel. In addition the authors Sanne van Raaij, Rachel van Swelm, Marius C. van den Heuvel and Harry van Goor were incorrectly indexed.