BACKGROUND/AIMS:It is still unclear what happens in the glomerulus when proteinuria starts. Using puromycin aminonucleoside nephrosis (PAN) rats, we studied early ultrastructural and permeability changes in relation to the expression of the podocyte-associated molecules nephrin, α-actinin, dendrin, and plekhh2, the last two of which were only recently discovered in podocytes.METHODS:Using immune stainings, semiquantitative measurement was performed under the electron microscope. Permeability was assessed using isolated kidney perfusion with tracers. Possible effects of ACE inhibition were tested.RESULTS:By day 2, some patchy foot process effacement, but no proteinuria, appeared. The amount of nephrin was reduced in both diseased and normal areas. The other proteins showed few changes, which were limited to diseased areas. By day 4, foot process effacement was complete and proteinuria appeared in parallel with signs of size barrier damage. Nephrin decreased further, while dendrin and plekhh2 also decreased but α-actinin remained unchanged. ACE inhibition had no significant protective effect.CONCLUSIONS:PAN glomeruli already showed significant pathology by day 4, despite relatively mild proteinuria. This was preceded by altered nephrin expression, supporting its pivotal role in podocyte morphology. The novel proteins dendrin and plekhh2 were both reduced, suggesting roles in PAN, whereas α-actinin was unchanged.

BACKGROUNDDendrin is an 81-kD cytosolic protein hitherto described in the brain, where it is associated with the actin cytoskeleton. Recently, we found dendrin in foot processes of mouse glomerular podocytes. Here we describe its expression both during mouse glomerulogenesis and in the normal and diseased human kidney for the first time.METHODSDendrin expression was characterized using RT-PCR and immunohistochemistry and semi-quantified using immunoelectron microscopy.RESULTSIn glomerulogenesis, dendrin mRNA and protein appeared first at the early capillary loop stage. It was concentrated to the pre-podocytes on the basal side of podocalyxin, an apical cell membrane marker. In human tissue, dendrin transcripts were detected in the brain and kidney. In the mature kidney dendrin localized solely in the podocytes, close to the filtration slit diaphragms. A comparison with the slit-associated protein zonula occludens-1 (ZO-1) was done in minimal change nephrotic syndrome (MCNS). Dendrin and ZO-1 were re-distributed from slit regions to the podocyte cytoplasm in areas with foot process effacement (FPE). In areas without FPE, dendrin and ZO-1 distributions were unchanged compared to controls. The total amounts of dendrin or ZO-1 markers were unchanged. This differs from nephrin that, according to our previous results, is also decreased in non-effaced areas.CONCLUSIONSThe expression of dendrin during glomerulogenesis and in the normal human kidney is similar to that previously shown for nephrin, which suggests that dendrin associates with the slit diaphragm complex. In MCNS patients, dendrin and ZO-1 are re-distributed within the podocytes. Whether this is a cause or a consequence of FPE remains unclear.

Impaired angiogenic function has been reported in patients with kidney failure. During vascular damage, endothelial cells may detach from the site of inflammation and be released into the peripheral blood. With the use of Wegener's granulomatosis as a study model, whether circulating inflammatory endothelial cells (IEC) can (1) be used as a disease activity marker and (2) contribute to sustained vascular damage by inducing endothelial progenitor cell (EPC) dysfunction were examined. IEC-defined as endothelial cells that express the two inflammatory-associated markers vascular-adhesion protein-1 (VAP-1) and MHC class I-related chain A (MICA)-were increased significantly in patients with active disease as compared with those in remission. IEC expressed high levels of inducible nitric oxide synthase and neutrophil-activating chemokines, such as macrophage inflammatory protein-1alpha, growth-related oncogene-alpha, epithelial neutrophil activating peptide-78, and IL-8, and induced increased neutrophil migration. IEC levels significantly correlated with C-reactive protein and extent of organ involvement. Patients with active disease had decreased numbers of EPC colony-forming units and a high expression of VAP-1 and MICA in kidney endothelium. EPC did not express VAP-1 or MICA. IEC significantly inhibited proliferation, migration, and endothelial nitric oxide synthase expression in EPC. Thus, apart from being a new disease activity marker, IEC may contribute to vascular damage by impairing the functional capacity for repair by EPC. IEC may provide a unique in vitro system to study pathogenesis of kidney and vascular diseases.

