Expression of apolipoprotein B in the kidney attenuates renal lipid accumulation

Abstract

The ability to produce apolipoprotein (apo) B-containing lipoproteins enables hepatocytes, enterocytes, and cardiomyocytes to export triglycerides. In this study, we examined secretion of apoB-containing lipoproteins from mouse kidney and its putative impact on triglyceride accumulation in the tubular epithelium. Mouse kidney expressed both the apoB and microsomal triglyceride transfer protein genes, which permit lipoprotein formation. To examine de novo lipoprotein secretion, kidneys from human apoB-transgenic mice were minced and placed in medium with (35)S-amino acids. Upon sucrose gradient ultracentrifugation of the labeled medium, fractions were analyzed by apoB immunoprecipitation. (35)S-Labeled apoB100 was recovered in approximately 1.03-1.04 g/ml lipoproteins (i.e. similar to the density of plasma low density lipoproteins). Immunohistochemistry of kidney sections suggested that apoB mainly is produced by tubular epithelial cells. ApoB expression in the kidney cortex was reduced approximately 90% in vivo by treating wild type mice with apoB-antisense locked nucleic acid oligonucleotide. Inhibition of apoB expression increased fasting-induced triglyceride accumulation in the kidney cortex by 20-25% (p = 0.008). Cholesterol stores were unaffected. Treatment with control oligonucleotides with 1 or 4 mismatching base pairs affected neither the triglyceride nor the cholesterol content of the kidney cortex. The results suggest that mammalian kidney secretes apoB100-containing lipoproteins. One biological effect may be to dampen excess storage of triglycerides in proximal tubule cells.

FIGURE 1.

Expression of mouse apoB, human apoB, and MTP mRNA in the kidney cortex, liver, duodenum, and heart of human apoB transgenic mice. Data are expressed as percent of the value in the liver and are means ± S.E., n = 4–5. Note that the human apoB transgene, as described previously (23), does not confer apoB mRNA expression in the intestine but robust expression in the kidney.

FIGURE 2.

ApoB100-containing lipoproteins are produced by mouse kidney. A, laser confocal microscopy (combined fluorescent and differential interference contrast micrograph) showing human apoB protein staining (red) in kidney tubular epithelial cells in a human apoB-transgenic mouse. B, control-stained section without primary antibody. C, kidney tissue from a human apo-B transgenic mouse was incubated with ³⁵S-labeled amino acids. The medium was subsequently subjected to sucrose density gradient ultracentrifugation. ApoB was isolated from each density fraction by immunoprecipitation and analyzed by SDS-PAGE and filmless autoradiographic analysis. ³⁵S-ApoB100 was seen in the d ∼1.03–1.04 g/ml lipoprotein fractions.

FIGURE 3.

ApoB-antisense LNA oligonucleotide reduces apoB mRNA expression in kidney and liver. C57Bl/6 mice received an intraperitoneal injection of apoB-antisense LNA oligonucleotide (filled bars) or saline (open bars). Four days later, the RNA was isolated from the kidney cortex and a liver biopsy and used for mouse apoB mRNA quantification. Data are expressed as percent of the average value in the saline group and are means ± S.E. The numbers of mice in each group are shown in the figure. ***, p < 0.0001.

FIGURE 4.

ApoB-antisense LNA oligonucleotide reduces plasma triglycerides and cholesterol. Plasma triglycerides (A), cholesterol (B), and free fatty acids (C) were determined in C57Bl/6 mice 4 days after an intraperitoneal injection of apoB-antisense LNA oligonucleotide (filled bars) or saline (open bars). Mice were fasted for 12 h or allowed free access to chow prior to collecting blood for plasma analyses in the morning. Values are means ± S.E. The numbers of mice in each group are shown in the figure. ***, p < 0.0001.

FIGURE 5.

ApoB-antisense LNA oligonucleotide reduces kidney triglyceride stores in fasted mice. Triglycerides (A, C, and E) and cholesterol (B, D, and F) were determined in kidney cortex (A and B), liver (C and D), and heart (E and F) 4 days after an intraperitoneal injection of apoB-antisense LNA oligonucleotide (filled bars) or saline (open bars). Mice were fasted for 12 h or allowed free access to chow prior to removal of tissues. Values are mean ± S.E. The numbers of mice in each group are shown in the figure. *, p < 0.05; **, p < 0.01; ***, p < 0.0001.

FIGURE 6.

Fasting induces neutral lipid accumulation in kidney tubule epithelial cells. Histological sections of kidneys from 12-h fasted (A) and a fed (B) mice were stained with Oil-red-O visualizing neutral lipids (red) in tubular epithelial cells (white arrow) but not in glomeruli (black arrow) of fasted mice.

FIGURE 7.

Effect of apoB-antisense LNA oligonucleotide and fasting on kidney expression of genes involved in fatty acid metabolism. Kidney cortex mRNA expression of lipoprotein lipase (LPL) (A), cytosolic acyl-CoA thioesterase 1 (CTE1) (B), fatty acid transport protein (FATP)1 (C), FATP4 (D), heart-type fatty acid-binding protein (H-FABP) (E), and long chain acyl-CoA dehydrogenase (LCAD) (F) was measured 4 days after an intraperitoneal injection of apoB-antisense LNA oligonucleotide (filled bars) or saline (open bars). Mice were fasted for 12 h or allowed free access to chow prior to removal of tissues. Values are means ± S.E. The numbers of mice in each group are shown in the figure. *, p < 0.05; **, p < 0.01; ***, p < 0.0001.