Hidekazu Ikeuchi

Revised classification of lupus nephritis is valuable in predicting renal outcome with an indication of the proportion of glomeruli affected by chronic lesions

OBJECTIVES To determine if the International Society of Nephrology/Renal Pathology Society (ISN/RPS) 2003 classification of lupus nephritis (LN) is helpful in predicting renal outcome. METHODS A total of 92 patients with LN who underwent renal biopsy in our hospital were re-classified according to the ISN/RPS 2003 criteria. RESULTS The mean patient age was 36.8 yrs and the median observation period was 65 months. The relative frequency for each class was as follows: Class I (minimal mesangial LN) 0%, Class II (mesangial proliferative LN) 13%, Class III (focal LN) 17%, Class IV (diffuse LN) 60% and Class V (membranous LN) 10%. Within Class IV, diffuse segmental (Class IV-S) was 25% and diffuse global (Class IV-G) 75%. During the observation period, renal function was more likely to deteriorate in Class IV-G cases than in Class IV-S cases. Importantly, when Class IV-G was subdivided into cases involving active lesion alone [IV-G (A)] or chronic lesion [IV-G (A/C)], the majority of cases in IV-G (A) was nephrotic, but responded well to therapy. In contrast, renal function declined only in IV-G (A/C) cases. Patients with Class IV-G (A/C) had persistent proteinuria in spite of intensified therapies. Moreover, the higher proportion of chronic lesions was related with the deterioration of renal function. CONCLUSIONS This study showed that in Class IV-G cases, renal outcome differed in the presence of chronicity. Chronicity could be a critical factor in predicting outcome. Thus, the revised classification of LN is clinically valuable in identifying different renal outcomes among patients with diffuse LN.

Expression of IL-19 and its receptors in RA: potential role for synovial hyperplasia formation

OBJECTIVE IL-19 is a novel cytokine of the IL-10 family. In this study, we sought to examine whether IL-19 plays a role in the pathogenesis of RA. METHODS Expression of IL-19, IL-20 receptor 1 (IL-20R1) and IL-20R2 was examined by RT-PCR and immunohistochemical analysis in rheumatoid synovium. The effects of IL-19 on synovial cells established from rheumatoid synovium (RASCs), with regard to IL-6 production and signal transducers and activators of transcription3 (STAT3) activation, were examined by ELISA and western blot analysis, respectively. The effect of IL-19 on RASC apoptosis was examined by Hoechst staining, flow cytometry analysis of annexin V binding and caspase-3 activity. RESULTS IL-19, IL-20R1 and IL-20R2 mRNA were detected by RT-PCR in synovial tissues from RA patients. Immunohistochemical analysis showed IL-19 was predominantly expressed in the hyperplastic lining layers of RA synovial tissues. The majority of IL-19-positive cells were vimentin-positive and CD68-positive synovial cells, serving as markers of fibroblasts and macrophages, respectively. IL-20R1 and IL-20R2 (IL-20Rs) were expressed in both the lining and sublining layers of RA synovium. In RASC, IL-19 was induced by lipopolysaccharide stimulation and constitutive expression of IL-20Rs was observed, suggesting IL-19 has an autocrine action. In terms of this function, IL-19 induced STAT3 activation and increased IL-6 production by RASC above the medium control. Moreover, IL-19 significantly reduced RASC apoptosis induced by serum starvation. CONCLUSIONS These data suggest that IL-19, produced by synovial cells, promotes joint inflammation in RA by inducing IL-6 production and decreasing synovial cell apoptosis.

Increased levels of serum sulfite in patients with acute pneumonia.

