Extrahepatic expression and regulation of protein C in the mouse

Abstract

Activated protein C (APC) acts as an anticoagulant by inhibiting coagulation factors Va and VIIIa. Although the liver appears to be the primary site of protein C (PC) synthesis, the demonstration that other components of this system are produced extrahepatically raises the possibility that PC itself is synthesized in other tissues. We therefore used quantitative reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemistry to screen various murine tissues for PC expression. Relatively high levels of PC mRNA were detected in the kidney (35% of liver) and testis (22% of liver). PC mRNA and antigen were demonstrated in tubular epithelial cells in the renal cortex, in spermatogenic cells in the testis, and in epithelial cells in the epididymis. Low but significant levels of PC mRNA were detected in the epididymis (1.7% of the level in liver), brain (1.1% of liver), and lung (0.8% of liver). PC antigen was demonstrated in bronchial epithelial cells in the lung, in pyramidal neurons in the cerebrum, and in Purkinje cells in the cerebellum. The extrahepatic expression of PC mRNA (ie, in the kidney) was significantly decreased in mice with renal disease (eg, in MRL lpr/lpr mice with autoimmune lupus nephritis, in db/db mice with diabetic nephropathy, and in endotoxin-treated mice with acute renal injury). The decreased renal expression of PC may contribute to the increased procoagulant potential of the kidney during septic and inflammatory processes and to the progression of kidney disease associated with these conditions.

Figure 1.

Distribution of PC mRNA in tissues of the normal mouse. The concentration of PC mRNA in various tissues from adult CB6 mice was determined by quantitative RT-PCR as described in Materials and Methods. Data are means (bars, SD) of three different experiments. The numbers above each bar represent the relative percentage of PC mRNA in that tissue compared with the level in the liver. Epidid.: Epididymis.

Figure 2.

Histological analysis of PC mRNA and antigen in the kidney and male reproductive organs of the normal mouse. Tissue sections from adult CB6 mice were analyzed by in situ hybridization for PC or u-PA mRNA and by immunohistochemical staining for PC antigen. A and B: Analysis of kidney sections by regional in situ autoradiography for PC (A) or u-PA (B) mRNAs. The slides were incubated on X-ray film in the dark at room temperature and developed after 2 weeks of exposure. C and D: High-resolution in situ hybridization analysis of kidney sections for PC mRNA (magnification, ×400). E and F: Immunohistochemical staining for PC antigen in kidney sections (magnification: E, ×400; F, ×200). In F, PC antigen is indicated by the red-brown stain. G and H: In situ hybridization for PC mRNA in male reproductive organs. G, testis (magnification, ×400); H, epididymis (magnification, ×400). Slides for C and D were exposed for 8 weeks at 4°C, and those for E and F were exposed for 12 weeks at 4°C. The slides were then stained with hematoxylin and eosin. C to H: G, glomeruli; T, tubules; Co, cortex; Me, medulla.

Figure 3.

Histological analysis of PC antigen in the lung and brain of the normal mouse. Tissue sections from adult CB6 mice were analyzed by immunohistochemistry for PC antigen (red-brown stain) as described in Materials and Methods. A: Lung (magnification, ×100). Br denotes bronchioles. B: Cerebral cortex (magnification, ×400). Arrows indicate typical pyramidal neurons. C and D: Hippocampal area in the cerebrum. Arrows indicate positive staining in neurons in the pyramidal layer of the hippocampal cortex. Magnification: C, ×100; D, ×400. E and F: Cerebellar cortex. Arrows indicate positive staining in Purkinje cells lining the outside granular layer in the cerebellar cortex. In control experiments, parallel sections were analyzed using a preimmune (normal) rabbit immunoglobulin G instead of anti-PC antibody. No staining was observed in those sections (data not shown). Magnification: E, ×100; F, ×400.

Figure 4.

Changes in PC mRNA levels in the kidneys and livers of mice with renal disease. Total kidney and liver RNAs were extracted from female MRL lpr/lpr mice and their controls (MRL +/+) at 2, 5, and 6 months of age; from male db/db mice and their normal counterparts (C57BL/KsJ +/?) at 2, 4, and 6 months of age; and from control (0 time point) and LPS (50 μg)-treated normal CB6 mice at 2, 4, 8, and 24 hours after injection. PC mRNA was determined using quantitative RT-PCR analysis as described in Materials and Methods. A and B: Kidneys (A) and livers (B) from control (open bar) and female MRL lpr/lpr (closed bar) mice. C and D: Kidneys (C) and livers (D) from control (open bar) and db/db (hatched bar) mice. E and F: Kidneys (E) and livers (F) from LPS-treated mice. Data are means (bars, SD) of three different experiments.

Figure 5.

High-resolution in situ hybridization analysis of PC mRNA in kidneys and livers of mice with renal disease. Kidney sections from control (MRL +/+; A) and lupus-prone (female, MRL lpr/lpr; B) mice of 5 months of age, from control (C57BL/KsJ +/?; C) and db/db (D) mice 6 months of age, and from control (saline-treated; E) and LPS-treated (8 hours; F) mice were analyzed by high-resolution in situ hybridization as described in Materials and Methods. Liver sections (G and H) from control (G) and LPS-treated (8 hours; H) mice were also examined by high-resolution in situ hybridization analysis. G, glomeruli; T, tubules. Slides for A to F were exposed for 8 weeks at 4°C, whereas those for (G) and (H) were exposed for 4 weeks at 4°C. The slides were then stained with hematoxylin and eosin; magnification, ×400.