Variations in the complement regulatory genes factor H (CFH) and factor H related 5 (CFHR5) are associated with membranoproliferative glomerulonephritis type II (dense deposit disease).

INTRODUCTION: Membranoproliferative glomerulonephritis type II or dense deposit disease (MPGN II/DDD) causes chronic renal dysfunction that progresses to end stage renal disease in about half of patients within 10 years of diagnosis. Deficiency of and mutations in the complement factor H (CFH) gene are associated with the development of MPGN II/DDD, suggesting that dysregulation of the alternative pathway of the complement cascade is important in disease pathophysiology. SUBJECTS: Patients with MPGN II/DDD were studied to determine whether specific allele variants of CFH and CFHR5 segregate preferentially with the MPGN II/DDD disease phenotype. The control group was compromised of 131 people in whom age related macular degeneration had been excluded. RESULTS: Allele frequencies of four single nucleotide polymorphisms in CFH and three in CFHR5 were significantly different between MPGN II/DDD patients and controls. CONCLUSION: We have identified specific allele variants of CFH and CFHR5 associated with the MPGN II/DDD disease phenotype. While our data can be interpreted to further implicate complement in the pathogenesis of MPGN II/DDD, these associations could also be unrelated to disease pathophysiology. Functional studies are required to resolve this question.

Localisation of clusterin in normal human sperm by immunogold electron microscopy.

In the human male reproductive tract, two forms of clusterin have been detected: the conventional heterodimeric form and a novel acrosomal form. On human sperm the novel form of clusterin is present in the acrosomal region of acrosome intact sperm only. The aim of this study was to determine the site of localisation of the acrosomal form of clusterin using immunogold electron microscopy on normal human sperm. Using the E5 anticlusterin mAb and a preembedding technique, acrosomal clusterin was localised in the acrosomal contents. Immunogold particles were detected on ethanol fixed spermatozoa that were subjected to Triton X-100 permeabilisation treatment. These sperm had lost their plasmalemma and outer acrosomal membrane. Specific immunogold labeling was present over the surface mainly of the acrosomal contents exposed by the loss of the plasma-lemma and outer acrosomal membrane. Immunogold particles were also detected in the equatorial segment of the sperm. These data confirm that the acrosomal form of clusterin is associated with the contents of the acrosome.

Cutaneous necrosis from calcific uremic arteriolopathy.

Calcific uremic arteriolopathy (calciphylaxis) is an uncommon complication of chronic renal failure that is associated with high morbidity and mortality. We report 16 patients (13 female) who presented between 1985 and 1996. All patients developed painful livido reticularis that progressed to cutaneous necrosis and ulceration (11 cases on the proximal extremities and five cases on the distal extremities). Two patients with predominately distal leg disease survived; the cause of death in the other 14 patients was sepsis (six patients), withdrawal from dialysis (three), cardiac arrest (three), and gastrointestinal hemorrhage (two). Mesenteric ischemia from intestinal vascular calcification occurred in two cases. Clinical factors identified included the use of warfarin therapy in seven cases and significant weight loss (>10% body weight) in seven cases in the 6 months preceding the development of calcific uremic arteriolopathy. Skin pathology was studied in 12 cases, with all showing calcific panniculitis and small vessel calcification. Electron microscopic spectral analysis of the mineral content of the calcific lesions in the subcutaneous tissue showed only calcium and phosphorous. In two cases, substitution of low molecular weight heparin for warfarin therapy resulted in clinical improvement. Current theories of pathogenesis and treatment are reviewed. This study confirms the high morbidity and mortality of calcific uremic arteriolopathy producing ischemic tissue necrosis while drawing attention to significant weight loss and warfarin therapy as risk factors for the development of ischemic tissue necrosis. Hyperbaric oxygen therapy warrants further study.