BACKGROUNDAntiendothelial cell antibodies (AECA), usually detected using human umbilical vein endothelial cells (HUVEC), are frequently observed in systemic vasculitis, but their pathogenic role is unclear. Heterogeneity of endothelial cells necessitates use of clinically relevant endothelial cells for elucidation of the role of AECA in systemic vasculitis involving small blood vessels of specific organs.METHODSHuman endothelial cells were isolated from normal tissue specimens from the nose, kidney, lung, liver, and umbilical vein. Using flow cytometry, AECA were detected against both unstimulated and cytokine-stimulated [tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] endothelial cells. Functional capacity of AECA was determined by complement fixation assay. Sera from patients with Wegener's granulomatosis (16), limited Wegener's granulomatosis (8), renal limited disease (4), microscopic polyangiitis (MPA) (5), rheumatoid arthritis (10), and systemic lupus erythematosus (SLE) (9), and from healthy controls (20) were analyzed.RESULTSCompared with controls (1) Wegener's granulomatosis is significantly associated with noncytotoxic AECA that selectively bind surface antigens on unstimulated nasal, kidney, and lung endothelial cells; (2) binding of Wegener's granulomatosis AECA to kidney and nasal endothelial cells in particular was lost upon treatment with IFN-gamma and TNF-alpha; (3) the two cytokines per se were cytotoxic (30%) to nasal and lung endothelial cells and lysis was further increased (60%) by addition of systemic vasculitis serum; and (4) Wegener's granulomatosis serum caused agglutination of cytokine-stimulated nasal endothelial cells.CONCLUSIONBased on these findings we suggest that AECA may be one factor involved in the initiation of Wegener's granulomatosis. Antigen identification and elucidation of the pathogenic roles of AECA and inflammatory cytokines in systemic vasculitis using these cells will be particularly important.

The past few years have witnessed a major breakthrough in the understanding of the molecular mechanisms and ultrastructural changes behind the development of proteinuria. The discovery of several proteins in the glomerular podocyte and slit diaphragm, where mutations lead to disease, has revealed the importance of this cell with its diaphragm as the major filtration barrier as opposed to the glomerular basement membrane (GBM) previously ascribed this function. Furthermore, accumulating clinical as well as experimental evidence points to the harmful effects of proteinuria, irrespective of the original damage. The purpose of this review is to shed light on what we know today about the two sides of this 'coin', the causes and the consequences of proteinuria.

BACKGROUND:The primary systemic vasculitides usually associated with autoantibodies to neutrophil cytoplasmic antigens include Wegener's granulomatosis and microscopic polyangiitis. We investigated whether exposure to cyclophosphamide in patients with generalized vasculitis could be reduced by substitution of azathioprine at remission.METHODS:We studied patients with a new diagnosis of generalized vasculitis and a serum creatinine concentration of 5.7 mg per deciliter (500 micromol per liter) or less. All patients received at least three months of therapy with oral cyclophosphamide and prednisolone. After remission, patients were randomly assigned to continued cyclophosphamide therapy (1.5 mg per kilogram of body weight per day) or a substitute regimen of azathioprine (2 mg per kilogram per day). Both groups continued to receive prednisolone and were followed for 18 months from study entry. Relapse was the primary end point.RESULTS:Of 155 patients studied, 144 (93 percent) entered remission and were randomly assigned to azathioprine (71 patients) or continued cyclophosphamide (73 patients). There were eight deaths (5 percent), seven of them during the first three months. Eleven relapses occurred in the azathioprine group (15.5 percent), and 10 occurred in the cyclophosphamide group (13.7 percent, P=0.65). Severe adverse events occurred in 15 patients during the induction phase (10 percent), in 8 patients in the azathioprine group during the remission phase (11 percent), and in 7 patients in the cyclophosphamide group during the remission phase (10 percent, P=0.94 for the comparison between groups during the remission phase). The relapse rate was lower among the patients with microscopic polyangiitis than among those with Wegener's granulomatosis (P=0.03).CONCLUSIONS: In patients with generalized vasculitis, the withdrawal of cyclophosphamide and the substitution of azathioprine after remission did not increase the rate of relapse. Thus, the duration of exposure to cyclophosphamide may be safely reduced.

