Mutations in genes required for the glycosylation of α-dystroglycan lead to muscle and brain diseases known as dystroglycanopathies. However, the precise structure and biogenesis of the assembled glycan are not completely understood. Here we report that three enzymes mutated in dystroglycanopathies can collaborate to attach ribitol phosphate onto α-dystroglycan. Specifically, we demonstrate that isoprenoid synthase domain-containing protein (ISPD) synthesizes CDP-ribitol, present in muscle, and that both recombinant fukutin (FKTN) and fukutin-related protein (FKRP) can transfer a ribitol phosphate group from CDP-ribitol to α-dystroglycan. We also show that ISPD and FKTN are essential for the incorporation of ribitol into α-dystroglycan in HEK293 cells. Glycosylation of α-dystroglycan in fibroblasts from patients with hypomorphic ISPD mutations is reduced. We observe that in some cases glycosylation can be partially restored by addition of ribitol to the culture medium, suggesting that dietary supplementation with ribitol should be evaluated as a therapy for patients with ISPD mutations.
Background. The optimal perfusate partial pressure of oxygen (Po 2) during hypothermic machine perfusion (HMP) is unknown. The aims of the study were to determine the functional, metabolic, structural, and flow dynamic effects of low and high perfusate Po 2 during continuous HMP in a pig kidney ischemia-reperfusion autotransplant model. Methods. The left kidneys of a ±40 kg pigs were exposed to 30 minutes of warm ischemia and randomized to receive 22-hour HMP with either low perfusate Po 2 (30% oxygen, low oxygenated HMP [HMPO2]) (n = 8) or high perfusate Po 2 (90% oxygen, HMPO2high) (n = 8), before autotransplantation. Kidneys stored in 22-hour standard HMP (n = 6) and 22-hour static cold storage (n = 6) conditions served as controls. The follow-up after autotransplantation was 13 days. Results. High Po 2 resulted in a 3- and 10-fold increase in perfusate Po 2 compared with low HMPO2 and standard HMP, respectively. Both HMPO2 groups were associated with superior graft recovery compared with the control groups. Oxygenation was associated with a more rapid and sustained decrease in renal resistance. While there was no difference in functional outcomes between both HMPO2 groups, there were clear metabolic differences with an inverse correlation between oxygen provision and the concentration of major central metabolites in the perfusion fluid but no differences were observed by oxidative stress and metabolic evaluation on preimplantation biopsies. Conclusions. While this animal study does not demonstrate any advantages for early graft function for high perfusate Po 2, compared with low perfusate Po 2, perfusate metabolic profile analysis suggests that aerobic mechanism is better supported under high perfusate Po 2 conditions.
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