Urinary properdin excretion is associated with intrarenal complement activation and poor renal function.

BACKGROUND: Proteinuria predicts progressive renal failure. Next to being a progression marker, non-selective proteinuria itself is thought to be toxic to the tubulointerstitium. In proteinuric states, activation of filtered or locally produced complement is toxic for renal tubular cells and likely contributes to the progression of renal failure. Recent experimental evidence suggests an important role for properdin in promoting intrarenal complement activation. We measured properdin in proteinuric urine and assessed its relation with urinary SC5b-9 levels, the soluble form of the effector phase of complement activation. METHODS: Seventy patients with renal disease of different origin but all with a protein excretion of at least 1 g/day were studied. Urinary properdin and SC5b-9 levels were measured using an ELISA technique. RESULTS: Properdin was detectable in the urine of 37 patients (53%). These subjects had higher urinary SC5b-9 levels {median 0.50 U/ml [interquartile range (IQR) 0.13-1.81] versus 0.049 U/ml (IQR 0.024-0.089), P < 0.001}. When adjusted for proteinuria and renal function, properdin excretion was strongly associated with increased urinary SC5b-9 levels (odds ratio 16.2, 95% confidence interval 3.6-74.4). Properdin excretion was associated with worse renal function. CONCLUSION: Our results suggest that urinary properdin excretion enhances intrarenal complement activation and thus may contribute to the progression of renal damage in proteinuric states.

CD40L stimulation of rat dendritic cells specifically favors the IL-12/IL-10 ratio resulting in a strong T cell stimulatory capacity.

Dendritic cells (DC) play an important role in immune responses and have been studied extensively in human and mouse models. CD40 triggering of DC has a pivotal role in their maturation process, obtaining the unique capacity to induce strong CD4 and CD8 T cell activation. Although rat models are frequently used for the understanding of the underlying mechanism of human diseases, relatively little is known about rat DC. To investigate the effect of CD40 triggering on rat DC, we cloned the rat CD40L gene and generated murine fibroblasts with stable expression (L-rCD40L). DC stimulated by L-rCD40L cells exhibited a strong T cell stimulatory capacity, associated with higher amounts of IFN-gamma as compared to LPS-stimulated DC. Analysis of cytokine production showed that LPS induced both IL-12 and IL-10 production, whereas CD40L induced high amounts of IL-12, but little IL-10 production by rat DC. This implies that the difference found in T cell stimulatory capacity by the stimulated DC is due to the cytokine profile of the DC at the time of T cell activation.

Generation and characterization of a novel anti-rat CD40L antibody with inhibitory activities in vitro and in vivo.

The CD40-CD40L interaction plays a critical role in cell-mediated immune responses. Blocking this interaction has been shown to be beneficial in the treatment of various diseases studied in murine models. Although rats are widely used to test therapeutic strategies in several disease models, a monoclonal antibody (mAb) to block the CD40-CD40L interaction in rats is not broadly available. In the present study we generated Armenian hamster fibroblasts expressing rat CD40L and used these to generate a novel anti-rat CD40L mAb (AS1). In vitro studies showed that AS1 was able to block CD40L-induced DC maturation and B cell proliferation. Most importantly, in vivo, AS1 inhibited B cell responses in a dose-dependent fashion, as measured by the production of OVA specific antibodies after subcutaneous immunization with OVA. AS1 was shown to be a powerful tool to modulate Ag presentation in vitro and in vivo. Elucidating the effect of AS1 in various rat models for human diseases will provide more insight into blocking the CD40-CD40L interaction as a therapeutic strategy to prevent human diseases.

Connective tissue growth factor and igf-I are produced by human renal fibroblasts and cooperate in the induction of collagen production by high glucose.

