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Abnormal proximal tubule apical membrane protein composition in X-linked hypophosphatemic mice
1Department of Pediatrics, University of Colorado Health Sciences Center, Denver.
Abstract:
The hypophosphatemic (Hyp) mouse is characterized by an isolated X-linked defect in proximal tubular phosphate (Pi) reabsorption associated with a decreased maximum velocity (Vmax) and a normal affinity (Km). To directly investigate the underlying cellular defect proximal tubular brush-border membranes (BBM) from normal control (Con) and Hyp male littermates were examined for differences in cholesterol content, total and individual phospholipid composition, phospholipid incorporation rates, membrane fluidity, and by two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide protein electrophoresis. The cholesterol content, total and individual phospholipid profiles, phospholipid incorporation rates, and membrane fluidity of Con and Hyp BBM samples were comparable. However, the two-dimensional gel electrophoreses of Con and Hyp BBM proteins, run simultaneously under identical conditions, revealed a protein with an apparent abnormal isoelectric migration pattern in Hyp BBM samples. This protein had an apparent molecular weight 56,000 and an apparent pI of 7.2 and was consistently evident on Hyp gels (n = 3) but not on Con gels (n = 3). The appearance of this protein band was associated with a diminution in staining of a control protein of comparable apparent molecular weight but markedly lower apparent pI.
Insights
Hyp mice exhibit X-linked hypophosphatemia due to impaired phosphate reabsorption. A specific abnormal protein was identified in their kidney brush-border membranes, suggesting a novel cellular defect in phosphate transport.
Area of Science:
- Nephrology
- Molecular Biology
- Biochemistry
Background:
- Hypophosphatemic (Hyp) mice display an X-linked defect in renal phosphate (Pi) reabsorption.
- This defect is characterized by reduced maximum velocity (Vmax) without altering affinity (Km).
Purpose of the Study:
- To investigate the cellular basis of the defect in proximal tubular phosphate transport in Hyp mice.
- To identify molecular differences in brush-border membranes (BBM) between normal and Hyp mice.
Main Methods:
- Comparative analysis of brush-border membranes (BBM) from normal control (Con) and Hyp male littermates.
- Assessment of membrane composition (cholesterol, phospholipids), fluidity, and protein profiles using two-dimensional SDS-polyacrylamide gel electrophoresis.
Main Results:
- No significant differences were observed in cholesterol content, phospholipid profiles, phospholipid incorporation rates, or membrane fluidity between Con and Hyp BBM.
- Two-dimensional gel electrophoresis revealed a unique protein (MW 56,000, pI 7.2) in Hyp BBM, absent in Con BBM.
- The presence of this abnormal protein correlated with a decrease in a control protein's staining intensity.
Conclusions:
- The identified abnormal protein in Hyp mouse BBM suggests a specific molecular alteration contributing to the phosphate transport defect.
- This finding points towards a potential novel cellular mechanism underlying X-linked hypophosphatemia.