Induction therapy with short-term high-dose intravenous cyclophosphamide followed by mycophenolate mofetil in proliferative lupus nephritis.

BACKGROUND: For decades, high-dose intravenous cyclophosphamide (ivCY) given for 24-30 months was regarded as the standard therapy for proliferative lupus nephritis, despite serious side effects. Our aim was to evaluate the effect of induction therapy with short-term high-dose ivCY followed by mycophenolate mofetil (MMF) on disease parameters, mortality and health-related quality of life (HRQoL) in patients with proliferative lupus nephritis. METHODS: Between January 2003 and November 2006, 71 patients with biopsy-proven proliferative lupus nephritis were included in the second Dutch Lupus Nephritis Study. All patients were treated with ivCY (750 mg÷m2, six monthly pulses) plus oral prednisone, followed by MMF (2000 mg÷day) plus oral prednisone for 18 months, and then azathioprine (2 mg÷kg÷day) plus oral prednisone. Study endpoints included the occurrence of renal relapse, end-stage renal disease (ESRD) and mortality. RESULTS: After a median follow-up of 3.8 years (range 0.1-4.5), four (5.6%) of the 71 patients had a renal relapse, one (1.4%) failed treatment, one (1.4%) reached ESRD, and two (2.8%) died. Systemic lupus erythematosus (SLE) Disease Activity Index, serum creatinine, proteinuria and antibodies against anti-dsDNA decreased significantly during treatment and serum levels of complement factor 3 and 4 increased significantly. Furthermore, six of eight domains of the Short Form-36 as well as the number of symptoms and total distress level according to the SLE Symptom Checklist improved significantly over time. CONCLUSIONS: This open-label study shows that induction therapy with short-term (six monthly pulses) high-dose ivCY followed by MMF is effective in preventing renal relapses, ESRD and mortality and improving HRQoL in patients with proliferative lupus nephritis.

Idiopathic giant oesophageal ulcer and leucopoenia after renal transplantation.

A 45-year-old male recipient of a renal allograft was admitted because of a giant oesophageal ulcer coinciding with leucopoenia. An extensive workup revealed no explanation for the ulcer and leucopoenia. Our final diagnosis by exclusion was an idiopathic giant oesophageal ulcer and late-onset neutropenia as consequences of rituximab induction therapy given during the transplant procedure. The patient fully recovered after treatment with prednisone. However, after four months, the ulcer and leucopoenia recurred and again successfully responded to treatment with prednisone.

Dutch guidelines for diagnosis and therapy of proliferative lupus nephritis.

Proliferative lupus nephritis is a strong predictor of morbidity and mortality in patients with systemic lupus erythematosus. Despite improvements in the management of lupus nephritis, a significant number of the patients do not respond to immunosuppressive therapy and progress to end-stage renal failure. In order to optimise the diagnostic strategy and treatment of patients with proliferative lupus nephritis, guidelines are needed. In this review, the Dutch Working Party on Systemic Lupus Erythematosus provides recommendations regarding four important areas in patients with proliferative lupus nephritis: I) indications for a first renal biopsy, II ) definitions of treatment response, III ) selection of treatment options, and IV) indications for a repeat biopsy.

Receptor for advanced glycation end products (RAGE) polymorphisms are associated with systemic lupus erythematosus and disease severity in lupus nephritis.

OBJECTIVE: Interaction of advanced glycation end products (AGEs) with their receptors (RAGE) plays an important role in inflammation in auto-immune diseases. Several functional polymorphisms of RAGE have been described. In this study we analysed the role of RAGE polymorphisms in disease susceptibility for systemic lupus erythematosus (SLE). In addition, we investigated whether these polymorphisms in SLE are associated with serum levels of soluble RAGE (sRAGE), renal involvement (lupus nephritis (LN)) and its outcome. METHODS: For this cross-sectional study DNA samples of 97 SLE patients, 114 LN patients and 429 healthy controls (HC) were genotyped for four RAGE polymorphisms: -429 T/C, -374 T/A, 2184 A/G and Gly82Ser. Differences in genotype frequencies and allele frequencies were tested between patients and HCs. In SLE patients, sRAGE was measured by enzyme-linked immunosorbent assay (ELISA). In addition, association of genotypes with sRAGE and disease severity in LN was analysed. RESULTS: The C allele of -429 T/C, the T allele of -374 T/A and the G allele of 2184 A/G were significantly more prevalent in SLE and LN compared with HC. In LN, the C allele of RAGE -429 T/C, the A allele of -374 T/A and the G allele of RAGE 2184 A/G polymorphism were significantly associated with more proteinuria and worse renal function during the first two years of treatment. No association of genotype with sRAGE was found. CONCLUSION: RAGE polymorphisms are associated with susceptibility to SLE and LN. In addition, some of these polymorphisms are likely to be associated with disease severity and initial response to treatment in LN.