Heparin-binding epidermal growth factor-like growth factor, an immediate-early gene for mesangial cells, is up-regulated in the Thy-1.1 model.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is one of five well-described growth factors which bind to and activate the EGF receptor. Since cultured mesangial cells synthesize HB-EGF and this cytokine is a potent mitogen for smooth muscle cells (SMC), a cell type similar to mesangial cells, we attempted to determine (1) whether HB-EGF mRNA is present in the proliferative phase of experimental mesangial proliferative glomerulonephritis, and (2) some of the factors which regulate its synthesis by mesangial cells. In this study we demonstrate that cultured rat mesangial cells (RMC) express HB-EGF mRNA and that transcript levels are markedly increased by serum with maximal induction occurring within 2 h. Stimulation with individual cytokines (EGF, TGF-alpha, PDGF, TGF-beta and TNF-alpha), by contrast, had only a minor effect. The increase in HB-EGF mRNA levels following addition of serum was rapid, transient and independent of protein synthesis, features characteristic of immediate-early genes. In normal rat kidneys, there was no detectable glomerular expression of HB-EGF mRNA as determined by in situ hybridization, although occasional tubular cross sections were positive. Within 30 min after induction of the Thy-1.1 model, however, cells within the glomerulus and an increased number of tubules were positive. The number of positive glomerular and tubular cells increased progressively at days 1 and 4 post-induction, but declined by day 9 and had returned to background at day 15. Within the glomerulus, HB-EGF was expressed by cells of Bowman's capsule and cells within the glomerular tuft. Using (a) combined in situ hybridization and immunohistochemical staining of the same section and (b) staining of sequential sections by immunohistochemistry or in situ hybridization, it was found that intrinsic glomerular cells which did not stain with the macrophage marker ED-1 expressed HB-EGF mRNA. Although some were probably glomerular epithelial cells because of their peripheral location, it was uncertain whether mesangial cells were also positive. Future studies will be directed towards firmer identification of the glomerular cells expressing HB-EGF mRNA and to define the functional role of HB-EGF in the Thy-1.1 model.

Immunocytochemical colocalization of clusterin in apoptotic photoreceptor cells in retinal degeneration slow rds mutant mouse retinas.

In the rds mutant mouse the photoreceptor cells differentiate normally for the first few postnatal days, with the inner segments projecting an extended cilium. However, outer segments fail to form and only rudimentary disks and opsin-laden vesicles assemble at the tip of the cilium. These are shed into the interphotoreceptor space where they are phagocytosed by the retinal pigment epithelial cells. In this animal model, the photoreceptors undergo a slow degeneration via apoptosis leading to eventual loss of the entire photoreceptor population. Since increased expression of clusterin has been implicated in apoptosis, we studied the expression of clusterin in the rds mutant mouse retina and compared it to normal BALB/ c retinas. Small intestinal microvillus epithelium was used as a positive control tissue for apoptosis. Immunocytochemistry revealed the presence of clusterin in the ganglion cell, inner nuclear and outer plexiform layers and in the retinal pigment epithelium of both the rds and the BALB/c retinas. Interestingly, scattered clusterin-positive cells were observed in the outer nuclear layer (onl) of dystrophic retinas. Since the increased presence of clusterin protein in the onl of dystrophic retina may indicate dying photoreceptor cells due to apoptosis, we utilized a co-localization procedure for apoptotic nuclei and clusterin. For apoptosis we utilized an in situ 3' end labeling of fragmented DNA (TUNEL) and immunohistochemistry for clusterin using brown and red colored substrates respectively. Small intestine tissue sections were also included as positive controls for apoptosis. Our results show that clusterin is co-localized with apoptotic nuclei both in the onl of rds mutant retinas as well as in the small intestine epithelial cells undergoing cell turnover and exfoliation. These results are of interest since overexpression of clusterin is also observed in other neuro-degenerative diseases such as Alzheimer's and Pick's disease.

The use of anticlusterin monoclonal antibodies for the combined assessment of human sperm morphology and acrosome integrity.

