Regulated expression of complement factor B in the human kidney.

We have previously demonstrated regulated expression of C3 in the proximal renal tubular epithelial cells of humans. To test the hypothesis that local alternative pathway complement activation could contribute to the tubulointerstitial component of chronic renal disease, we examined factor B gene expression in human kidneys. 35S riboprobes were generated from a human factor B cDNA. By in situ hybridization, proximal tubular factor B message was seen in 17 kidneys with various nephropathies. The expression was most intense in organs with evidence of interstitial inflammation, and its localization paralleled the inflammation. As was the case with C3 and C4, there was never any evidence of glomerular factor B message, nor was any seen in infiltrating inflammatory cells. In eight normal kidney tissues, factor B expression was either absent or restricted to rare foci of interstitial infiltration. The proximal renal tubular epithelium of humans appears to express the genes for both components of the alternative pathway convertase, C3 and factor B. These locally produced components may be important mediators of the interstitial inflammation that is common to all progressive nephritides.

Glomerulonephritis associated with complete deficiency of the fourth component of complement. Response to intravenous immunoglobulin.

A 15-year-old girl with complete C4 deficiency and a lupus-like disorder developed evidence of nephritis after 4 years of followup. Renal biopsy demonstrated an immune complex glomerulonephritis, with deposits in the capillary loops, the paramesangium, and the mesangial matrix. Renal function was normal. The patient was treated with monthly infusions of intravenous immunoglobulin for 6 months. The treatment was well tolerated, and resulted in resolution of the rash and hematuria. Followup biopsy showed less proliferation and fewer loop deposits. In light of the serious risk of infections that is associated with complement deficiency, approaches to glomerulonephritis that do not include immunosuppression should be considered.

Differential expression of complement C3 and C4 in the human kidney.

Complement activation is associated with a variety of immunologically-mediated renal diseases. Proximal tubular epithelial cells in situ constitutively express messenger RNA for C4 of the complement system. These same epithelial cells in culture have been reported to contain message for C3 and to secrete this protein when stimulated by IL-2. The present study compared the in situ localization of C3 and C4 message in parallel in a variety of renal biopsy and nephrectomy specimens. All adequate tissue samples (n = 23) had C4 mRNA throughout in the cortical tubular epithelium. Although C3 message was also expressed in tubular epithelial cells, there was much greater variation in its distribution. mRNA for C3 was not detected in histologically normal specimens (n = 4) either by in situ or Northern hybridization. Focal C3 message correlated with focal histologic abnormalities (e.g., focal glomerulosclerosis), while more generalized C3 signal occurred in specimens with more diffuse inflammatory processes (e.g., SLE). Infiltrating inflammatory cells and cells of the glomeruli were uniformly negative for C3 (and C4) message. Tubular C3 and C4 mRNA appeared to be translated, since selected specimens showed cytoplasmic staining by monoclonal antibodies to C3c and C4c. These observations are consistent with the hypothesis that local production of inflammatory mediators could induce C3 synthesis in the renal interstitium, with the possibility that subsequent complement activation could enhance the pathogenic process.

DP polymorphism in HLA-A1,-B8,-DR3 extended haplotypes associated with membranoproliferative glomerulonephritis and systemic lupus erythematosus.

We and others have shown an association between autoimmune disorders and the major histocompatibility complex extended haplotype HLA-A1,-B8,-SCO1,-DR3. The primary gene or genes within this haplotype conferring such susceptibility, however, have not been defined. In this study, we tested the hypothesis that linkage disequilibrium in this haplotype extends through the DP locus, and that DP type may be linked to membranoproliferative glomerulonephritis (MPGN) and systemic lupus erythematosus (SLE). DP and DQ typing was performed by restriction fragment length polymorphism analysis for 43 chromosomes (19 healthy controls, 9 SLE, 15 MPGN) bearing the -A1,-B8,-SCO1,-DR3 extended haplotype. Although all were DQw2, a variety of DP types were identified (DPw1, 0.26; DPw2, 0.09; DPw3, 0.14; DPw4, 0.44). Although DPw1 was represented on extended haplotypes with greater frequency than on 113 non-A1,-B8,-SCO1,-DR3 haplotypes (0.26 vs. 0.03; P < 0.001), there were no significant differences between healthy individuals with this haplotype and those with autoimmune disease. We conclude that the strong linkage disequilibrium of this haplotype breaks down between the DQ and DP loci. Loci important to disease susceptibility, therefore, are more likely to occur telomeric to DP.

Detection and cellular localization of human C4 gene expression in the renal tubular epithelial cells and other extrahepatic epithelial sources.

Although the liver is the major source of most complement proteins, recent reports have characterized extrahepatic expression of the genes for some of these components in a variety of tissues. In most cases, however, the specific cell type responsible for the extrahepatic complement expression has not been determined in situ. The authors studied expression of the fourth component of complement (C4) in a variety of human tissues by Northern analysis and by in situ hybridization. The C4 gene was found to be expressed at high levels in liver and both normal and diseased kidneys. In addition, evidence of C4 gene expression was found in the small intestine and brain. By in situ hybridization, the renal C4 gene expression was exclusively localized to tubular epithelial cells. C4 expression was also identified in hepatocytes, thyroid follicular epithelial cells, and ductal epithelial cells of the submandibular salivary gland. Although it is unlikely that local expression of the C4 gene contributes to glomerulonephritis, these results indicate that some components of complement may have a physiologic role in epithelial cell function.

Uniparental isodisomy 6 associated with deficiency of the fourth component of complement.