Sulfite, a common air pollutant, is toxic for humans, causing hypersensitivity or chronic airway diseases. We previously reported that sulfite is actively produced from neutrophils by stimulation with bacterial endotoxin, lipopolysaccharide (LPS). We also found that the serum sulfite concentration is increased in a rat model of sepsis induced by systemic injection of LPS. However, information on sulfite metabolism in human inflammatory conditions is limited. In the current study, the serum concentration of sulfite was determined in 25 patients with acute pneumonia. Serum sulfite concentration in pneumonia patients was significantly higher than that in control subjects (3.75 ± 0.88 vs. 1.23 ± 0.48 &mgr;M, respectively, P < 0.05). Among 20 patients, serum sulfite was serially determined before and after antibiotic therapy. The levels of serum sulfite were significantly reduced during the recovery phase compared with those during the acute phase (1.34 ± 0.56 vs. 3.65 ± 0.92 &mgr;M, respectively, P < 0.05). Moreover, neutrophils obtained from three patients during the acute phase of pneumonia spontaneously produced higher amounts of sulfite in vitro than those obtained after recovery. There was a close positive correlation (r = 0.71, P < 0.05) between serum sulfite and C-reactive protein (CRP) in patients with pneumonia. Taken together, the current findings suggest that serum sulfite increases during systemic inflammation in humans. Activated neutrophils might be responsible, at least in part, for the up-regulation of sulfite. Given various biological effects reported previously, sulfite may act as a mediator in inflammation.

Interleukin-6 promotes destabilized angiogenesis by modulating angiopoietin expression in rheumatoid arthritis

OBJECTIVE To examine whether IL-6 promotes angiogenesis by modulating angiopoietin (Ang) expression in RA. METHODS Synovial fibroblasts derived from RA patients (RASFs) and human umbilical vein endothelial cells (HUVECs) were co-cultured for 6 days with or without recombinant IL-6, VEGF or Ang-1. HUVECs were stained with anti-CD31 antibody and their growth was determined by quantifying the CD31-positive area. SFs were collected from RA (n = 25) and OA (n = 7) patients. RESULTS In the co-culture system, IL-6 and VEGF significantly enhanced HUVEC growth to a similar extent. However, the morphology of proliferating cells was distinct between IL-6- and VEGF-stimulated HUVEC. HUVEC stimulated with IL-6 exhibited small, loose clusters surrounded by dispersed single cells, suggesting destabilized angiogenesis by IL-6. In the supernatants, IL-6 up-regulated VEGF compared with controls and Ang-2, while it down-regulated Ang-1. In contrast, down-regulation of Ang-1 was not observed with VEGF stimulation. Consistent with the destabilized morphology, stimulation with IL-6 decreased cell surface expression of vascular endothelial cadherin (VE-cadherin) on HUVEC, presumably by inducing internalization. Interestingly, adding recombinant Ang-1 partially inhibited IL-6-induced morphological changes in HUVEC including a destabilized morphology with small, loose clusters and internalization of VE-cadherin. In SFs from RA patients, VEGF was negatively correlated with Ang-1 (r = -0.559, P=0.004). CONCLUSION IL-6 not only enhances VEGF expression but also inhibits Ang-1 signalling by directly down-regulating Ang-1 expression and up-regulating Ang-2, an antagonist of Ang-1. These synergistic effects may play a critical role in destabilized angiogenesis in RA.

Interleukin-1β measurement in stimulated whole blood cultures is useful to predict response to anti-TNF therapies in rheumatoid arthritis

OBJECTIVE In RA, response to TNF blockers may be associated with a profile of cytokine production unique to each patient. This study sought to predict the response to biologic agents by examining pro-inflammatory cytokine synthesis in stimulated whole blood cultures (WBCs). METHODS We measured the concentration of TNF-α, IL-1β and IL-6 in supernatants of lipopolysaccharide (LPS)-stimulated WBCs obtained from RA patients (n = 41) before anti-TNF therapy (infliximab, 13; etanercept, 26; and adalimumab, 2) and from healthy controls (n = 12). At 24 weeks after biologics, whole bloods were again drawn from 14 of 41 patients. Response was defined by the European League Against Rheumatism response criteria after 24 weeks of therapy. RESULTS Among 41 patients, 32 were responders (good 14/moderate 18), while 9 were non-responders. All cytokines measured were significantly lower in RA patients than in controls. In RA, IL-1β production was lower in non-responders than in responders [median (interquartile range): 3.5 (1.5-9.4) vs 10.0 (5.1-93.1) pg/ml, P = 0.048]. The area under the curve from a receiver operating characteristic curve analysis for the prediction of response using IL-1β was 0.717 (95% CI 0.520, 0.914). The sensitivity and specificity of IL-1β (cut-off value 4.84 pg/ml) was 78.1 and 77.8%, respectively. All cytokines were significantly higher 6 months later compared with their respective baseline. CONCLUSION IL-1β measurement in LPS-stimulated WBC is useful to predict responsiveness to anti-TNF agents. Cytokine production capacities in LPS-stimulated WBCs are up-regulated by biologics.