Clusterin is an abundant protein in the human male reproductive tract which appears to be produced by the testis, epididymis and the seminal vesicles. Using monoclonal antibodies and an amplified immunoperoxidase technique, we have identified two apparently biochemically distinct forms of clusterin on human spermatozoa. Morphologically abnormal spermatozoa have an extensive surface coating of conventional 80 kDa native clusterin, but this form of clusterin is not detectable on normal spermatozoa. Normal spermatozoa, however, contain within the acrosomal cap a different form of clusterin, reactive with an anticlusterin alpha-chain antibody. Agglutinated spermatozoa, most of which are grossly abnormal, were intensely labelled with the antibody against conventional 80 kDa clusterin, suggesting that the 'clustering' properties of this protein may play a role in the aggregation of abnormal spermatozoa. Anticlusterin monoclonal antibodies may be useful for semen analysis. Staining spermatozoa with anticlusterin monoclonal antibodies is a technically simple method which provides a visually obvious means of assessing spermatozoa morphology and acrosome status simultaneously. The current data also suggest that different functions of clusterin in the reproductive tract may be attributed to different molecular forms of the protein.

Immunohistological localization of clusterin in the male genital tract in humans and marmosets.

Clusterin is a multifunctional protein, first described in the reproductive tracts of the rat and the ram. It is produced by several cell types and exists in at least two differentially glycosylated forms. The aim of this study was to extend knowledge of clusterin expression in the primate (human and marmoset) male reproductive tracts by means of clusterin-specific immunohistochemical techniques. In both normal and abnormal testicular tissue, clusterin was found in association with Sertoli cells, lumenal sperm, proacrosomal Golgi complexes, residual bodies, and degenerating germ cells. The major differences observed between the two groups were attributable primarily to morphological differences rather than to clusterin expression specifically. There was no correlation between testicular clusterin content and the cause and severity of spermatogenic disorders. Within normal epididymides, regional differences in clusterin staining similar to those reported in the rat were observed. The seminal vesicles contained large amounts of positive clusterin staining, whereas normal human prostate was completely negative. Low levels of clusterin expression were observed in the marmoset prostate. This study suggests that clusterin is an important and widespread product in the human and marmoset reproductive tracts and is likely to have a role in spermatogenesis.

Clusterin depletion enhances immune glomerular injury in the isolated perfused kidney.

Clusterin is a normal plasma protein, shown to be an inhibitor of reactive complement hemolysis and a component of the fluid phase SC5b-9 terminal complement complexes. It is a component of glomerular immune deposits in human and experimental glomerulonephritis. Using the complement-dependent isolated perfused rat kidney model of autologous phase passive Heymann nephritis, we have studied the effect of clusterin depletion of perfused plasma on the development of glomerular injury. Kidneys with planted glomerular sheep anti-rat Fx1A antibody were perfused with human plasma either depleted of clusterin to < or = 30%, or control plasma depleted of plasma fibronectin. Glomerular injury was then initiated by the addition of guinea pig anti-sheep immunoglobulins to the perfusate. Kidneys perfused with clusterin depleted plasma developed significantly greater proteinuria at all time points when compared to control kidneys. Glomerular antibody binding and C3 deposition were similar in the two groups, but terminal complement components were deposited in larger amounts in the clusterin depleted group. These data support a possible role for clusterin in vivo in the protection of complement-induced glomerular injury.

Clusterin levels increase during neuronal development.

The expression of clusterin has been shown to be elevated in several models of experimentally induced programmed cell death and in association with a number of neurodegenerative conditions. In order to test whether this protein is expressed in neurons during development, the expression of clusterin was examined in the developing nervous system, using immunohistochemistry and mRNA analysis. Clusterin expression was observed in the earliest neurons of the cortical plate on embryonic day (E) 12. Thereafter, the intensity of clusterin staining continued to increase in an age-dependent manner, with the greatest intensity of staining being found in the postnatal mature brain. Virtually all neurons were clusterin-positive and there was no evidence for the appearance of clusterin-positive cells specifically during epochs of programmed neuronal death in the embryo. This study suggests that clusterin has a role in neuronal maturation and it is unlikely to be associated exclusively with neuronal cell death.

