No abstract

Mesangial cell proliferation and matrix accumulation, driven by platelet-derived growth factor (PDGF), contribute to many progressive renal diseases. In a novel approach to antagonize PDGF, we investigated the effects of a nuclease-resistant high-affinity oligonucleotide aptamer in vitro and in vivo. In cultured mesangial cells, the aptamer markedly suppressed PDGF-BB but not epidermal- or fibroblast-growth-factor-2-induced proliferation. In vivo effects of the aptamer were evaluated in a rat mesangioproliferative glomerulonephritis model. Twice-daily intravenous (i.v.) injections from days 3 to 8 after disease induction of 2.2 mg/kg PDGF-B aptamer, coupled to 40-kd polyethylene glycol (PEG), led to 1) a reduction of glomerular mitoses by 64% on day 6 and by 78% on day 9, 2) a reduction of proliferating mesangial cells by 95% on day 9, 3) markedly reduced glomerular expression of endogenous PDGF B-chain, 4) reduced glomerular monocyte/macrophage influx on day 6 after disease induction, and 5) a marked reduction of glomerular extracellular matrix overproduction (as assessed by analysis of fibronectin and type IV collagen) both on the protein and mRNA level. The administration of equivalent amounts of a PEG-coupled aptamer with a scrambled sequence or PEG alone had no beneficial effect on the natural course of the disease. These data show that specific inhibition of growth factors using custom-designed, high-affinity aptamers is feasible and effective.

The anaphylatoxic peptide C3a is part of a basic immunological defense mechanism, the complement system. Research on the human C3a receptor and signal transduction is hampered by the lack of a suitable human cell or cell line. We screened tumor cell lines and human blood cells for a C3a-dependent increase in cytosolic Ca2+ ([Ca2+]i) and analyzed this reaction in a fura-2/AM fluorescence assay for cells in suspension. U937 cells, when differentiated with dibutyryl-cAMP (Bt2cAMP), and purified human neutrophils reacted in a dose-dependent fashion to C3a and a C3a analogue synthetic peptide. We found complete homologous desensitization of this response and no heterologous desensitization to human C5a. Pertussis toxin totally blocked the increase in [Ca2+]i, indicating the possible involvement of a G-protein. Single-cell analysis by digital imaging fluorescence microscopy indicated that neutrophilic granulocytes responded to C3a. In binding studies with Bt2cAMP-differentiated U937 cells and human granulocytes, the 125I-C3a binding was displaced by C3a, yielding one class of C3a binding sites with dissociation constants (Kd) in the low nanomolar range. We identified myo-inositol 1,4,5-trisphosphate (IP3) as the second messenger possibly causing the [Ca2+]i increase and the release of N-acetyl-beta-D-glucosaminidase as one secretory cell response. By functional and binding studies we demonstrated the expression of the C3a receptor on Bt2-cAMP-differentiated U937 cells and human neutrophils and characterized parts of the C3a signal pathway. Our data support a physiological concept in which C3a might be more important than presently thought.

In the search for a serologic marker of disease activity, we measured concentrations of activated C3 (actC3, that is, neoantigens developing after C3 activation on breakdown products), C4-C3 complexes and soluble C5b-9 (sC5b-9) in one or two plasma samples from adult patients with IgA nephropathy (IgAN, N = 50) or Henoch-Schönlein purpura (HSP, N = 4). As controls, 20 patients with non-immune renal disease, but comparable age, degree of proteinuria, renal dysfunction and prevalence of hypertension were studied. Compared to controls, actC3 levels were elevated in 30% of the patients with IgAN and one of the HSP patients. C4-C3 complexes were elevated in only 8% of the IgAN patients, and sC5b-9 levels were within the control range in all IgAN and HSP patients. In IgAN patients with elevated actC3 levels, proteinuria and hematuria were more pronounced than in those with normal levels. Elevated plasma concentrations of actC3 at the first presentation correlated with subsequent deterioration of renal function both in patients with initially normal and already impaired renal function (r = -0.56, N = 44, P = 0.003). The five IgAN patients with elevated actC3 on both occasions of obtaining plasma showed the most rapid loss of renal function. We conclude that mainly alternative pathway complement activation can be demonstrated in patients with IgAN and HSP. In IgAN patients the presence of complement activation is associated with more severe renal disease. Further studies are warranted to examine the clinical usefulness of actC3 as a predictor of the subsequent course of IgAN.

Polyomavirus-mediated nephropathy is an increasingly recognized complication in renal transplant recipients, but data on the status of viral activity in the native kidneys of non-renal solid organ recipients are limited.Thirteen native kidney biopsies of heart transplant recipients with significant renal impairment were evaluated for the evidence of polyomavirus reactivation by immunohistochemistry and PCR.One case of BK virus-mediated nephropathy in a cardiac transplant recipient exposed to high levels of immunosuppressive drugs was identified. Clinical and histopathological findings of this patient progressing to terminal renal failure are discussed in detail.In conclusion, polyomavirus reactivation in native kidneys of heart transplant recipients can cause significant renal impairment and should be considered in the differential diagnosis in this patient cohort.

SDMA might be a valuable and early marker of change in GFR in the clinical and experimental setting. Future studies will have to clarify whether sensitivity, specificity and temporal resolution of SDMA make it an attractive candidate for the assessment of renal function in both the experimental and clinical setting.

No abstract

Mesangial cell proliferation and matrix overproduction characterize many progressive glomerular diseases. Based on currently available data, the role of interleukin-6 (IL-6) in mediating mesangial cell proliferation and matrix production is controversial. The present study attempts to clarify this issue by showing that: (1) IL-6 knock out mice develop a normal glomerular architecture and in particular a normal mesangium. (2) Mesangioproliferative glomerulonephritis induced by Habu snake venom is equally severe in IL-6 knock out mice as in control mice. (3) A continuous seven-day intraperitoneal infusion of 50 micrograms recombinant human IL-6 into rats with a prior minimal (subnephritogenic) injury to mesangial cells does not induce glomerular cell activation, cell proliferation, matrix production, leukocyte influx, platelet influx or proteinuria. (4) A continuous seven-day IL-6 infusion into rats with mesangioproliferative nephritis (anti-Thy 1.1 nephritis) increases matrix protein transcription in the absence of detectable effects on matrix protein accumulation and otherwise has no effect on the natural course of the disease. We conclude from these findings that IL-6 is not an important mediator of mesangial cell proliferation and matrix overproduction in vivo, and that currently little rationale exists to advocate anti-IL-6 therapy in mesangioproliferative disease states.