Karl Lhotta

Fibroblast Growth Factor 23 (FGF23) Predicts Progression of Chronic Kidney Disease: The Mild to Moderate Kidney Disease (MMKD) Study

It has not been firmly established whether disturbed calcium-phosphate metabolism affects progression of chronic kidney disease (CKD) in humans. In this cohort study of 227 nondiabetic patients with CKD, we assessed fibroblast growth factor 23 (FGF23) plasma concentrations in addition to other variables involved in calcium-phosphate metabolism, and we followed 177 of the patients prospectively for a median of 53 months to assess progression of renal disease. In the baseline cohort, we found a significant inverse correlation between glomerular filtration rate and both c-terminal and intact FGF23 levels (both P < 0.001). The 65 patients who experienced a doubling of serum creatinine and/or terminal renal failure were significantly older, had a significantly lower glomerular filtration rate at baseline, and significantly higher levels of intact parathormone, c-terminal and intact FGF23, and serum phosphate (all P < 0.001). Cox regression analysis revealed that both c-terminal and intact FGF23 independently predict progression of CKD after adjustment for age, gender, GFR, proteinuria, and serum levels of calcium, phosphate, and parathyroid hormone. The mean follow-up time to a progression end point was 46.9 (95% CI 40.2 to 53.6) months versus 72.5 (95% CI 67.7 to 77.3) months for patients with c-terminal FGF23 levels above or below the optimal cut-off level of 104 rU/mL (derived by receiver operator curve analysis), respectively. In conclusion, FGF23 is a novel independent predictor of progression of renal disease in patients with nondiabetic CKD. Its pathophysiological significance remains to be elucidated.

Mutations in complement C3 predispose to development of atypical hemolytic uremic syndrome

Atypical hemolytic uremic syndrome (aHUS) is a disease of complement dysregulation. In approximately 50% of patients, mutations have been described in the genes encoding the complement regulators factor H, MCP, and factor I or the activator factor B. We report here mutations in the central component of the complement cascade, C3, in association with aHUS. We describe 9 novel C3 mutations in 14 aHUS patients with a persistently low serum C3 level. We have demonstrated that 5 of these mutations are gain-of-function and 2 are inactivating. This establishes C3 as a susceptibility factor for aHUS.

Results of a Nationwide Screening for Anderson-Fabry Disease among Dialysis Patients

Anderson-Fabry disease is possibly underdiagnosed in patients with end-stage renal disease. Nationwide screening was therefore undertaken for Anderson-Fabry disease among dialysis patients in Austria. Screening for alpha-galactosidase A (AGAL) deficiency was performed by a blood spot test. In patients with a positive screening test, AGAL activity in leukocytes was determined. Individuals with decreased leukocyte AGAL activity were subjected to mutation testing in the GLA gene. Fifty (90.9%) of 55 Austrian hemodialysis centers participated in this study; 2480 dialysis patients (80.1% of the Austrian dialysis population) were screened. In 85 patients, the screening test was positive (85 of 2480, 3.42%; women, 3.32%; men, 3.50%). Among these 85 patients, 4 men (in 3 of whom Anderson-Fabry disease was already known before screening) had a severely decreased and 11 subjects had a borderline low AGAL activity. Genetic testing revealed mutations associated with Fabry disease in all four men with severely decreased AGAL activity resulting in a prevalence of 0.161% for the entire study population. A nationwide screening of dialysis patients permitted detection of a hitherto unknown man with Anderson-Fabry disease. The overall prevalence among dialysis patients was at least ten times higher as compared with recent registry data. Screening programs among patients with end-stage renal disease, especially men, should be put in place to identify families with Anderson-Fabry disease who probably may benefit from specific clinical care, and perhaps from enzyme replacement therapy. In dialysis patients, however, there is no evidence to support enzyme replacement therapy at present.

Elevated plasma concentrations of lipoprotein(a) in patients with end-stage renal disease are not related to the size polymorphism of apolipoprotein(a).