Wegener's granulomatosis: clinical features and prognosis in 37 patients.

Thirty-seven patients (21 female, 16 male) with Wegener's granulomatosis (WG) were reviewed. Patients were followed for a mean six years after diagnosis; 14 were followed for more than seven years. The clinical features were similar to those in previous studies. In this series, only 13 patients (35%) had renal disease at presentation and the cumulative incidence of renal involvement was 51%. Thirty-one patients received treatment which included cyclophosphamide (CP). The case fatality rate of the six patients not treated with CP was 83% (five deaths). By contrast, all CP treated patients improved and 21 (68%) had complete remissions. Nine (29%) were in complete remission for a mean 4.9 years after discontinuing all treatment. Two were disease free for over ten years. The actuarial probability of survival for these patients was 97% at one year and 71% at ten years. Only three CP treated patients (10%) progressed to end-stage renal disease. The case fatality rate was 26% (eight patients) and sepsis was the cause of death in five. Fourteen patients (45%) treated with CP had at least one relapse of vasculitis and seven (23%) had multiple (two or more) relapses. These data indicate that CP is effective in inducing remissions and prolonging survival in patients with WG; however, relapses are frequent.

Non-dilated urinary tract obstruction.

OBJECTIVE: To describe the occurrence of obstructive uropathy in the absence of dilatation of the urinary tract. CLINICAL FEATURES: Five cases of non-dilated obstructive nephropathy are described. All patients were uraemic on presentation. Obstruction was caused by retroperitoneal malignancy in two patients and uric acid lithiasis in the remaining three. All patients had at least one ultrasound examination. Isotope renography and computed tomography were performed in three and four patients respectively. None of these imaging techniques suggested obstruction in any of the cases. Radionuclide scans were characterised by unusually poor perfusion and parenchymal phase images. INTERVENTION AND OUTCOME: An immediate diuresis and a rapid return of normal renal function occurred after relief of the obstruction in all cases. CONCLUSION: The absence of dilatation in obstructive nephropathy is uncommon but may be responsible for delayed diagnosis and management of a readily treatable cause of acute renal failure.

Regional localization of the gene for clusterin (SP-40,40; gene symbol CLI) to human chromosome 8p12-->p21.

The human clusterin (SP-40,40) gene, designated CLI (complement lysis inhibitor) by the Human Gene Nomenclature Committee, has previously been assigned to chromosome 8. In situ hybridization allowed us to map the locus at 8p12-->p21.

Antineutrophil cytoplasm antibody (ANCA) associated vasculitis.

In 1982 we first reported the presence of antineutrophil cytoplasm antibodies (ANCA) in 8 patients with systemic vasculitis and segmental necrotizing glomerulonephritis. The results of long-term follow-up are described. Screening of 7,500 serum samples revealed positive ANCA in 9 additional patients with vasculitis. Eighty-eight other patients with vasculitis were ANCA negative, including 7 with microscopic polyarteritis nodosa (MPAN) and 8 with Wegener's granulomatosis (WG). Conversely, ANCA were never detected in the absence of vasculitis. Fourteen patients presenting with glomerulonephritis and ANCA were followed for a median of 6.3 years. Eleven patients had MPAN and 3 WG. Remissions were obtained with immunosuppressive therapy in all patients. Clinical relapse was associated with the reappearance of ANCA. Five-year survival was 89% and 5-year dialysis free survival was 77%. ANCA are specific markers for a sub-group of patients with vasculitis and are sensitive markers of disease activity. Glomerulonephritis associated with ANCA positive vasculitis has a favorable outcome with immunosuppressive therapy.