Pregnancy, chimerism and lupus nephritis: a multi-centre study.

Chimerism occurs twice as often in the kidneys of women with lupus nephritis as in normal kidneys and may be involved in the pathogenesis of systemic lupus erythematosus. Pregnancy is considered the most important source of chimerism, but the exact relationship between pregnancy, the persistence of chimeric cells and the development of systemic lupus erythematosus has not been investigated. Renal biopsies and clinical data from patients in the First Dutch Lupus Nephritis Study were used. Chimeric cells were identified by in-situ hybridization of the Y chromosome. A questionnaire was used to obtain detailed reproductive data including pregnancy history and miscarriages. Chimerism was found in 12 of 26 (46%) renal biopsies. Of the 12 chimeric women, 5 reported a pregnancy; of 14 women who were not chimeric, 8 reported a pregnancy. Chimeric women who had been pregnant reported significantly more pregnancies than non-chimeric women who had been pregnant (P=0.04). The median age of the youngest child was higher in chimeric women (19 years) than in non-chimeric women (6 years). Despite the attention given to pregnancy histories with respect to chimerism, this study shows that in patients with systemic lupus erythematosus, a clear-cut relationship is not apparent. A considerable number of chimeric women did not report a pregnancy: in these women, other sources of chimerism must be considered. Our data support the theory that only certain subsets of chimeric cells persist into the maternal circulation after pregnancy.

Heparanase induces a differential loss of heparan sulphate domains in overt diabetic nephropathy.

AIMS/HYPOTHESIS: Recent studies suggest that loss of heparan sulphate in the glomerular basement membrane (GBM) of the kidney with diabetic nephropathy is due to the increased production of heparanase, a heparan sulphate-degrading endoglycosidase. Our present study addresses whether heparan sulphate with different modifications is differentially reduced in the GBM and whether heparanase selectively cleaves heparan sulphate with different domain specificities. METHODS: The heparan sulphate content of renal biopsies (14 diabetic nephropathy, five normal) were analysed by immunofluorescence staining with four anti-heparan sulphate antibodies: JM403, a monoclonal antibody (mAb) recognising N-unsubstituted glucosamine residues; two phage display-derived single chain antibodies HS4C3 and EW3D10, defining sulphated heparan sulphate domains; and anti-K5 antibody, an mAb recognising unmodified heparan sulphate domains. RESULTS: We found that modified heparan sulphate domains (JM403, HS4C3 and EW3D10), but not unmodified domains (anti-K5) and agrin core protein were reduced in the GBM of kidneys from patients with diabetic nephropathy, compared with controls. Glomerular heparanase levels were increased in diabetic nephropathy kidneys and inversely correlated with the amounts of modified heparan sulphate domains. Increased heparanase production and loss of JM403 staining in the GBM correlated with the severity of proteinuria. Loss of modified heparan sulphate in the GBM as a result of degradation by heparanase was confirmed by heparan sulphate staining of heparanase-treated normal kidney biopsy specimens. CONCLUSIONS/INTERPRETATION: Our data suggest that loss of modified heparan sulphate in the GBM is mediated by an increased heparanase presence and may play a role in the pathogenesis of diabetes-induced proteinuria.

Reduction of anionic sites in the glomerular basement membrane by heparanase does not lead to proteinuria.

