The role of ferroptotic cell death in diabetic nephropathy

PhD Student: Francesca Maremonti
Supervisor at TUD:  Andreas Linkermann
Supervisor at KCL:  Christer Hogstrand, Francesco Rubino
Start Date: 01.04.2020

Type I Diabetes Mellitus (T1D) is a chronic autoimmune disease that eventually lead to the disruption of pancreatic beta cells. The increasing loss of beta cells, due to the autoimmune process, finally cause an insulin deficiency and an increase in blood glucose levels (hyperglycaemia). The persistent glycaemic dysregulation associated with this condition is the cause of a series of chronic complications at level of many organ systems, accountable for the majority of morbidity and mortality associated with this disease. The mechanisms by which chronic hyperglycaemia leads to diabetic nephropathy is unknown. An important signalling pathway of tubular damage in the progression of diabetic nephropathy is referred to as ferroptosis, but so far little is known about its role in diabetic nephropathy. Ferroptosis is a regulated necrotic cell death pathway that results from iron-catalysed lipid peroxidation and further on in the rupture of the plasma membrane. Another important feature of ferroptosis is its capability to affect functional units rather than single cells, causing a “wave-of-death”-like cell death propagation phenotype termed synchronized regulated necrosis (SRN).

Given i) the susceptibility of diabetic patients to acute kidney injury and ii) the emerging role of ferroptosis in the pathophysiology of tubular necrosis, we aim to investigate the relative contribution of ferroptosis-induced cell death to kidney disease (DK), especially in T1D. We hypothesize, that diabetic tissues and/or the presence of hyperglycaemia predispose and/or sensitize to SRN and ferroptosis. In particular, we aim to test different ferroptosis inducers in kidney tubules derived from mice, as well as in primary kidney tubular cells.

Induction of ferroptosis can be achieved in several ways by small molecules referred to as ferroptosis-inducing agents (FINs). These molecules are classified in Type I-IV FINs, each group targeting a different pathway to induce ferroptosis. In order to acquire confidence with cell death induction and its rescue, a panel of different ferroptosis inducers (e.g. erastin, RSL3, ferroptocide) were employed to incubate ferroptosis-sensitive cell lines. The induced cell death was assessed with flow cytometry analysis (FACS). Cells were stained with annexin V, marker for phosphatidylserine exposure, and 7-AAD, a vital dye. To confirm the necrotic type cell death to be ferroptotic, inhibitors of ferroptosis, e. g. ferrostatin-1 (Fer-1) was added as co-treatment at 1 µM concentration. Reversal of necrosis was interpreted as an indirect detection of ferroptosis.

In future,we are looking forward to investigating the influence of type 1 diabetes mellitus to the threshold of ferroptosis in murine tubules and primary cells. In addition, this project holds promise to potentially translate findings to clinically identified susceptible individuals who might be at risk for SRN and dialysis.

Publication:

Dysfunction of the key ferroptosis-surveilling systems hypersensitizes mice to tubular necrosis during acute kidney injury. W. Tonnus, C. Meyer, C. Steinebach, A. Belavgeni, A. von Mässenhausen, N.Z. Gonzalez, F. Maremonti, F. Gembardt, N. Himmerkus, M. Latk, S. Locke, J. Marschner, W. Li, S. Short, S. DollS, I. Ingold, B. Proneth, C. Daniel, N. Kabgani, R. Kramann, S. Motika, P.J. Hergenrother, S.R. Bornstein, C. Hugo, J.U. Becker, K. Amann, H.J. Anders, D. Kreisel, D. Pratt, M. Gütschow, M. Conrad, A. Linkermann. Nat Commun. 2021;12(1):4402.