Patients with terminal renal insufficiency suffer from an increased incidence of atherosclerotic diseases. Elevated plasma concentrations of lipoprotein(a) [Lp(a)] have been established as a genetically controlled risk factor for these diseases. Variable alleles at the apo(a) gene locus determine to a large extent the Lp(a) concentration in the general population. In addition, other genetic and nongenetic factors also contribute to the plasma concentrations of Lp(a). We therefore investigated Apo(a) phenotypes and Lp(a) plasma concentrations in a large group of patients with end-stage renal disease (ESRD) and in a control group. Lp(a) concentrations were significantly elevated in ESRD patients (20.1 +/- 20.3 mg/dl) as compared with the controls (12.1 +/- 15.5 mg/dl, P < 0.001). However, no difference was found in apo(a) isoform frequency between the ESRD group and the controls. Interestingly, only patients with large size apo(a) isoforms exhibited two- to fourfold elevated levels of Lp(a), whereas the small-size isoforms had similar concentrations in ESRD patients and controls. Beside elevated Lp(a) concentrations, ESRD patients had lower levels of plasma cholesterol and apolipoprotein B. These results show that elevated Lp(a) plasma levels might significantly contribute to the risk for atherosclerotic diseases in ESRD. They further indicate that nongenetic factors related to renal insufficiency or other genes beside the apo(a) structural gene locus must be responsible for the high Lp(a) levels.

The Intricate Role of Complement Component C4 in Human Systemic Lupus Erythematosus

It was observed about 50 years ago that low serum complement activity or low protein concentrations of complement C4 concurred with disease activities of systemic lupus erythematosus (SLE). Complete deficiencies of complement components C4A and C4B, albeit rare in human populations, are among the strongest genetic risk factors for SLE or lupus-like disease, across HLA haplotypes and racial backgrounds. However, whether heterozygous or partial deficiency of C4A (C4AQ0) or C4B (C4BQ0) is a predisposing factor for SLE has been a highly controversial topic. In this review we critically analyzed past epidemiologic studies on deficiency of C4A or C4B in human SLE. Cumulative results from more than 35 different studies revealed that heterozygous and homozygous deficiencies of C4A were present in 40-60% of SLE patients from almost all ethnic groups or races investigated, which included northern and central Europeans, Anglo-Saxons, Caucasians in the US, African Americans, Asian Chinese, Koreans and Japanese. In addition, French SLE and control populations had relatively low frequencies of C4AQ0, but the difference between the patient and control groups was statistically significant. The relative risk of C4AQ0 in SLE varied between 2.3 and 5.3 among different ethnic groups. In Caucasian and African SLE patients, the two major causes for C4AQ0 are (1) the presence of a mono-S RCCX (RP-C4-CYP21-TNX) module with a single, short C4B gene in the major histocompatibility complex; and (2) a 2-bp insertion into the sequence for codon 1213 at exon 29 of the mutant C4A gene. Both mono-S structures and 2-bp insertion in exon 29 are absent or extremely rare in the C4AQ0 of Oriental SLE patients. The highly significant association of C4AQ0 with SLE across multiple HLA haplotypes and ethnic groups, and the presence of different mechanisms leading to a C4A protein deficiency among SLE patients suggested that deficiency or low expression level of C4A protein is a primary risk factor for SLE disease susceptibility per se. On the other hand, Spanish, Mexican, Australian Aborigine SLE patients had increased frequencies of C4B deficiency instead of C4A deficiency. Such observations underscore the importance of both C4A and C4B proteins in the fine control of autoimmunity. Different racial and genetic backgrounds could change the thresholds for the requirement of C4A or C4B protein levels in immune tolerance and immune regulation. Most past epidemiological studies of C4 in human SLE did not consider the polygenic and gene size variations of C4A and C4B. In addition, many studies were overly dependent on phenotypic observations or methods that did not distinguish differential C4A and C4B protein expression caused by unequal gene number or different gene size from the absence of a functional C4A or C4B gene. For further longitudinal studies on clinical manifestations of SLE, it would be informative to stratify the patients with accurately defined C4A and C4B genotypes. Likewise, elucidation of epistatic genetic factors interacting with C4AQ0 would provide important insights into the intricate roles of C4 in SLE disease susceptibility and pathogenesis.

Apolipoprotein(a) phenotypes predict the risk for carotid atherosclerosis in patients with end-stage renal disease.

