Identification of membrane-bound CR1 (CD35) in human urine: evidence for its release by glomerular podocytes.

Abstract

SummaryComplement receptor 1 (CR1) is present on erythrocytes (E-CR1), various leucocytes, and renal glomerular epithelial cells (podocytes). In addition, plasma contains a soluble form of CR1 (sCR1) By using a specific ELISA, CR1 was detected in the urine (uCR1) of normal individuals (excretion rate in 12 subjects, 3.12 + 1.15 #g/24 h). Contrary to sCR1, uCR1 was pelleted by centrifugation at 200,000 g for 60 min. Analysis by sucrose density gradient ultracentrifugation showed that uCR1 was sedimenting in fractions larger than 19 S, whereas sCR1 was found as expected in fractions smaller than 19 S. The addition of detergents reduced the apparent size of uCR1 to that of sCR1. After gel filtration on Sephacryl-300 of normal urine, the fractions containing uCR1 were found to be enriched in cholesterol and phospholipids. The membrane-association of uCR1 was demonstrated by analyzing immunoafhnity purified uCR1 by electron microscopy which revealed membrane-bound vesicles. The apparent molecular mass of uCR1 was 15 kD larger than E-Clkl and sCR1 when assessed by SDS-PAGE and immunoblotting. This difference in size could not be explained on the basis of glycosylation only, since pretreatment with N-glycosidase F reduced the size of all forms of CR1; however, the difference in regular molecular mass was not abrogated. The structural alleles described for E-CR1 were also found for uCR1. The urine of patients who had undergone renal transplantation contained alleles of uCR1 which were discordant with E-CR1 in 7 of 11 individuals, indicating that uCR1 originated from the kidney, uCR1 was shown to bind C3b-coated immune complexes, suggesting that the function of CR1 was not destroyed in urine. A decrease in uCR1 excretion was observed in 3 of 10 patients with systemic lupus erythematosus, corresponding to the three who had severe proliferative nephritis, and in three of three patients with focal sclerosis, but not in six other patients with proteinuria. Taken together, these data suggest that glomerular podocytes release CRl-coated vesicles into the urine. The function of this release remains to be defined, but it may be used as a marker for podocyte injury.

Soluble complement receptor type 1 (sCR1) in chronic liver diseases: serum levels at different stages of liver diseases

“…It is also expressed in renal glomerular podocytes [2]. CR1 binds C3b-coated particles and serves as a cofactor for the degradation of C3b by factor I.…”

Cr1, CD35 IN synovial fluid from patients with inflammatory joint diseases

“…Characteristics of SF CR1 compared with other forms of CR1. Previous experiments have shown that serum sCRl is not pelleted by ultracentrifugation for 60 minutes at 200,OOOg (5). By contrast, all urinary CR1, which is known to be a transmembrane molecule on urinary vesicles, is pelleted.…”

“…Thus, the cellular release of CR1 in vivo may occur via 2 different mechanisms, enzymatic cleavage (ie., shedding of the extracellular portion of the molecule) and cellular microvesiculation (5,6). PMN that invade the synovium in patients with inflammatory joint conditions resemble in vitroactivated cells (9), which are known to release a soluble form of CR1.…”

Increased levels of soluble complement receptor 1 in serum of patients with liver diseases

“…The size and solubility of sCR1 were assessed by ultracentrifuat 260,000g at room temperature, and 24 fractions of 200 mL were collected from the bottom. 10 The immunoglobulin M position was gation and chromatography studies. First, serum samples from 4 patients with cirrhosis and 2 normal controls were centrifuged at determined using radiolabeled monoclonal immunoglobulin M. sCR1 was measured in the fractions by ELISA as described above.…”

“…Data from 3 such patients are presented in liver disease was not aggregated, fragmented, or bound to cell fragments or vesicles released from cells. 10,16 Thus, the Fig. 5.…”