Article Figures & Data
Figures
- FIGURE 1.
Megalin/cubilin-dependent renal accumulation of 99mTc-DMSA. Whole-body scintigram of mice 6 h after injection of 99mTc-DMSA shows accumulation in kidneys of control mice (A) but no accumulation in kidneys of megalin/cubilin-deficient mice (B). In megalin/cubilin-deficient mice, evidence of accumulation of tracer in bladder was seen. Images shown are representative of similar analyses (n = 3 in each group).
- FIGURE 2.
Renal uptake and urinary excretion of 99mTc-DMSA by megalin/cubilin-deficient and control mice. Renal uptake (A), urinary excretion (B), and plasma levels (C) of 99mTc-DMSA in megalin/cubilin-deficient mice are compared with controls. Columns represent mean of each group (n = 7) ± SD. *Significantly different from control mice (P < 0.0001).
- FIGURE 3.
Urinary excretion of 99mTc-MAG3 by megalin/cubilin-deficient and control mice. Columns represent mean of each group (n = 7) ± SD.
- FIGURE 4.
Urinary excretion of protein-bound 99mTc-DMSA by megalin/cubilin-deficient mice. (A) SDS-PAGE separation and autoradiography of urine from megalin/cubilin-deficient mice and controls injected with 99mTc-DMSA. A single, labeled band of approximately 27 kDa is observed in urine collected from megalin/cubilin-deficient mice only. (B) Urine profile analysis by SDS-PAGE and silver staining of urine from megalin/cubilin-deficient mice and controls. A distinct protein band of 27 kDa was identified in urine from megalin/cubilin-deficient mice (*). This band was excised and protein identified by MALDI MS as α1-microglobulin (Table 2). (C) Immunoblotting identifying α1-microglobulin as an approximate 27-kDa band in urine from megalin/cubilin-deficient mice but not from controls. Data shown are representative of 2 (autoradiography and MALDI MS) or 3 (SDS-PAGE, silver staining, and immunoblotting) independent experiments.
- FIGURE 5.
Megalin/cubilin-dependent renal uptake of α1-microglobulin. Immunohistochemical staining is shown for megalin (green) and α1-microglobulin (red) in kidney cortex of control and megalin/cubilin-deficient mice. In megalin/cubilin-deficient mice, a few profiles of megalin-expressing proximal tubule cells can be identified (*) because of mosaic expression. Merged images showed that α1-microglobulin cannot be identified in proximal tubular cells lacking megalin.
- FIGURE 6.
Mechanism for renal uptake of 99mTc-DMSA. (A) α1-microglobulin–bound 99mTc-DMSA is freely filtered by glomeruli and accumulates in renal proximal tubules by endocytosis mediated by multiligand-binding megalin/cubilin receptors (B). (C) Free 99mTc-DMSA and trace amounts of α1-microglobulin–bound 99mTc-DMSA are excreted in urine. Consequently, megalin/cubilin dysfunction leads to abolishment of renal uptake and increased urinary excretion of α1-microglobulin–bound 99mTc-DMSA.
Tables
Molecular weight (kDa) Control Megalin/cubilin-deficient >100 4 8 30–100 9 43 10–30 4 9 3–10 3 2 <3 20 19 Values are from mixed urines (n = 3) and expressed as percentage of injected dose.
- TABLE 2
Mass Spectrometry (MS/MS) Analysis of Trypsin-Cleaved 27-kDa Band from Urine Separated by SDS-PAGE
Protein Measured peptide mass (Da) Peptide sequence identified by MS/MS α1-microglobulin* 1,131.6 K.LYGREPQLR.D 1,354.6 R.GVCEEITGAYQK.T 1,510.7 R.RGVCEEITGAYQK.T 1,809.8 K.WYNLAVGSTCPWLSR.I 2,090.0 K.DVALNVGISENSIIFMPDR.G ↵* Mascot score, 393.
