The role of genetical and pharmacological blockade of necroptosis in translational models of acute kidney injury and systemic inflammatory response syndrome

For some decades, apoptosis was thought to represent the sole form of regulated cell death, whereas necrosis was thought to be unregulated and accidental. However, differnet pathways of regulated necrosis have been recently established. Necroptosis was identified first and depends on formation of the necrosome, a cytosolic complex containing RIPK3, which phosphorylates MLKL, the effector of necroptosis.

Regarding Nec-1, the prototypical necroptosis blocking agent (necrostatin), and RIPK3-deficient mice protection in pre-clinical models of TNFα-induced systemic inflammatory response syndrome (SIRS, sterile septic shock) and renal ischemia/reperfusion injury (IRI, acute kidney injury) has been demonstrated. Nevertheless, it remains unclear whether this protection is due to blockade of necroptosis or differing outcomes of RIPK1/ RIPK3 signaling.

Thus, we aimed to verify necroptotic involvement by testing MLKL^-/--mice in these models and to identify drugs already in clinical use as off-label inhibitors of necroptosis. We hypothesized that ion currents mediate osmotic swelling in necroptotic cell death and, thus, ion channel blocking agents could be necrostatins.

In vivo, we utilized genetically modified mice as well as small molecules in above mentioned mouse models of IRI and TNFα-shock to test necroptotic involvement. Primary endpoints for IRI included histopathological severity of kidney damage and serum levels of creatinine and urea. Regarding SIRS, overall survival was the primary endpoint. Complementary, immunohistochemistry of pMLKL was performed in human kidney biopsies. Furthermore, TNFα-dependent necroptosis was induced in cell culture and effects of small molecules were analyzed by Western blot and FACS. Furthermore, TNFα-independent necroptosis was induced to elucidate mechanisms of inhibition by small molecules.

In TNFα-shock we found MLKL^-/--mice to be significantly stronger protected than RIPK3^-/--mice, but unlike the complete protection seen in Caspase-8/RIPK3d^-/--mice. For RIPK3^-/--mice being known to be protected in renal IRI over wildtypes, we found MLKL^-/- to yield even stronger protection in histology and serum levels of creatinine and urea.

Human kidney biopsies stained positive for pMLKL in areas of acute tubular necrosis, demonstrating a possible involvement of necroptosis in acute kidney injury. To evaluate the potential of pMLKL as a biomarker, we induced necroptosis in HT29 cells and found phosphorylation of MLKL to be required, but not sufficient to drive necroptosis.

Screening FDA-approved ion channel blocking agents, we found antiepileptic drug phenytoin to be a necrostatin. To elucidate the mechanism of phenytoin action, we utilized a chemical dimerizer of RIPK3 to induce TNFα-independent necroptosis. Falsifying our initial hypothesis, phenytoin failed to block necroptosis in this system, pointing to a mechanism upstream of pMLKL. Immunoprecipitation demonstrated Phenytoin to bind RIPK1 and RIPK3, inhibiting necrosome formation and, simultaneously, weakly inhibiting kinase activity of RIPK1 as demonstrated in a cell-free system. In vivo, treatment with phenytoin prolonged survival of wildtype mice in TNFα-shock significantly over vehicle-treated ones. Along these lines, Phenytoin reduced histopathological damage and levels of serum creatinine and urea in renal IRI. Unlike RIPK3^-/-- or MLKL^-/--mice Phenytoin-treated wildtypes did not display changes in renal perfusion compared to vehicle-treated ones. Interestingly, Phenytoin and Nec-1 both are hydantoin derivates. Testing the hypothesis that this structural overlap might be responsible for protection, we challenged wildtypes in IRI with 5-benzyl hydantoin, a hydantoin with a benzyl ring instead of Nec-1´s indole. Compared to vehicle treatment, this hydantoin also demonstrated protection.