Tubulointerstitial fibrosis is an important component in the development of diabetic nephropathy. Various renal cell types, including fibroblasts, contribute to the excessive matrix deposition in the kidney. Although transforming growth factor-beta (TGF-beta) has been thought to play a major role during fibrosis, other growth factors are also involved. Here we examined the effects of connective tissue growth factor (CTGF) and IGF-I on collagen type I and III production by human renal fibroblasts and their involvement in glucose-induced matrix accumulation. We have demonstrated that both CTGF and IGF-I expressions were increased in renal fibroblasts under hyperglycemic conditions, also in the absence of TGF-beta signaling. Although CTGF alone had no effect on collagen secretion, combined stimulation with IGF-I enhanced collagen accumulation. Furthermore, IGF-I also had a synergistic effect with glucose on the induction of collagens. Moreover, we observed a partial inhibition in glucose-induced collagen secretion with neutralizing anti-CTGF antibodies, thereby demonstrating for the first time the involvement of endogenous CTGF in glucose-induced effects in human renal fibroblasts. Therefore, the cooperation between CTGF and IGF-I might be involved in glucose-induced matrix accumulation in tubulointerstitial fibrosis and might contribute to the pathogenesis of diabetic nephropathy.

NF-kappaB mediated IL-6 production by renal epithelial cells is regulated by c-jun NH2-terminal kinase.

Tubular epithelial cells (TEC) play an important role in tubulointerstitial inflammation, a hallmark of most renal diseases, via production of cytokines and chemokines. In this study, the role of mitogen-activated protein kinases (MAPK) in regulation of the proinflammatory cytokine IL-6 in cultured human TEC in response to the leukocyte-derived factors IL-1, TNF-alpha, IL-17, and CD40L was investigated. IL-6 production induced by IL-1, TNF-alpha, and IL-17 was specifically inhibited by the c-jun NH(2)-terminal kinase (JNK) inhibitor SP600125, but not by a selective inhibitor of p38 MAPK, and was moderately increased when the ERK1/2 pathway was inhibited. Also for CD40L stimulation, inhibition of JNK resulted in a pronounced inhibition of IL-6 production. Although stimulation of TEC induced activation of activator protein-1 (AP-1), the down-stream target of JNK, reporter assays demonstrated that mutation of the AP-1 binding site in the IL-6 promoter did not affect gene transcription. Furthermore, IL-1-induced transcriptional activation of the IL-6 promotor was repressed by SP600125 or by co-transfection of a dominant-negative expression plasmid of c-jun even in the absence of a functional AP-1 binding site. This suggests that IL-6 production by renal epithelial cells is regulated by JNK, via a mechanism, however, independent of the AP-1 binding site. The data rather suggest that the JNK pathway may interfere with other signaling pathways, involving NF-kappaB and possibly ERK.

Secretion of collagen type IV by human renal fibroblasts is increased by high glucose via a TGF-beta-independent pathway.

BACKGROUND: Tubulointerstitial fibrosis is an important component of diabetic nephropathy, which is characterized by increased expression of interstitial extracellular matrix components and aberrant expression of the basement membrane component collagen type IV. The present study examined the effect of high ambient glucose and transforming growth factor-beta1 (TGF-beta1) on collagen secretion by human renal fibroblasts and proximal tubular epithelial cells (PTECs). METHODS: Human renal fibroblasts (TK173) and PTECs (HK2) were used to examine the effects of high glucose (25 mM d-glucose) and TGF-beta1 (1 ng/ml) on collagen type I, III and IV secretion compared with control medium (5.5 mM glucose). Matrix components were measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Renal fibroblasts are the main producers of the interstitial components collagen type I and type III, while collagen type IV was secreted predominantly by PTECs. However, renal fibroblasts were also able to secrete collagen type IV. Secretion of collagen type IV by fibroblasts was increased upon stimulation with TGF-beta1, reaching levels comparable with those secreted by TGF-beta1-induced PTECs. Moreover, high glucose stimulated increased collagen type IV secretion. Importantly, this could not be attenuated by neutralizing pan-specific anti-TGF-beta antibodies. CONCLUSIONS: These data show that renal fibroblasts secrete collagen type IV, which can be increased by high glucose independent of endogenous TGF-beta. This suggests that as well as the increased expression of interstitial components, renal fibroblasts can contribute to the increased expression of the basement membrane component collagen type IV in tubulointerstitial fibrosis observed during diabetic nephropathy.