Enhancement of in vitro human tubulogenesis by endothelial cell-derived factors: implications for in vivo tubular regeneration after injury

Renal proximal tubular epithelium can regenerate after various insults. To examine whether the tubular repair process is regulated by surrounding peritubular capillaries, we established an in vitro human tubulogenesis model that mimics in vivo tubular regeneration after injury. In this model, HGF, a potent renotropic factor, dose dependently induced tubular structures in human renal proximal tubular epithelial cells cultured in gels. Consistent with regenerating tubular cells after injury, HGF-induced tubular structures expressed a developmental gene, Pax-2, and a mesenchymal marker, vimentin, and formed a lumen with aquaporin-1 expression. Electron microscopic analysis showed the presence of microvilli on the apical site of the lumen, suggesting that these structures are morphologically equivalent to renal tubules in vivo. When cocultured with human umbilical vein endothelial cells (HUVEC), HGF-induced tubular formation was significantly enhanced. This could not be reproduced by the addition of VEGF, basic FGF, or PDGF. Protein array revealed that HUVEC produced various matrix metalloproteinases (MMPs). The stimulatory effects of coculture with HUVEC or HUVEC-derived conditional medium were almost completely abolished by addition of the tissue inhibitor of metalloproteinase (TIMP)-1 or TIMP-2. These data suggest that endothelial cell-derived factors including MMPs play a critical role in tubulogenesis and imply a potential role of peritubular capillary endothelium as a source of factor(s) required for tubular recovery after injury.

Fluvastatin Reduces Renal Fibroblast Proliferation and Production of Type III Collagen: Therapeutic Implications for Tubulointerstitial Fibrosis

Background: Accumulating evidence suggests that hydroxymethylglutaryl-CoA reductase inhibitors have many biological effects beyond reducing cholesterol synthesis. In a mouse model of renal interstitial fibrosis induced by unilateral ureteral obstruction, fluvastatin, one of the lipophilic hydroxymethylglutaryl-CoA reductase inhibitors, was shown to ameliorate fibrosis. Methods: In the present study, we examined the direct effects of fluvastatin on proliferation, matrix and growth factor production by rat kidney fibroblasts (NRK-49F cells). Results: Treatment with fluvastatin reduced proliferation of NRK-49F cells in a dose-dependent manner. The addition of mevalonate or geranylgeranyl pyrophosphate but not farnesyl pyrophosphate to the culture medium almost completely abolished the effect of fluvastatin. Moreover, fluvastatin treatment decreased the expression of activated Rho in NRK-49F cells suggesting that fluvastatin may decrease cell growth through blocking the activation of Rho. The majority of fluvastatin-treated cells were arrested at the G1 phase, associated with down-regulation of cyclin A and up-regulation of cyclin-dependent kinase inhibitor p27kip1, indicating that cell cycle modulation is an important mechanism. Fluvastatin significantly decreased messenger RNA expression of type III collagen and connective tissue growth factor. Conclusions: Taken together, it is suggested that fluvastatin may prevent tubulointerstitial fibrosis in a variety of progressive renal diseases by inhibiting proliferation of interstitial fibroblasts and their matrix synthesis.