BACKGROUND:Nephrin is a cell-adhesion protein that is defective in congenital nephrotic syndrome of the Finnish type (CNF). Nephrin is synthesized by the podocytes and is localized to the slit membrane between individual foot processes of the podocytes. Although nephrin is apparently pivotal in the development of CNF, the role of nephrin in other causes of nephrotic syndrome is not fully understood.METHODS:Renal biopsy specimens from patients with minimal change nephrotic syndrome (MCNS) were investigated. Nephrin distribution was studied with immunohistochemical and semiquantitative immunoelectron microscopic techniques and the results were related to the degree of foot process effacement found under the electron microscope.RESULTS:In normal kidney, immunofluorescence revealed a linear staining along the capillary basement membranes, corresponding to the localization of nephrin in the slit membranes. In the biopsies from patients with MCNS, the nephrin pattern had become granular. The degree of granularization corresponded to the degree of foot process effacement. Ultrastructural semiquantification showed the amount of nephrin to be reduced both in areas with and without foot process effacement compared with the control specimens. The concentration of nephrin was lowest in the areas with foot process effacement and there was redistribution from the slits into the cytoplasm.CONCLUSIONS:These findings demonstrate that nephrin expression is altered in MCNS. Whether this reflects a pathogenetic role for nephrin in MCNS or a phenomenon secondary to other causes of foot process effacement remains to be elucidated.

The role of the Fas/FasL system in ANCA-associated vasculitis is unclear. We therefore assessed levels of soluble Fas (sFas) in sera and Fas expression on mononuclear cells from patients with ANCA-positive vasculitis and compared the results with those found in other rheumatic diseases. Serum levels of sFas were determined by ELISA. The ANCA-positive vasculitis patients studied included 29 at onset, 17 in first remission while on therapy, and 12 in quiescence. For comparison, 10 patients with Sjögren’s syndrome (SS), 14 patients with systemic lupus erythematosus (SLE), 29 patients with rheumatoid arthritis (RA), 7 patients on dialysis (DP), and 26 healthy controls (HC) were studied. In addition, Fas expression in mononuclear cells was examined at the mRNA level using reverse transcriptase (RT)-PCR in 6 vasculitis patients at onset and in first remission. The expression of CD95 on the surface of leukocytes was determined by flow cytometry in 6 vasculitis patients at onset of the disease, in 6 patients in clinical remission, and in 6 HC. Expression of Fas and FasL in renal biopsy specimens was studied using immunohistochemistry. Patients with vasculitis had high sFas levels irrespective of disease phase. Both vasculitis patients and patients with RA and SLE had significantly increased sFas levels compared with healthy controls. All patient groups had sFas levels, which correlated with raised serum creatinine values. However, the sFas levels in vasculitis patients in first remission and in quiescence were increased despite a lower serum creatinine compared with onset. Some of the vasculitis patients showed an increased mRNA expression of Fas in mononuclear cells after treatment, suggesting that Fas production fluctuates with the intensity of the disease. The expression of CD95 on leukocytes was slightly decreased in vasculitis patients compared to healthy controls. No alterations of Fas and FasL expression were seen in renal biopsy specimens. These results show that ANCA-positive vasculitis patients have high sFas levels and that the levels remain elevated even in clinical remission. The findings indicate that perturbations in the Fas/Fas ligand system may play a role in the disease process in ANCA vasculitis.

Gene therapy of Alport syndrome (hereditary nephritis) aims at the transfer of a corrected type IV collagen α chain gene into renal glomerular cells responsible for production of the glomerular basement membrane (GBM). A GBM network composed of type IV collagen molecules is abnormal in Alport syndrome which leads progressively to kidney failure. The most common X-linked form of the disease is caused by mutations in the gene for the α5(IV) chain, the α5 chain of type IV collagen. Full-length human α5(IV) cDNA was expressed in HT1080 cells with an adenovirus vector, and the recombinant α5(IV) chain was shown to assemble into heterotrimers consisting of α3(IV) and α4(IV) chains, utilizing a FLAG epitope in the recombinant α5(IV) chain. The results indicate that correction of the molecular defect in Alport syndrome is possible. Previously, we had developed an organ perfusion method for effective in vivo gene transfer into glomerular cells. In vivo perfusion of pig kidneys with the recombinant adenovirus resulted in expression of the α5(IV) chain in kidney glomeruli as shown by in situ hybridization and its deposition into the GBM was shown by immunohistochemistry. The results strongly suggest future possibilities for gene therapy of Alport syndrome.