Heparan sulfate in the glomerular basement membrane has been considered crucial for charge-selective filtration. In many proteinuric diseases, increased glomerular expression of heparanase is associated with decreased heparan sulfate. Here, we used mice overexpressing heparanase and evaluated the expression of different heparan sulfate domains in the kidney and other tissues measured with anti-heparan sulfate antibodies. Glycosaminoglycan-associated anionic sites were visualized by the cationic dye cupromeronic blue. Transgenic mice showed a differential loss of heparan sulfate domains in several tissues. An unmodified and a sulfated heparan sulfate domain resisted heparanase action in vivo and in vitro. Glycosaminoglycan-associated anionic sites were reduced about fivefold in the glomerular basement membrane of transgenic mice, whereas glomerular ultrastructure and renal function remained normal. Heparanase-resistant heparan sulfate domains may represent remnant chains or chains not susceptible to cleavage. Importantly, the strong reduction of glycosaminoglycan-associated anionic sites in the glomerular basement membrane without development of a clear renal phenotype questions the primary role of heparan sulfate in charge-selective filtration. We cannot, however, exclude that overexpression of heparanase and heparan sulfate loss in the basement membrane in glomerular diseases contributes to proteinuria.

Selection and characterization of a unique phage display-derived antibody against dermatan sulfate.

Dermatan sulfate (DS) is a member of the glycosaminoglycan (GAG) family and is primarily located in the extracellular matrix. Using a modified phage display procedure, we selected 2 different antibodies against DS of which one antibody, LKN1, was specific for DS. LKN1 was especially reactive with 4/2,4-di-O-sulfated DS, and did not react with other classes of GAGs including chondroitin sulfate and heparan sulfate. Immunohistochemical analysis of kidney, skin and tendon showed a typical fibrillar staining pattern, co-localizing with type I collagen. Staining was abolished by specific enzymatic digestion of DS. Immunoelectron microscopy confirmed the association of the DS epitope with collagen fibrils. The location of DS did not follow the main banding period of collagen, which is in line with the current concept that the core protein rather than the DS moiety of DS-proteoglycans specifically binds to collagen fibrils. This unique anti-DS antibody and the availability of its coding DNA may be instrumental in studies of the structure and function of DS.

Azathioprine/methylprednisolone versus cyclophosphamide in proliferative lupus nephritis. A randomized controlled trial.

Until recently, intravenous cyclophosphamide pulses with oral corticosteroids were regarded standard therapy for proliferative lupus nephritis (LN). Azathioprine, a less toxic alternative, was never proven to be inferior. In the first Dutch lupus nephritis study (enrollment between 1995 and 2001), we randomized 87 proliferative LN patients to either cyclophosphamide pulses (750 mg/m(2), 13 pulses in 2 years) combined with oral prednisone (CY) or to azathioprine (2 mg/kg/day in 2 years) combined with intravenous pulses of methylprednisolone (3 x 3 pulses of 1000 mg) and oral prednisone (AZA). After a median follow-up of 5.7 years (interquartile range 4.1-7.2 years), doubling of serum creatinine was more frequent in the AZA group, although not statistically significant (relative risk (RR): 4.1, with 95% confidence interval (95% CI): 0.8-20.4). Relapses occurred more often in the AZA group (RR: 8.8, 95% CI: 1.5-31.8). Creatinine and proteinuria at last visit did not differ between the two treatment arms. Moreover, 88.4% of the patients in the AZA arm were still free of cyclophosphamide treatment. During the first 2 years, the frequency of remission was not different, but infections, especially herpes zoster virus infections (HZV) were more frequent in the AZA group. Parameters for ovarian function did not differ between the two groups. In conclusion, in this open-label randomized controlled trial, cyclophosphamide was superior to azathioprine with regard to renal relapses and HZV. At last follow-up, there were no differences in serum creatinine or proteinuria between the two groups. However, since our study lacked sufficient power, longer follow-up is needed to reveal putative differences.

Reactive oxygen species deglycosilate glomerular alpha-dystroglycan.