Nephrotic Syndrome Caused by Immune-Mediated Acquired LCAT Deficiency

Lecithin-cholesterol acyltransferase (LCAT) is an enzyme involved in maintaining cholesterol homeostasis. In familial LCAT deficiency (FLD), abnormal lipid deposition causes renal injury and nephrotic syndrome, frequently progressing to ESRD. Here, we describe a 63-year-old Japanese woman with no family history of renal disease who presented with nephrotic syndrome. The laboratory data revealed an extremely low level of serum HDL and undetectable serum LCAT activity. Renal biopsy showed glomerular lipid deposition with prominent accumulation of foam cells, similar to the histologic findings of FLD. In addition, she had subepithelial electron-dense deposits compatible with membranous nephropathy, which are not typical of FLD. A mixing test and coimmunoprecipitation study demonstrated the presence of an inhibitory anti-LCAT antibody in the patients serum. Immunohistochemistry and immunofluorescence detected LCAT along parts of the glomerular capillary walls, suggesting that LCAT was an antigen responsible for the membranous nephropathy. Treatment with steroids resulted in complete remission of the nephrotic syndrome, normalization of serum LCAT activity and HDL level, and disappearance of foam cell accumulation in renal tissue. In summary, inhibitory anti-LCAT antibody can lead to glomerular lesions similar to those observed in FLD.

Age-related decline in label-retaining tubular cells: implication for reduced regenerative capacity after injury in the aging kidney

Recovery after acute kidney injury is impaired in the elderly, but the precise mechanism for such age-related incompetence remains unclear. By in vivo bromodeoxyuridine (BrdU) labeling, renal progenitor cells (label-retaining cells; LRCs) were identified in tubules of normal rat kidney and were shown to be the origin of proliferating cells after injury. In the present study, the involvement of LRCs in the age-related decline of tubular recovery after injury was examined. After 1 wk of BrdU labeling followed by a 2-wk chase period, ischemia-reperfusion injury was induced in 7-wk-, 7-mo-, and 12-mo-old rats. Age-related decreases in DNA synthesis and cell proliferation in renal tubules after injury were found. The number of LRCs also significantly declined with age. At 24 h after reperfusion, the number of LRCs significantly increased in all ages of rats tested. There was no significant difference in the ratio of LRC division among rats of different ages. The area of the rat endothelial cell antigen (RECA)-1-positive capillary network declined with age. When renal tubules isolated from rats treated with BrdU label were cocultured with human umbilical vein endothelial cells (HUVEC), the number of LRCs significantly increased compared with tubules cultured without HUVEC. These data suggest that the reduced capacity of tubular regeneration in the aging kidney is partly explained by the shortage of LRC reserves. The size of the LRC pool might be regulated by the surrounding peritubular capillary network.

Efficacy and safety of multi-target therapy using a combination of tacrolimus, mycophenolate mofetil and a steroid in patients with active lupus nephritis

Abstract Objectives. To examine the efficacy and safety of multi-target therapy using tacrolimus (TAC), mycophenolate mofetil (MMF) and a steroid as initial treatment for active lupus nephritis (LN). Methods. We conducted a retrospective analysis of the data of 16 consecutive patients who received the multi-target therapy for active Classes III–V LN at our department. We also compared the outcomes of the multi-target therapy with those of TAC therapy (TAC + steroid), a study of which we had conducted previously in 13 patients with active LN (TAC group). Results. All the patients treated with multi-target therapy achieved complete remission (CR) (mean, 4.6 ± 3.8 months; range, 1–15 months). The clinical profiles of the patients of the multi-target group were similar to those of the TAC group at baseline, except for a significantly higher level of proteinuria (4.6 ± 2.8 vs. 2.5 ± 2.1 g/gCr, p = 0.033) in the former. The CR rate at 6 months was significantly higher in the multi-target group as compared with that in the TAC group (81% vs. 38%, p = 0.018). Two cases of serious adverse events were associated with cytomegalovirus infection in the multi-target group, namely gastric ulcer and pancytopenia, both of which were successfully treated by antiviral therapy. Conclusions. Multi-target therapy was effective as initial treatment for active LN, with CR achieved early and in a high percentage of patients. Although this therapy was generally well tolerated, it is important to bear in mind the associated risk of cytomegalovirus infection.