In summary, we partially confirmed the initial hypothesis of necroptosis blockade being the mechanism of protection by Nec-1 or RIPK3-deficiency in renal IRI or TNFα-shock. However, further mechanisms seem to participate, especially demonstrated by the complete protection of Caspase-8/RIPK3dko-mice. The hypothesis of differing renal perfusion as a protective mechanism in RIPK3^-/-´s could not be falsified by us, as MLKL^-/-´s demonstrated the same phenotype. On the other hand, phenytoin did not change perfusion, but was also protective in IRI; thus, not all effects can be explained by differing perfusion. We conclude that necroptosis is involved in pathogenesis of these models, which is further underpinned by positive staining for pMLKL in human tubular necrosis.

Hypothesizing that ion channels mediate necroptosis terminally, we identified phenytoin in a cellular screen as a necrostatin. Phenytoin was capable of blocking necroptosis in vitro but failed to do so when necroptosis was induced in a TNFα-independent manner, falsifying the initial hypothesis of ion channels as anti-necroptotic target. Concomitantly, blockade of calcium currents in vitro did not alter necroptotic kinetics. Further workup demonstrated binding to RIPK1 and RIPK3 and, by doing so, inhibiting RIPK1 kinase activity and necrosome formation as mechanism of phenytoin action. However, phenytoin was protective in vivo, demonstrating effects in TNFα-shock and renal IRI. Thus, we demonstrate necroptosis inhibition to be effective by genetic and pharmacological means. The structural analogies between Nec-1 and phenytoin led to the new hypothesis of the hydantoin being responsible for action. In line with this, 5-benzyl hydantoin also demonstrated protection in IRI. This led to the conclusion of hydantoins blocking necroptosis per se, which, however, might be problematic as hydantoins also interfere with ferroptosis. To which extend this interfered with our results is, currently, unclear.

Publications:

A.S. Greve, …, W. Tonnus, et.al. Front Cell Infect Microbiol. 2017;7:113.

S. Martens, M. Jeong, W. Tonnus, et.al.Cell Death Dis. 2017;8:e2904.

W. Tonnus, F. Gembardt, C. Hugo, A. Linkermann. Oncotarget. 2017;8:41790-41791.

W. Tonnus, C. Hugo, A. Linkermann. Kidney Int. 2017;91:267-269.

W. Tonnus, A. Linkermann. Immunol Rev. 2017;277:128-149.

M. Sarhan, W.G. Land, W. Tonnus, C.P. Hugo, A. Linkermann. Physiol Rev. 2018;98:727-780.

W. Tonnus, M. Al-Mekhlafi, F. Gembardt, C. Hugo, A. Linkermann. Methods Mol Biol. 2018;1857:145-151.

W. Tonnus, M. Al-Mekhlafi, C. Hugo, A. Linkermann. Methods Mol Biol. 2018;1857:135-144.

A. von Massenhausen*, W. Tonnus*, et.al.Cell Death Dis. 2018;9:359.

A. von Massenhausen, W. Tonnus, A. Linkermann. Nephron. 2018;140:144-147.

A. Belavgeni,…, W. Tonnus, et.al. Proc Natl Acad Sci U S A. 2019;116:22269-22274.

W. Tonnus, A. Belavgeni, Y. Xu, A. Linkermann. Kidney Int. 2019;95:736-738.

W. Tonnus, et.al. Cell Death Differ. 2019;26:68-82.

W. Tonnus, A. Linkermann. Kidney Int. 2019;96:1061-1063.

W. Tonnus, A. Linkermann. Saunders, 2019, pp. 197-205.e191.

W. Tonnus, et.al. J Pathol. 2019;247:697-707.

H. Henneicke, W. Tonnus, L.C. Hofbauer. Lancet Diabetes Endocrinol. 2020;8:456.

B. Demarco,…, W. Tonnus, et.al. Sci Adv. 2020;6:eabc3465.

J. Stumpf, W. Tonnus, et.al.Transplantation. 2021. Epub ahead of print.

W. Tonnus, et.al.Nat Commun. 2021;12(1):4402.

W. Tonnus, et.al. Nat Rev Endocrinol. 2021;17(8):497-510.