BACKGROUND AND AIMS:Patients with vasculitic disease and autoantibodies to neutrophil cytoplasmic antigens (ANCA) generally respond to immunosuppressive therapy with a reduction of the inflammation and lowering of the ANCA titre. However, most patients experience relapses, sometimes after years of quiescence. In the present study we addressed the question whether the relapsing nature of this disease could be dependent on an underlying T cell activation. Patients were analyzed at disease onset, in remission while on treatment, and in quiescence.PATIENTS AND METHODS:Blood lymphocyte subsets and the expression of molecules associated with T cell activation were analyzed by flow cytometry and soluble interleukin-2 receptor (sIL2r) levels in serum by ELISA. Three patient categories (la, 1b and 2) were studied and compared with age-matched healthy controls (1a: 16 patients at onset of the disease before therapy, 1b: 10 patients from group 1a, re-analyzed after first remission, 2: 11 other patients in quiescence, 2-10 years after debut).RESULTS:All patient groups, 1a, 1b and 2, showed signs of T cell activation such as reduced CD28 on CD3+ and increased of the early T cell activation marker CD69 on CD3+, as well as of CD38 on CD8+ T cells. The sIL2r levels were significantly raised in all patient categories (la: 4280, 1b: 1844, 2: 2882 ng/ml) compared with the controls (923 ng/ml).CONCLUSION:Patients with ANCA-positive vasculitis show an increased expression of T cell activation markers irrespective of immunosuppressive therapy or disease phase. Such memory cells may form the basis for the remitting course of vasculitides and would be a rational target for new strategies of therapy.

In patients receiving cyclosporine A (CyA) – based immunosuppressive therapy, Ca2+ channel blockers (CCBs) prevent the development of CyA – related nephrotoxicity in which increased Ca2+ content plays an important role. We evaluated the dynamics of the intracellular (erythrocytes) and extracellular (plasma) Ca2+ levels and the influence of the CCB, Phenihydine, on this process during the conversion from Sandimmun (S) to Sandimmun Neoral (SN). Forty-two patients were enrolled. The conversion from S to SN normalized the elevated CA2+ level of erythrocytes in groups with Phenihydine (n = 20) and without Phenyhidine (n = 12) 4 weeks after the switch (P < 0.05); this level remained stable until the end of study. Therefore we suggest that the switch from S to SN is effective in reducing elevated intracellular Ca2+ levels. The decrease of Ca2+ content in erythrocytes was similar in all groups switched to SN (with or without Phenihydine). The last effect should be an important argument to focus the further long-term investigations on the ability of CCBs to act as cytoprotective and nephroprotective agents during immunosuppressive protocols with the new microemulsion formulation of CyA.

Approximately 85% of patients with Alport syndrome (hereditary nephritis) have been estimated to have mutations in the X chromosomal COL4A5 collagen gene; the remaining cases are autosomal with mutations in the COL4A3 or COL4A4 genes located on chromosome 2. In the present work, the promoter sequence and previously unknown intron sequences flanking exons 2 and 37 of COL4A5 were determined. Furthermore, intron sequences flanking the other 49 exons were expanded from 35 to 190 to facilitate mutation analysis of the gene. Using this information, all 51 exons and the promoter region were PCR-amplified and sequenced from DNA of 50 randomly chosen patients with suspected Alport syndrome. Mutations were found in 41 patients, giving a mutation detection rate of 82%. Retrospective analysis of clinical data revealed that two of the cases might be autosomal. Although it could not be determined whether the remaining seven cases (14%) were autosomal or X chromosome-linked, it is likely that some of them were autosomal. It is concluded that PCR amplification and direct DNA sequencing of the promoter and exons is currently the best procedure to detect mutations in COL4A5 in Alport syndrome.

Thirty years since its discovery, IgA nephropathy (IgAN) has turned out to be the commonest primary glomerulonephritis around the world. The major presenting symptom is haematuria, with or without proteinuria. IgAN was initially considered a benign disease, but long-term follow-up studies have shown the course to be slowly progressive with up to 50% of the patients developing terminal renal failure after several decades. The aetiology and pathogenesis are still unclear, but IgAN seems to be associated with a dysregulation of the immune response which is not very well understood. There is today no specific treatment. Effective blood pressure control is the cornerstone of therapy.

Alport syndrome, also termed hereditary nephritis, was initially described in 1927 by A.C. Alport [1] as an inherited kidney disease characterized by hematuria and sensorineural deafness. Later, ocular lesions were also associated with the syndrome and, with the introduction of the electron microscope, irregularities and disruptions in the glomerular basement membrane (GBM) were shown to be typical for this disorder as well [2, 31. The disease is highly progressive and usually leads to renal failure during adolescence or before middle age. The disease is primarily inherited as an X-chromosome linked dominant trait with an estimated gene frequency of 1:5,000 to 1:10,000, but autosomal forms also exist [2, 3]. The defective gene in X-linked Alport syndrome was located in 1988 and 1989 to the long arm of the X chromosome [4—61. In 1990, a gene encoding a novel basement membrane (type IV) collagen a5 chain was discovered and localized to the Alport gene region on chromosome X, and this was soon followed by identification of mutations in this gene in Alport patients [7, 8]. More recently, mutations have also been reported in the genes for the a3 and a4 type IV collagen chains in the rarer autosomal forms of Alport syndrome

