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Broeker 2014 Thesis

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Publications in the MiPMap
Broeker C (2014) Mitochondriopathien als Ursache des renalen Fanconi-Syndroms. Dissertation 85pp.

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Broeker C (2014) Dissertation

Abstract: Renal Fanconi syndrome is defined as an impaired transport capacity of the proximal tubule and can be caused by a variety of factors. Besides secondary Fanconi syndromes caused by another disease or as side effects of medication primary forms exist as a consequence of a mutation. Here, two distinct forms of renal Fanconi syndrome are described which are both inherited in an autosomal dominant fashion. Firstly, EHHADH, a protein involved in peroxisomal β-oxidation, and secondly FAP2, a component of creatine synthesis.

EHHADH was already known to be expressed in the late proximal tubule. This was confirmed by staining with two commercial antibodies, one staining for EHHADH and another staining for the peroxisomal protein D-amino acid oxidase. In both cases, a positive staining was visible in the kidney cortex in the late proximal tubule (S2/S3 segment). The observed granular staining was consistent with peroxisomal localization. I therefore concluded that EHHADH is expressed in peroxisomes of the late proximal tubule of the kidney. Previous experiments indicated that mutated EHHADH is also imported into the mitochondria. Intramitochondrial localization was evaluated electron microscopically using the ReAsH method. In EHHADHWT-transfected cells only the peroxisomes were positive. No electron-dense precipitates were visible in mitochondria. In contrast, in EHHADHMUT-transfected cells both peroxisomes and mitochondria were positive. In my diploma thesis I could already determine that mitochondrial import of EHHADHMUT leads to a decreased activity of oxidative phosphorylation. To evaluate if this affects transcellular transport α-D-methyl glucoside was added to the medium of cells which were grown on filters. This glucose derivative is transported but not metabolized by cells. Transport in EHHADHMUT-expressing cells was reduced to 25% of the capacity of EHHADHWT-expressing cells.

These data reveal new insights into the pathogenesis of this form of Fanconi syndrome. Mutated EHHADH is mistargeted to the mitochondria where it interferes with mitochondrial β-oxidation. This in turn leads to a decreased ATP production and subsequently to an impaired transepithelial transport capacity in the proximal tubule of affected patients. Keywords: Physiology, Tubulopathy, Mitochondria, Pig proximal tubulus LLC PK1 cells

O2k-Network Lab: DE Regensburg Renner-Sattler K


Labels: MiParea: Respiration, Genetic knockout;overexpression  Pathology: Other 

Organism: Pig  Tissue;cell: Kidney, Other cell lines  Preparation: Permeabilized cells 


Coupling state: LEAK, OXPHOS, ET  Pathway: N, CIV, NS, ROX  HRR: Oxygraph-2k 

2016-05 

Abstract continued

By contrast, FAP2 is a genuine mitochondrial protein. Expression in human kidney could be narrowed down to the proximal tubule where it was localized in the mitochondria. Late proximal tubules as well as medullary regions were negative for FAP2. A mutation in the gene coding for FAP2 was investigated closer using a stably transfected cell line. Cells overexpressing wildtype FAP2 showed a mild phenotype. Mitochondria appeared bloated and fragmented, which is most likely caused by overexpression and the resulting increased mitochondrial import of the protein. This was also confirmed by electron microscopy. Mitochondria of cells overexpressing FAP2MUT clearly showed elongated mitochondria throughout the entire cell. Electron microscopy revealed filament-like intramitochondrial deposits. Immuno-gold labelling revealed FAP2MUT to be a component of these filaments. Half-life of FAP2MUT was greatly increased indicating an impaired degradation of FAP2MUT-containing filaments. Despite the severe changes in mitochondrial morphology no difference was observed in respiratory measurements of the oxidative phosphorylation between FAP2WT and FAP2MUT cells. The observed Fanconi syndrome doesn’t appear to be caused by an impaired energy provision.

Furthermore, a biopsy of a patient’s kidney was examined. HE staining revealed well preserved glomeruli and distal segments. In contrast, proximal segments showed signs of tubular damage. Electron microscopical images showed evidence of giant mitochondria with intramitochondrial filament-like inclusions. These results suggest that these filaments are no cell culture artefacts but are rather involved in the in vivo pathogenesis. The observed Fanconi syndrome and subsequent renal failure does not appear to be caused by decreased oxidative phosphorylation. However, the underlying pathomechanism has yet to be elucidated.

Both forms of Fanconi syndrome described in this thesis develop due to mitochondriopathies. While in the case of mutated EHHADH a decreased energy provision causes the disease, the underlying pathomechanisms of FAP2MUT are more complex and require further investigation.