We identified an extremely rare condition, isolated complete deficiency of the fourth component of complement, in a child with systemic lupus erythematosus. The genes for C4 are located within the major histocompatibility complex (MHC) on the short arm of chromosome 6. The patient expressed only paternal phenotypes for proteins encoded by the MHC (HLA and GLO), yet was 46XX with no detectable 6p deletion. Genomic DNA from patient, parents, and sibling was digested with restriction enzymes, and blots were probed for five chromosome 6 markers. At all loci, maternal and paternal RFLPs could be distinguished, and the patient showed only paternal bands. RFLP analysis of markers from four other chromosomes showed maternal and paternal contribution. The data are consistent with uniparental isodisomy 6 (inheritance of two identical chromosome 6 haplotypes from the father and none from the mother). Direct analysis of genetic material from both parents, as well as detection of multiple protein polymorphisms encoded on chromosome 6, clearly demonstrates this novel mechanism for the expression of a recessive genetic condition.

C4B deficiency: a risk factor for bacteremia with encapsulated organisms.

The fourth component of complement (C4) is crucial to the activation of the classical complement pathway, a key defense against invading microorganisms. The two isotypes of C4, C4A and C4B, have very different in vitro activities. An increased incidence of total C4B deficiency was found in white patients with Streptococcus pneumoniae, Haemophilus influenzae, or Neisseria meningitidis infection (14% of bacteremic children vs. 2% of race-matched controls, P = .02). In black patients, however, there was no difference in incidence of C4B deficiency between bacteremic patients and race-matched controls (7% and 5%, respectively, P greater than .5). These data suggest that, at least in whites, total C4B deficiency is a risk factor for invasive disease with these three encapsulated organisms.

Molecular genetics of C4B deficiency in IgA nephropathy.

The fourth component of complement (C4) occurs in two functionally distinct isotypes, C4A and C4B. The two closely linked genes are located on chromosome 6p, between HLA-B and -DR. Several reports have established complete C4B deficiency as the major genetic risk factor for IgA nephropathy (RR = 6.5; p = 0.0004). It is not clear whether this association derives from immune dysfunction related to the absent isotype or from another disease susceptibility gene closely linked to C4B. To help distinguish between these mechanisms, we examined the molecular basis of complete C4B deficiency in five patients with IgA nephropathy and eight healthy individuals. C4 and Bf protein typing were performed by immunofixation electrophoresis of plasma. Genomic DNA was digested with several restriction enzymes, chosen to produce informative restriction fragment length polymorphisms (RFLPs). After electrophoresis and Southern blotting, digests were hybridized to a series of cDNA probes specific to the 5' and 3' ends of the C4 genes, the C4d region, and the adjacent 21-hydroxylase genes. Availability of DNA from family members allowed assignment of RFLPs to specific haplotypes. The 10 C4B-deficient IgA nephropathy-associated haplotypes displayed seven different protein phenotype/RFLP patterns. Three haplotypes consisted of the common C4B/21-hydroxylase deletion on the Bf*S, C4A*3, C4B*Q0 complotype. Two haplotypes were characterized by the C4A*3,2 duplication, with two C4 genes present but a C4A protein being produced by the gene at the usual C4B locus. All of the remaining haplotypes had unique Bf, C4, and 21-hydroxylase patterns. C4B-deficient IgA nephropathy patients display a variety of molecular genetic bases for their protein deficiency. This observation speaks against linkage of C4B deficiency with a locus encoding disease susceptibility and supports a primary role for the complement abnormality in this disease.

Major histocompatibility complex extended haplotypes in systemic lupus erythematosus.

In a study of 32 white patients with systemic lupus erythematosus from 28 families, 60 unique chromosome 6 haplotypes were defined. The MHC extended haplotype HLA-B8, -DR3, SC01, GL02 was strongly disease-associated (0.09 patients, 0.02 controls, RR = 4.5, C.I. = 1.6-12.4, P less than 0.05), while the corresponding haplotype with the GL01 specificity was not increased in frequency (0.05 in both patients and controls). In the present data, the increase in the haplotype bearing GL02 accounted entirely for the association between HLA-DR3 and SLE. Furthermore, the phenotype of complete C4A deficiency was also strongly disease-associated (patients 0.14, controls 0.02, RR = 8.5, C.I. = 1.8-37.0, P less than 0.05). The only other MHC association in these patients was an increased occurrence of the HLA-B17, -DR7, SC61 haplotype (patients 0.07, controls 0.01, RR = 6.0, C.I. = 1.8-20.6, P corr. less than 0.05). The relationship between MHC markers and autoimmune disease appears to be a result of an association with MHC extended haplotypes and complete complement component deficiencies rather than with individual alleles. It is important that future studies include family members so that such haplotypes can be defined.

C4 isotype deficiency in IgA nephropathy.

C4 and factor B typing were performed in 37 pediatric patients with primary IgA nephropathy. Null alleles for C4B occurred with a frequency of 26% in patients, as compared to 15% in healthy controls (NS). The phenotype of C4B deficiency (homozygous C4B null), however, was found in 16% of patients and 4% of controls (P less than 0.05). Comparison of observed C4B phenotypes with those predicted from the Hardy-Weinberg equilibrium also confirmed an excess of C4B deficiency (P less than 0.0005). In contrast, there was no evidence of distortion in the frequencies of the C4A null allele or phenotype, or of the factor B alleles. The data suggest that C4B deficiency may be one of multiple interacting factors contributing to the development of this glomerulopathy.