Abstract
Nephronophthisis (NPH) is an autosomal recessive form of cystic kidney disease and the leading cause of hereditary kidney failure in children and young adults. Like other NPH proteins, the NPHP16/Anks6-interacting protein Anks3 has been identified to cause laterality defects in humans. However, the cellular functions of Anks3 remain enigmatic. We investigated the metabolic impact of Anks3 depletion in cultured murine inner medullary collecting duct cells via GC-MS profiling and LC-MS/MS analysis. Combined metabolomics successfully identified 155 metabolites; 48 metabolites were identified to be significantly altered by decreasing Anks3 levels. Especially, amino acid and purine/pyrimidine metabolism were affected by loss of Anks3. Branched-chain amino acids were identified to be significantly downregulated suggesting disrupted nutrient signalling. Tryptophan and 1-ribosyl-imidazolenicotinamide accumulated whereas NAD+ and NADP+ concentrations were diminished indicating disturbances within the tryptophan-niacin pathway. Most strikingly, nucleosides were reduced upon Anks3 depletion, while 5-methyluridine and 6-methyladenosine accumulated over time. Hence, elevated PARP1 and cleaved PARP1 levels could be detected. Furthermore, living cell number and viability was significantly declined. In combination, these results suggest that Anks3 may be involved in DNA damage responses by balancing the intracellular nucleoside pool.
Highlights
Recent findings revealed that a defectively mutated Anks[3] is causing laterality defects in an autosomal recessive manner[14]
It has been shown that both Anks[3] as well as Anks[6] are interacting with Bicaudal-C homologue 1 (Bicc1) which is mainly expressed in liver, pancreas and kidney functioning as negative regulator for canonical Wnt signalling[20,21,22,23]
Usage of gas-chromatography coupled to mass spectrometry (GC-MS) provides an excellent opportunity to monitor central metabolism in a global untargeted manner
Summary
Recent findings revealed that a defectively mutated Anks[3] is causing laterality defects in an autosomal recessive manner[14]. Recent studies revealed Anks[6] as a strong interaction partner of Anks[315]. Interaction of Anks[3] and Anks[6] with hypoxia-induced factor 1 alpha inhibitor (HIF1AN) was previously described[16,23]. Given that Anks[3] is required for ciliary motility and polarity[23] and interacting with important intracellular signalling cascades such as WNT signalling, we hypothesise that defects of Anks[3] might influence central cellular metabolism dependent on intact mitochondrial function for energy and biomass production. Targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses were conducted to monitor free amino acids, purine/pyrimidine metabolites as well as nucleosides
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