Several studies have demonstrated that atherosclerotic complications are the major cause of morbidity and mortality in hemodialysis patients. High lipoprotein(a) [Lp(a)] plasma concentrations are an independent risk factor for atherosclerosis. Patients with end-stage renal disease (ESRD) have elevated plasma concentrations of Lp(a), which are not explained by size variation at the apolipoprotein(a) [apo(a)] gene locus. The aim of our study was to investigate whether Lp(a) concentrations and/or apo(a) phenotypes are predictive of the degree of atherosclerosis in the extracranial carotid arteries in ESRD patients. Of 167 patients, 108 showed atherosclerotic plaques (65%). Univariate analysis showed that the plaque-affected group was significantly older and had a higher frequency of angina pectoris, previous myocardial infarction, or cerebrovascular accident. Furthermore, this group included significantly more patients with low-molecular-weight apo(a) isoforms (26.9% versus 8.5%, P < .005) and had significantly higher mean Lp(a) plasma concentrations (29.3 +/- 31.0 versus 19.7 +/- 25.7 mg/dL, P < .05). Lp(a) plasma concentration increased significantly with the number of affected arterial sites, from 19.7 mg/dL in patients without plaques to 40.1 mg/dL in patients with seven or eight affected sites. In patients with low-molecular-weight phenotypes, significantly more arterial sites were affected (3.62 versus 2.08, P < .001). Multivariate regression analysis showed that age, angina pectoris, and the apo(a) phenotype were the only significant predictors of the degree of atherosclerosis. We conclude that, besides age, the apo(a) phenotype is the best predictor of carotid atherosclerosis in ESRD patients and may be used for assessment of general atherosclerosis risk in this patient group.

Reinforced intradermal hepatitis B vaccination in hemodialysis patients is superior in antibody response to intramuscular or subcutaneous vaccination

Since 1960, hepatitis B virus-associated chronic liver disease has been considered an important problem in dialysis units in both Europe and North America. Separate dialysis facilities for hepatitis B-infected patients, the implementation of universal precautions for the prevention of transmission, and the active immunization against hepatitis B have now reduced the yearly incidence to less than 0.05% in Western countries. However, only 50% to 60% of patients with renal insufficiency develop sufficient immune response after intramuscular hepatitis B vaccination. The aim of the current study was to determine whether the mode of vaccine application plays a role in vaccination response and whether increasing the vaccine dose of primary intradermal hepatitis B vaccination can reduce the number of vaccine injections in hemodialysis patients. We designed a prospective, randomized study of antibody responses to hepatitis B vaccine given intradermally, subcutaneously, or intramuscularly in 81 hemodialysis patients. Outcome measures were rates of seroconversion, mean levels of anti-Hbs antibodies, and antibody levels 8 years after vaccination. The results show that intradermal hepatitis B vaccination response with a higher vaccination dose than previously used in hemodialysis patients is superior to conventional intramuscular and subcutaneous vaccination and is also well tolerated. Five intradermal injections of 20 microg each induced the development of sufficient anti-Hbs antibody titer, which persisted in 70% of the patients over 3 years.

Apolipoprotein(a) phenotype-associated decrease in lipoprotein(a) plasma concentrations after renal transplantation.

High lipoprotein(a) [Lp(a)] plasma concentrations are an independent risk factor for atherosclerosis. In the general population, Lp(a) levels are primarily determined by allelic variation at the apolipoprotein(a) [apo(a)] gene locus. Apo(a) isoforms of various sizes are associated with different Lp(a) concentrations. Patients with end-stage renal disease (ESRD) have elevated plasma concentrations of Lp(a), which are not explained by the size variation at the apo(a) gene locus. To further investigate the origin of the elevated Lp(a) plasma concentrations, we examined Lp(a) concentrations and apo(a) phenotypes in 154 ESRD patients undergoing renal transplantation. In a prospective longitudinal study we observed a rapid normalization of Lp(a) levels from an average concentration of 25.9 +/- 28.7 mg/dL before to 17.9 +/- 25.5 mg/dL 3 weeks after renal transplantation (P < .0001). Only patients with high-molecular-weight phenotypes had a significant decrease in Lp(a) plasma concentrations. This study demonstrates the nongenetic origin of elevated Lp(a) concentrations in ESRD patients, which is obviously caused by the disease. It further confirms a phenotype-associated elevation of Lp(a) concentrations in ESRD.