In the kidney, dystroglycan (DG) has been shown to cover the basolateral and apical membranes of the podocyte. alpha-DG is heavily glycosilated, which is important for its binding to laminin and agrin in the glomerular basement membrane. Furthermore, alpha-DG is negatively charged, which maintains the filtration slit open. Reactive oxygen species (ROS) are known to degrade and depolymerize carbohydrates, and to play a role in several glomerular diseases. Therefore, we evaluated the effect of ROS on the glycosilation of glomerular alpha-DG. By using specific antibodies directed against the core protein or glyco-epitopes of alpha-DG, this was studied in a solid-phase assay, in situ on kidney sections, and in vivo in adriamycin nephropathy. A ligand overlay assay was used to study binding of alpha-DG to its ligands. Exposure to ROS leads to a loss of carbohydrate epitopes on alpha-DG both in vitro and on kidney sections. In the in vitro assays, a decreased binding of deglycosilated alpha-DG to laminin and agrin was found. In adriamycin nephropathy, where radicals play a role, we observed a loss of alpha-DG carbohydrate epitopes. We conclude that deglycosilation of glomerular alpha-DG by ROS leads to disruption of the agrin-DG complex, which in vivo may lead to the detachment of podocytes. Furthermore, loss of negative charge in the filtration slit may lead to foot process effacement of podocytes.

Glomerular hyperfiltration in type 1 diabetes mellitus results from primary changes in proximal tubular sodium handling without changes in volume expansion.

BACKGROUND: Glomerular hyperfiltration plays a role in the pathophysiology of diabetic nephropathy. An increase in the glomerular filtration rate (GFR) could result from primary actions at the glomerular/vascular level or could be the consequence of a primary increase in proximal tubular sodium reabsorption resulting in systemic volume expansion. Recently it was hypothesized that an increase in sodium reabsorption may lead to glomerular hyperfiltration through the tubulo-glomerular feedback mechanism (tubular-hypothesis) without volume expansion. DESIGN: We have studied 54 normoalbuminuric patients with type 1 diabetes. The GFR was measured by inulin clearance. Proximal and distal sodium reabsorption were calculated according to standard formulas using the free water clearance technique. Plasma volume, measured by the (125)I-albumin method, atrial natriuretic peptide (ANP) and the second messenger cyclic guanosine-3,5-monophosphate (c-GMP) were used as markers of extracellular volume expansion. RESULTS: Glomerular hyperfiltration (GFR >or= 130 mL min(-1) 1.73 m(-2)) was present in 14 out of 55 patients with diabetes (25%). There were no differences in plasma volume between normo-(NF) and hyper-filtrating (HF) patients (2933 +/- 423 in NF vs. 3026 +/- 562 mL in HF, NS). Also plasma ANP and c-GMP levels were not significantly different between the groups. The fractional proximal reabsorption of sodium was significantly increased in HF [fPRNa(+) (%) 90.1 +/- 2.0 vs. 91.5 +/- 1.6, P = 0.02]. There were no differences in distal sodium reabsorption or distal sodium load (approximately macula densa concentration of NaCl) in both groups. CONCLUSIONS: Our data suggest that the primary event in diabetic glomerular hyperfiltration is an increase in proximal tubular sodium reabsorption. They do not support the hypothesis that systemic volume expansion or ANP mediate glomerular hyperfiltration in patients with normoalbuminuric type 1 diabetes. As such, changes in tubular sodium handling most probably influence tubulo-glomerular feedback.

Extranuclear detection of histones and nucleosomes in activated human lymphoblasts as an early event in apoptosis.

OBJECTIVE: To evaluate the presence of histones and nucleosomes in cell lysates of freshly isolated peripheral blood mononuclear cells (PBMC), fully activated lymphoblasts, or lymphoblasts after induction of apoptosis. METHODS: Each histone class (H1, H2A, H2B, H3, and H4) was detected by western blot analysis with specific antibodies. Annexin V/propidium iodide (PI) staining was performed for each treatment to distinguish viable, early, and late apoptotic, and necrotic cells. DNA degradation was evaluated by PI staining in a hypotonic buffer. RESULTS: All five histones were detected in cell lysates of activated lymphoblasts in higher concentrations than in lysates of PBMC. An additional significant increase of H2A, H2B, H3, H4, and complete nucleosomes in cell lysates of lymphoblasts was found during interleukin (IL)2 deprivation for 8 or 24 hours. The content of these histones or nucleosomes in cell lysates decreased after delayed IL2 readdition. H1 content in cell lysates was not affected by IL2 deprivation or addition. Quantities of H2A, H2B, H3, and H4 in cell lysates correlated significantly with signs of early apoptosis. UV-B light exposure or etoposide treatment of lymphoblasts resulted in a distinct increase for each histone class in cell lysates compared with standard curves. No bands for post-translationally modified histones were detected in cell lysates in contrast with signals in nuclear preparations. CONCLUSION: Extranuclear accumulation of histones and nucleosomes is an early event of apoptosis in human lymphoblasts. Dysregulation of early apoptosis might support the induction of autoimmunity against nuclear components.