Objective: To assess the predictive value of different renal laboratory tests for development of superimposed preeclampsia in women with chronic hypertension.Methods: Thirty-seven pregnant patients with chronic hypertension and 14 healthy pregnant women were examined at 11-13, 19-21, 27-29, and 31-33 weeks with the following laboratory tests: plasma concentrations of creatinine, urea and uric acid, urinary excretion of albumin, and calcium; erythrocyte excretion was investigated only at 11-13 and 27-29 weeks.Results: Among the patients who developed superimposed preeclampsia (30%), a pathologic urinary albumin excretion, i.e., microalbuminuria, was seen in 30% at 31-33 weeks pregnancy, as compared to none among patients with chronic hypertension who did not develop this complication and 7% in the normotensive women (P < 0.05, chi-square test). There was no difference between the groups at the earlier gestational ages. Plasma urea concentration tended to be lower among the normotensive women than in the other two groups only at 19-21 and 27-29 weeks (P < 0.05, P = 0.065, respectively, one-way ANOVA). Plasma urea levels tended to increase with gestational length in those with superimposed preeclampsia, whereas it decreased in the other two groups (P<0.05, ANOVA with repeated measures). The other laboratory parameters differed in no way between the three groups.Conclusion: Blood and urine sampling every 4 to 8 weeks for assessment of renal function in pregnant women with chronic hypertension seems to be of limited value for the prediction of superimposed preeclampsia.

IgA nephropathy (IgAN), the most common form of glomerulonephritis, is characterized by normal to elevated levels of serum IgA. In order to understand the molecular mechanism(s) involved in the production of IgA in IgAN, peripheral blood mononuclear cells (PBMC) from these patients were analysed in this study. IL-10, transforming growth factor-beta 1 (TGF-beta 1) and CD40 have previously been shown to be involved in IgA production. We show here that CD40L expression was increased three-fold in these patients. However, expression of TGF-beta 1 in serum levels was comparable to controls. In vitro stimulation of PBMC with a polyclonal activator resulted in a three-fold increase in synthesis of both IgA subclasses, with a preference for IgA1 RNA. In situ hybridization studies also showed a three-fold increase in the numbers of IgA1- and IgA2-producing cells, but the subclass distribution was similar to the controls. Furthermore, using the nested primer polymerase chain reaction (PCR) for amplifying switch (S mu/S alpha) breakpoints we could demonstrate that in unstimulated PBMC the switch frequency did not differ from that of control donors. Sequence analysis of the amplified switch breakpoints and the I alpha regulatory region from patients showed no structural abnormality. Although we have previously demonstrated a correlation to in vivo germ-line RNA expression and class switching, no I alpha transcripts were detected in unstimulated PBMC from these patients. However, stimulation of PBMC with TGF-beta 1 resulted in I alpha production. Taken together, results from in vivo and in vitro studies suggest that increased cytokine production and hyperresponsiveness to polyclonal stimulation may play an important role in the increased synthesis of IgA. The preference for IgA1 is due to increased production of IgA1 per cell, and the absence of I alpha RNA indicates that additional defect(s) in immune regulation may play an important role in the pathogenesis of IgAN.

The incidence of hypertension 2 years after renal transplantation and the possible causes of hypertension were studied retrospectively. A group of 93 patients treated with cyclosporin (CyA), azathioprine (Aza), and/or prednisolone (Pred) were compared to a group of 31 patients treated with Aza and Pred. There were more patients with hypertension in the CyA group (73%) than in the Aza group (58%). Hypertension before transplantation predisposed to hypertension after transplantation. After transplantation, hypertension was most common among patients with polycystic kidney disease (46%), chronic glomerulonephritis (67%), and diabetes (71%). The accumulated immunosuppressive medication (CyA/Pred) did not affect the occurrence of hypertension. Hypertensive patients had significantly poorer graft function than did nor-motensive patients (serum creatinine level 229 μmol/l vs 162 μmol/l, P < 0.01). The 10-year graft survival was markedly impaired in the group with hypertension (42% vs 65% for normotensives, P < 0.05). The 10-year patient survival was 59% vs 79% (P= NS). The study further confirms the frequent finding that hypertension has a negative effect on graft and patient survival rates.