Fig 1.
Cystatin C binding to megalin and cubilin in surface plasmon analysis.
Surface plasmon resonance analysis showing binding of purified human cysC to immobilized megalin (A) and cubilin (B) at dilutions ranging from 20 nM to 500 nM. The calculated Kd was approximately 32 nM for binding to megalin and 24 nM for binding to cubilin. In (C) and (D) the flow of cystatin C (150 nM) was preceded by binding of RAP (5 μM) essentially abolishing the binding to megalin (C) and cubilin (D).
Fig 2.
Urinary cystatin C excretion in mice.
Western blot of urine samples, showing the presence of cysC in urine from cub -/-/meg-/- mice (lanes 1–4), cub -/- mice (lanes 5–8), and wildtype mice (lanes 9–12). CysC was identified in the urine from cub -/-/meg-/- mice only. The urines were loaded on the gel according to excretion rate.
Fig 3.
Renal cortical sections from mice.
Immunohistochemistry was used to identify megalin, cubilin, and cysC in kidney cortical sections from wildtype (A, E and F), cub -/- (G and H) and cub -/-/meg-/- mice (B, C and D). In proximal tubule brush border of wildtype mice the expression of cubilin (green A) and megalin (green B) co-localized with vesicular accumulation of cysC (red A and B). In Cub -/- mouse the labeling for cysC appeared similar to wildtype despite the absence of immuno-detectable cubilin (G). Megalin labeling appeared normal in Cub -/- (H). Note that megalin is also expressed in proximal tubule cells forming part of Bowman’s capsule with corresponding cysC uptake (arrows in H). In cub-/-/meg-/- mouse residual expression of megalin was observed only in a few mosaic tubular profiles (B and C (arrows indicating proximal tubules not expressing megalin)) when compared to wildtype (F) illustrating the high degree of conditional knockout. Uptake of cysC was observed only in the few proximal tubular cells with residual megalin expression selected in (C and D). Note that cells not expressing megalin do not reveal any cysC uptake (arrows in D). Scalebars in sections A, C, D, G and H correspond to 50 μm and scalebars in sections A and B correspond to 200 μm.
Table 1.
U-cysC excretion after I/R injury vs sham rats.
Fig 4.
Renal cortical sections of rats exposed to I/R injury.
HE stain of a cortical kidney section from I/R exposed rat (4A). Sham operated rat section is shown as insert in right corner for comparison. Evidence of acute tubular injury can be observed, consisting of tubular luminal distension (*), irregular nuclear size (#), interstitial inflammation (x) and loss of tubular cell adhesion to the basal membrane (arrow). Immunohistochemistry identified megalin (green) and cysC (red) in kidney cortical sections from rats two days after I/R injury (4B) or sham operation (4C). Normal, endocytic uptake of cysC was observed in most megalin expressing cells in sham operated rats (4C). In I/R operated rats, a clearly reduced, intracellular labeling for cysC was observed indicating a reduced endocytic uptake (4B), despite preserved expression of megalin in the proximal tubule brush border, Megalin, but not cysC, was identified in tubules with evidence of proximal tubule cell injury as indicated by *(4D). Scalebar = 50 μm.