#1546 Mitochondrial oxidative phosphorylation in human proximal tubular epithelial cells is impaired by iron deficiency

Abstract Background and Aims We recently reported that iron deficiency may precede decline in renal function in patients with diabetes who had normal renal function at baseline but had a subsequent annual rate of eGFR decline of 3.3% or more (J Diabetes Investig. 2023;14:874-882.). Mitochondria contain many mitochondrial respiratory chain (MRC) enzymes that require iron as an active center, and consequently iron deficiency is known to impair mitochondrial function. Since the proximal tubular epithelial cells (PTECs) of the kidney are the cells with the greatest number of mitochondria in the body, we investigated whether iron deficiency impairs the mitochondrial capacity of the PTECs. Method Human cultured PTECs were analyzed in ‘control group’, ‘iron-deficient group ‘(co-cultured with deferiprone (DFP), an iron chelator), and ‘rescue group’ (co-cultured with DFP and FeCl3 which saturates the chelating ability of DFP), each with n ≥ 4 (MRC activities n = 4-5, others n ≥ 5). All analyses were performed after 24 hours of incubation. Values are shown as averages ± SEM. Results A Seahorse Extracellular Flux Analyzer showed that the basal oxygen consumption rates (OCRs) of control, iron-deficiency and rescue groups were 30.5 ± 1.2 pmol/min, 11.5 ± 0.7 pmol/min, and 31.1 ± 1.2 pmol/min, respectively, which revealed that basal OCRs of PTECs significantly decreased in the iron-deficient group. In addition, the analyzer also revealed that the activities of MRC for complexes I, II, and III, which have iron-sulfur clusters as their active centers, were significantly decreased in citrate synthase ratio to 77.6 ± 6.2%, 75.1 ± 6.4%, and 72.8 ± 9.3% of the control group, respectively, while complex IV was not significantly different at 84.9% of the control group. These reductions in MRC activities were not observed in the rescue group. Western blotting results also revealed that complex I, II, and III subunit proteins, NDUF9, SDHA, and UQCR2, were significantly reduced in the iron-deficient group (reduced to 38 ± 5.8%, 87 ± 8.8%, and 55 ± 5.7% of the control group, respectively). On the other hand, lactate concentrations in the culture medium of control group, iron-deficient group and rescue group were 1.73 ± 0.13 mmol, 3.01 ± 0.15 mmol and 1.61 ± 0.12 mmol, which revealed that glycolytic system was increased in the iron-deficient group, presumably to compensate for decreased oxidative phosphorylation. In response to intracellular iron deficiency, the iron regulatory protein 2 (IRP2) was shown in Western blotting to be unchanged in the rescue group (123 ± 4.0% of the control group), but increased in the iron-deficient group (170 ± 20% of the control group). The qRT-PCR analyses showed that mRNA expression of iron-sulfur cluster assembly enzyme (ISCU) was significantly decreased in the iron-deficiency group compared with the control group (41.8%), but not in the rescue group (95.1%), which is presumed that IRP2 suppressed ISCU expression, as has been proved previously. These results should indicate that iron deficiency suppressed iron utilization in the mitochondria of PTECs. The number of viable PTECs in the iron-deficient group after 24 hours was 1.99 ± 0.09 × 10^5 cells/well, which was significantly lower than that of the control (3.15 ± 0.15 × 10^5 cells/well) and rescue (2.68 ± 0.18 × 10^5 cells/well) groups. On the other hand, the number of dead PTECs was 0.042 ± 0.019 × 10^5 cells in the iron-deficient group, which was not significantly different from the control (0.023 ± 0.005 × 10^5 cells) and the rescue (0.020 ± 0.011 × 10^5 cells) groups. These data suggest that cell growth should be suppressed by iron deficiency. Conclusion Mitochondrial oxidative phosphorylation through the MRC was impaired by iron deficiency in PTECs. This impairment of mitochondrial capacity in PTECs may lead to PTECs dysfunction, potentially inducing renal damage such as cell death and interstitial injury. Further analysis of the effects of this mitochondrial dysfunction on ATP production capacity and reactive oxygen species production is needed.