Uromodulin mutations causing Familial Juvenile Hyperuricaemic Nephropathy lead to protein maturation defects and retention in the endoplasmic reticulum

Familial juvenile hyperuricaemic nephropathy (FJHN), an autosomal dominant disorder, is caused by mutations in the UMOD gene, which encodes Uromodulin, a glycosylphosphatidylinositol-anchored protein that is expressed in the thick ascending limb of the loop of Henle and excreted in the urine. Uromodulin contains three epidermal growth factor (EGF)-like domains, a cysteine-rich region which includes a domain of eight cysteines and a zona pellucida (ZP) domain. Over 90% of UMOD mutations are missense, and 62% alter a cysteine residue, implicating a role for protein misfolding in the disease. We investigated 20 northern European FJHN probands for UMOD mutations. Wild-type and mutant Uromodulins were functionally studied by expression in HeLa cells and by the use of western blot analysis and confocal microscopy. Six different UMOD missense mutations (Cys32Trp, Arg185Gly, Asp196Asn, Cys217Trp, Cys223Arg and Gly488Arg) were identified. Patients with UMOD mutations were phenotypically similar to those without UMOD mutations. The mutant Uromodulins had significantly delayed maturation, retention in the endoplasmic reticulum (ER) and reduced expression at the plasma membrane. However, Gly488Arg, which is the only mutation we identified in the ZP domain, was found to be associated with milder in vitro abnormalities and to be the only mutant Uromodulin detected in conditioned medium from transfected cells, indicating that the severity of the mutant phenotypes may depend on their location within the protein. Thus, FJHN-causing Uromodulin mutants are retained in the ER, with impaired intracellular maturation and trafficking, thereby indicating mechanisms whereby Uromodulin mutants may cause the phenotype of FJHN.

Flow cytometry based detection of HLA alloantibody mediated classical complement activation

Complement-dependent cytotoxicity (CDC) panel reactive antibody (PRA) testing is used to assess recipient presensitization and post-transplant alloantibody formation in transplant recipients. However, CDC test results can be affected by false-positive reactions brought about by autoantibodies or antilymphocyte reagents. As an alternative to the CDC-PRA assay, detection of HLA alloantibodies using HLA antigen-coated microbeads (FlowPRA test) was recently established. FlowPRA testing, however, does not distinguish between (presumably more harmful) complement-fixing and noncomplement-fixing alloantibodies. In this study, we established a novel assay allowing flow cytometric detection of HLA alloantibody dependent classical complement activation using the FlowPRA test. For the detection of complement activation, FlowPRA beads were incubated with sera from highly sensitized dialysis patients (CDC-PRA reactivity >60%) and then stained for C4 (C4d, C4c) and C3 (C3d, C3c) fragments, as well as C1q deposition using indirect immunofluorescence. We demonstrate alloantibody induced induction of C4 fragment, and in parallel C1q deposition to HLA class I or class II beads. As shown by immunoblotting, C4 staining was not due to the presence of preformed C4 fragment-IgG/M complexes. Indeed, C4 fragment deposition in our in vitro system was demonstrated to result from de novo complement activation. First, inactivation of C4 by treatment of sera with methylamine, which inhibits cleavage of the internal thioester, completely abolished C4 fragment deposition. Second, C4 fragment deposition was not observed in the evaluation of C4-free immunoadsorption eluates obtained from highly sensitized dialysis patients. After supplementation with complement, however, eluates induced C4 deposition. Deposition of C4 split products and C1q was temperature-dependent with maximum binding after incubation at 4 degrees C for 60 min. In contrast, maximum C3 fragment deposition was found at 37 degrees C. At this temperature, C3 deposition occurred in an alloantibody and C4-independent fashion, presumably as a result of alternative complement activation. In summary, we describe a novel cell-independent and easy-to-perform PRA test that permits flow cytometry based detection of alloantibody induced classical complement activation. Future studies will have to evaluate its possible relevance as an alternative to CDC-PRA testing in clinical transplantation.