Untersuchung der pharmakologischen Modulation von ARNT-Homodimeren auf die Renoprotektion

The pharmacological effect of ARNT homodimers on renoprotection

by Gregor Christof Rapp
Doctoral thesis
Date of Examination:2023-10-11
Date of issue:2023-10-02
Advisor:Prof. Dr. Michael Zeisberg
Referee:Prof. Dr. Dr. Albrecht Neeße
Referee:Prof. Dr. Margarete Schön
Persistent Address: http://dx.doi.org/10.53846/goediss-10120

 

 

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Abstract

English

Chronic kidney disease is marked by progressive fibrosis and consecutive loss of function – the availability of specific therapies is limited. The bHLH-PAS transcription factor ARNT is able to form homodimeric complexes and to induce the expression of the renoprotective ALK3 in the tubular epithelium by binding to the ALK3 promoter. Pharmacologically, ARNT expression can be induced by a non-immunosuppressive dose of the pharmaceutical FK506 (low-dose) or by the small molecule GPI-1046. In the research literature, the protein phosphatase inhibitor okadaic acid is described as an inducer of ARNT homodimer formation. While the mechanisms of heterodimerisation of ARNT with other bHLH-PAS transcription factors are well known, the mechanism of homodimerization of ARNT is unknown by now. The aim of the thesis was to identify a mechanism of ARNT homodimer formation and to explore therapeutic effects by pharmacological modulation of ARNT homodimers on renal fibrosis in the context of chronic kidney disease. In transgenic HEK293 cells, it was demonstrated that the formation of ARNT homodimers is underlying PP2A inhibition. The specific PP2A inhibitor LB-100 induced the formation of ARNT homodimers in vitro and increased protein stability of Arnt. A transcriptional effect of LB-100 on Arnt expression was not observed by qRT-PCR. Endogenous ARNT homodimers were detected in MCT cells by Blue Native-PAGE and MALDI-TOF analysis. Serine-77 in ARNT was identified by mass spectrometry as a relevant phosphorylation site for the formation of ARNT homodimers. By using an aspartic acid point mutant of ARNT, the specific relevance of phosphorylation at serine-77 on the formation of ARNT homodimers has been proved in transgenic HEK293 cells. The antifibrotic effect of ARNT homodimers was investigated in the mouse model of unilateral ureteral obstruction (UUO). For this study, C57BL/6J wild-type mice were treated with LB-100, FK506, GPI-1046 as a monotherapy or in combination, FK506 + LB-100 and GPI-1046 + LB-100. Combination therapy showed an enhanced formation of ARNT homodimers, an increased expression of the Alk3 and additive antifibrotic effects in murine kidneys. The renoprotective effect of ARNT homodimers could be used in the future for the treatment of chronic kidney disease.
Keywords: ARNT; fibrosis; HIF-1β; kidney; CKD; pSMAD1/5/8; aryl hydrocarbon receptor nuclear translocator; ALK3 (BMPR1A); homodimerization; PP2A; Serine-77; renal fibrogenesis; GPI-1046; FK506; LB-100; bHLH-PAS
Schlagwörter: renale Fibrogenese; Homodimerisierung; Fibrose; Serin-77; Tubulusepithelzellen; chronische Nierenerkrankung; PP2A-Inhibition
 
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