Localization of terminal complement components S-protein and SP-40,40 in renal biopsies.

The terminal complement complex has been implicated in the development of glomerular injury in both experimental and, indirectly, in human glomerulonephritis. Recent data suggests that the terminal complement complex in human glomerulonephritis may be in the cytolytically inactive SC5b-9 form which also contains S-protein and a recently identified protein, SP-40,40. In this study renal biopsies were examined by immunofluorescence to determine the incidence and inter-relation of deposition of the SC5b-9 components C6, C9, S-protein and SP-40,40. All components of SC5b-9 were found in arteries and arterioles, along the tubular basement membrane and in areas of glomerulosclerosis in all biopsies. This deposition was sometimes associated with C3 but never immunoglobulin deposition and correlated with the degree of renal injury. In addition, in biopsies with glomerular deposition of immunoglobulin and C3, the SC5b-9, components co-localized with the immune deposits. Glomeruli without immune deposits or glomerulosclerosis contained none of the SC5b-9 components. The incidence and pattern of distribution of SP-40,40 was similar to that of S-protein, C6 and C9 in all of cases. These data confirm that the terminal complement complex in the kidney is, at least partly, in the SC5b-9 form both in the specific immune glomerular deposition and in the "non-specific" deposition in areas of renal injury. SP-40,40 is also found in the SC5b-9 complex in all forms of renal disease.

The effect of immunosuppression on vascularised allografts. A preliminary report.

Five vascularised allografts of the knee joint were performed in dogs immunosuppressed with cyclosporin A and azathioprine. Three survived with normal function for 3 to 4 months after operation. One of the unsuccessful grafts had a failed vascular anastomosis, the other an inadequate blood level of cyclosporin A. All three successful grafts healed well. In two, bone scans, radiographs and biopsies were indistinguishable from successful autografts; in the third the blood supply to the graft failed despite patent anastomoses but the graft healed well with good function. All three grafts were rejected within 2 to 3 weeks of withdrawal of cyclosporin A and azathioprine. In non-immunosuppressed dogs, allografts of the knee, both vascularised and non-vascularised, were rejected within a few days of operation. In two non-vascularised allografts, administration of cyclosporin and azathioprine had no apparent effect on the rate of rejection of the graft.

Absence of competitive interactions among axon terminals of regenerating motor neurons.

Competition among axon terminals is usually considered to contribute to the formation of patterned synaptic connections. During axonal regeneration of motor neurons in the cockroach, leg muscles initially become innervated by appropriate and inappropriate motor neurons. All axon terminals from inappropriate neurons eventually are eliminated, resulting in the reformation of the original innervation pattern. Destruction of an identified motor neuron by the intracellular injection of pronase did not prevent the elimination of inappropriate axon terminals in the muscle normally innervated by that motor neuron. Therefore, competition does not play a role in the reinnervation of the leg muscles. This indicates a major role for specific cell-cell recognition.

SP-40,40, a newly identified normal human serum protein found in the SC5b-9 complex of complement and in the immune deposits in glomerulonephritis.

We report herein the isolation and initial characterization of a novel protein, termed SP-40,40, which is present at moderate levels (35-105 micrograms/ml) in normal human serum. SP-40,40 is deposited in the renal glomeruli of patients with glomerulonephritis but is not found in normal glomeruli. The protein is a heterodimeric structure of relative molecular mass 80 kD, both chains of which are of a similar size (40 kD). The amino-terminal sequences of both chains are unrelated to one another and possess no significant homology to any known protein sequence. The tissue distribution of SP-40,40 closely resembles that of the terminal complement components and its physicochemical properties are similar to, but distinct from, those of the S protein of complement. We have identified SP-40,40 in the SC5b-9 complex of complement and have demonstrated incorporation of labeled SP-40,40 into this complex. These data suggest that SP-40,40 is an additional component of SC5b-9.