Decreased phagocytosis of apoptotic cells in diseased SLE mice.

Antibodies against nucleosomes are a serological hallmark of systemic lupus erythematosus (SLE). Apoptotic cells are the unique source of nucleosomes, which are formed through cleavage of chromatin by nucleases. These nucleosomes and other autoantigens targeted in SLE are expressed in apoptotic blebs or at the surface of apoptotic cells. Therefore, it is conceivable that circulating antibodies can influence apoptotic cell clearance. Using an in vitro phagocytosis assay, we analysed the phagocytic efficacy for apoptotic cells of resident peritoneal macrophages from pre-morbid and diseased lupus mice. The assay was carried out in the presence of autologous serum, using autologous apoptotic thymocytes as targets. Under these conditions macrophages from diseased MRL/lpr and NZBxNZW(F1) lupus mice, and from age-matched NZB mice showed a decreased phagocytic efficacy (decrease 47%, 48% and 37%, respectively compared to measurements in pre-morbid mice). The cause of this decrease resides in the serum, and is not due to an acquired defect of macrophages. In conclusion, during disease progression in murine SLE, apoptotic cell clearance becomes impaired, which might amplify further disease progression.

Lupus nephritis: consequence of disturbed removal of apoptotic cells?

In the last decade it has become clear that systemic lupus erythematosus (SLE) is an autoantigen driven T cell dependent autoimmune disease. The nucleosome has been identified as a major autoantigen. Nucleosomes are generated during apoptosis. Either an increased or delayed apoptosis or a reduced clearance of apoptotic cells (which are not mutually exclusive) leads to an increased exposure of (modified, more immunogenic) nucleosomes to the immune system. This generates the formation of nucleosome specific T cells and antinucleosome autoantibodies. After complex formation of antinucleosome or anti-double-stranded (ds)DNA antibodies with nucleosomes, these autoantibodies are targeted to basement membranes, especially the glomerular basement membrane (GBM). This nephritogenic potential is due to the binding of the positively charged histone components of the nucleosome to the negatively charged heparan sulphate (HS) within the GBM. This incites glomerular inflammation.

Health-related quality of life in patients with systemic lupus erythematosus: development and validation of a lupus specific symptom checklist.

Reliable and sensitive measures are needed to evaluate the quality of life (QoL) in patients with systemic lupus erythematosus (SLE). No lupus specific questionnaires are available. This study describes the development and validation of a disease-specific questionnaire for lupus patients, which assesses the presence and burden of 38 disease- and treatment-related symptoms: the SLE Symptom Checklist (SSC). Reliability and reproducibility were tested in respectively 87 and 28 stable SLE patients. The internal consistency (Cronbach's alpha coefficients 0.89) and test-retest reliability (Pearson product-moment correlation coefficient between 0.67 and 0.87) were satisfactory. Concurrent validity was supported by significant, but moderate correlations with other measures of subjective well-being and functional status. Responsiveness was measured in 17 patients with lupus nephritis treated with cyclophosphamide, at start of therapy and 1 year thereafter. A significant change in number of symptoms and total distress level was found. It is concluded that the SSC has satisfactory psychometric properties and appears suitable for both clinical and research purposes.

Prognostic factors for the vascular components of erectile dysfunction in patients on renal replacement therapy.

