Cubilin maintains blood levels of HDL and albumin

Abstract

Cubilin is an endocytic receptor highly expressed in renal proximal tubules, where it mediates uptake of albumin and filtered forms of apoA-I/HDL. Cubilin deficiency leads to urinary loss of albumin and apoA-I; however, the consequences of cubilin loss on the homeostasis of blood albumin and apoA-I/HDL have not been studied. Using mice heterozygous for cubilin gene deletion (cubilin HT mice), we show that cubilin haploinsufficiency leads to reduced renal proximal tubular uptake of albumin and apoA-I and significantly increased urinary loss of albumin and apoA-I. Moreover, cubilin HT mice displayed significantly decreased blood levels of albumin, apoA-I, and HDL. The levels of albumin and apoA-I protein or mRNA expressed in the liver, kidney, or intestine of cubilin HT mice did not change significantly. The clearance rate of small HDL3 particles (density>1.13 g/ml) from the blood increased significantly in cubilin HT mice. In contrast, the rate of clearance of larger HDL2 particles from the blood did not change significantly, indicating a decreased half-life for HDL particles capable of filtering through the glomerulus. On the basis of these findings, we conclude that cubilin deficiency reduces renal salvage and delivery back to the blood of albumin and apoA-I, which decreases blood levels of albumin and apoA-I/HDL. These findings raise the possibility that therapeutic increase of renal cubilin expression might reduce proteinuria and increase blood levels of albumin and HDL.

Figure 1.

Cubn^{+/delexon1–6;EGFP} mice have reduced expression of cubilin protein in the kidney and intestine compared with WT mice. (A) Anticubilin, antimegalin, and antiactin immunoblot analysis of detergent extracts of kidney cortex from WT and Cubn^{+/delexon1–6;EGFP} (HT) mice. (B) Anticubilin and anti-actin immunoblot analysis of detergent extracts of ileum from WT and Cubn^{+/delexon1–6;EGFP} mice. (C and D) Densitometric analyses of the anticubilin immunoblots shown in A and B, respectively. Horizontal lines in C and D indicate median values for each data group. The findings are representative of at least three experiments.

Figure 2.

Nuclear magnetic resonance analysis of serum lipoproteins shows reduced serum HDL levels in Cubn^{+/delexon1–6;EGFP} mice. (A) VLDL. (B) LDL. (C) HDL particle concentration. (D) HDL-C measured in fasted serum samples from age-matched adult male Cubn^{+/delexon1–6;EGFP} (HT) and WT mice. Horizontal lines in each panel indicate median values for each data group. P value calculations were based on Mann–Whitney U tests (n=9 WT, 8 HT).

Figure 3.

FPLC gel filtration analysis of serum shows reduced serum HDL levels in Cubn^{+/delexon1–6;EGFP} mice. (A) Cholesterol. (B) Cholesterol ester. (C) Phospholipids. (D) Triglyceride levels in fractions of pooled serum samples (6 WT and 6 HT) subjected to gel filtration using Superose 6 column chromatography. Serum samples were obtained from age-matched adult male Cubn^{+/delexon1–6;EGFP} (HT) and WT mice after a 15-hour fast.

Figure 4.

Cubn^{+/delexon1–6;EGFP} mice have reduced plasma apoA-I and increased urinary apoA-I levels, which are inversely correlated. (A and B) Levels of apoA-I in plasma (A) and urine (B) samples obtained from age-matched 1-year-old male Cubn^{+/delexon1–6;EGFP} (HT) and WT mice. Samples were analyzed by a two-antibody sandwich ELISA using purified apoA-I as standard. Horizontal lines in A and B indicate median values for each data group. P value calculations in A and B were based on Mann–Whitney U tests. (C) The inverse correlation of plasma apoA-I and urinary apoA-I levels from Cubn^{+/delexon1–6;EGFP} (HT) (Spearman r=−0.70, P=0.02; linear regression R²=0.47, P=0.04) and WT mice (Spearman r=−0.78, P=0.003; linear regression R²=0.37, P=0.05).

Figure 5.

Cubn^{+/delexon1–6;EGFP} mice have reduced plasma albumin and increased urinary albumin levels. (A and C) Levels of albumin in plasma (A) and urine (C) samples obtained from age-matched 1-year-old male Cubn^{+/delexon1–6;EGFP} (HT) and WT mice. Samples were analyzed by a two-antibody sandwich ELISA for albumin. (B) Plasma samples analyzed for total protein using a bicinchoninic acid protein assay. Horizontal lines in each panel indicate median values for each data group. P value calculations in each panel were based on Mann–Whitney U tests.

Figure 6.

Cubilin haploinsufficiency does not affect ApoA-I and Abca1 mRNA expression in the intestine, liver, and kidney. qPCR analysis of Cubn (A and F), apoA-I (B, D, and G), and Abca1 (C, E, and H) transcript levels in mRNA extracted from intestine, liver, and kidney of age-matched Cubn^{+/delexon1–6;EGFP} (HT) and WT mice. Horizontal line indicates median values for each data group. P value calculations in each panel were based on Mann–Whitney U tests.

Figure 7.

Cubn^{+/delexon1–6;EGFP} mice have increased fractional clearance of small HDL particles. (A) Fractional clearance of human HDL₃ (d>1.13 g/ml) supplemented with 6% purified apoA-I infused into Cubn^{+/delexon1–6;EGFP} (HT) (n=6) and WT (n=3) mice. Areas under the curves in A were statistically different (P=0.05). (B) Fractional clearance of human HDL₂ (d=1.11–1.13 g/ml) infused into WT and HT mice (n=3 and 5, respectively). Analysis showed no statistically significant difference between the two data sets in B (i.e., area under the curves, P=0.11).

Figure 8.

Cubn^{+/delexon1–6;EGFP} mouse renal proximal tubules show reduced apical levels of apoA-I. (A) Image of a WT mouse kidney section immunolabeled with anti–apoA-I (red) and anti-EGFP (green). Nuclei were stained with Draq5. (B) Cubn^{+/delexon1–6;EGFP} kidney section labeled similarly to A. Note that very little or no apoA-I is present on the apical aspects of tubules expressing EGFP. (C) Graph showing the percentage of anti–apoA-I immunolabeling positive proximal tubules counted from 109 low-magnification fields from four WT and five HT mice. Horizontal line indicates median values for each data group. The P value was calculated using a Mann–Whitney U test.