A total of 76 male patients on renal replacement therapy (RRT) were investigated. Erectile dysfunction (ED) was defined as insufficient erection during visual erotic stimulation (VES) or during sleep as measured with Rigiscan and Erectiometer. Data on medical history, physical examination, and laboratory variables were collected. Furthermore, penile pharmacological duplex ultrasonography (PPDU) was performed. Univariate and multivariate logistic regressions were used to determine prognostic values and to develop prognostic models. Independent prognostic factors for ED were the number of cardiovascular events, waist-hip ratio, body mass index, and acceleration time (AT) as measured with PPDU. Independent prognostic factors for an abnormal AT (>100 ms) were number of cardiovascular events, age category, and the presence of carotid bruits. Independent prognostic factors for insufficient veno-occlusion during PPDU were number of cardiovascular events and supine diastolic blood pressure. The vascular contribution to ED in patients on RRT is substantial. Data from medical history, limited physical examination, and PPDU contribute to the prediction of the vascular contribution to ED.

Sodium-lithium countertransport is increased in normoalbuminuric type 1 diabetes but is not related to other risk factors for microangiopathy.

BACKGROUND: It has been reported that sodium-lithium countertransport (Na/Li CT) activity is increased in patients with diabetes mellitus and that this increased Na/Li CT activity is associated with the development of diabetic nephropathy. It is unclear however, whether Na/Li CT is related to other pathophysiological factors in diabetic patients. We studied kinetic parameters of Na/Li CT activity together with other putative risk factors for microangiopathy in normoalbuminuric type 1 diabetic patients and matched control subjects. SUBJECTS AND METHODS: We measured maximum velocity (Vmax) and sodium affinity (Km) of Na/Li CT in 53 diabetic patients and 45 healthy controls. Endothelial function was assessed by monitoring forearm vascular response to intrabrachial infusion of acetylcholine. Blood samples were collected for measurement of HbA1c, glucose, insulin and lipids. Blood pressure was measured intra-arterially. Renal haemodynamics were measured by inulin/p-aminohippurate clearance. Urinary albumin was measured by enzyme-linked immunosorbent assay. Transcapillary escape of albumin (TERalb) was calculated by the disappearance curve of 125I-labelled albumin. RESULTS: Vmax was increased in diabetic patients (779 +/- 36 micromol Li+ h-1 L-1 erythrocytes vs. 623 +/- 35 in controls, P < 0.01), whereas Km was decreased (64 +/- 16 mmol L-1 vs. 76 +/- 27 in controls, P = 0.03). The ratio of Vmax : Km was 12.4 +/- 0.6 in diabetic patients and 8.9 +/- 0.9 in controls (P < 0.001). When comparing diabetic patients in the lowest and highest quartile of Vmax or Km there were no differences in blood pressure, renal haemodynamics, urinary albumin excretion, TERalb, endothelial function, HbA1c, glucose, insulin, or lipid profile. CONCLUSION: Na/Li CT is increased in uncomplicated type 1 diabetes and characterized by an increase in Vmax and a decrease in Km. The increase in Na/Li CT is not associated with changes in endothelial function, degree of metabolic control, blood pressure or renal haemodynamics.

Triggers for anti-chromatin autoantibody production in SLE.

The formation of autoantibodies against chromatin is the main feature of systemic lupus erythematosis (SLE), an autoimmune disease, which is T-cell dependent and autoantigen-driven. Historically, antibodies against dsDNA, one of the components of chromatin, are considered as a hallmark of SLE. However, dsDNA is poorly immunogenic. Nucleosome-specific T helper cells have been identified. These T cells propagate not only nucleosome-specific antibodies, but also anti-dsDNA antibodies. Nucleosomes are formed during apoptosis by cleavage of chromatin, and evidence of disturbed apoptosis has been found especially in certain murine models of lupus. In addition to an increased rate of apoptosis, autoimmunity against chromatin might also result from an impaired phagocytosis of apoptotic material, for which strong evidence has been provided by studies in certain knock-out mice (C1q, SAP, Dnase I). The induction of an immune response to nucleosomes could be enhanced by modifications of histones or DNA during apoptosis, altered presentation by antigen presenting cells or a viral infection. The release of nucleosomes and the formation of anti-chromatin autoantibodies result in formation of complexes, which bind to the glomerular basement membrane via heparan sulfate. This deposition incites glomerulonephritis, the most serious manifestation of SLE.