Ken-ichi Samejima

Decreased renal α-Klotho expression in early diabetic nephropathy in humans and mice and its possible role in urinary calcium excretion

Hypercalciuria is one of the early manifestations of diabetic nephropathy. We explored here the role of α-Klotho, a protein expressed predominantly in distal convoluted tubules that has a role in calcium reabsorption. We studied 31 patients with early diabetic nephropathy and compared them with 31 patients with IgA nephropathy and 7 with minimal change disease. Renal α-Klotho expression was significantly lower and urinary calcium excretion (UCa/UCr) significantly higher in diabetic nephropathy than in IgA nephropathy or minimal change disease. Multiple regression analyses indicated that α-Klotho mRNA was inversely correlated with calcium excretion. We next measured these parameters in a mouse model of streptozotocin (STZ)-induced diabetic nephropathy, characterized by glomerular hyperfiltration, as seen in early diabetic nephropathy. We also confirmed a reduction of renal α-Klotho mRNA down to almost 50% and enhanced calcium excretion in mice with STZ-induced diabetic nephropathy in comparison with nondiabetic mice. Hypercalciuria was exacerbated in heterozygous α-Klotho knockout mice in comparison with wild-type mice, each with STZ-induced diabetic nephropathy. Thus, α-Klotho expression was decreased in distal convoluted tubules in diabetic nephropathy in humans and mice. Renal loss of α-Klotho may affect urinary calcium excretion in early diabetic nephropathy.

Fractalkine expression and CD16 monocyte accumulation in glomerular lesions: association with their severity and diversity in lupus models

Fractalkine (Fkn) is expressed on injured endothelial cells and is a membrane-bound chemokine that attracts cells expressing its receptor, CX3CR1, including CD16(+) monocytes (CD16(+) Mos). To clarify the role played by Fkn in the development of glomerular lesions in lupus nephritis, we examined Fkn expression and CD16(+) Mo accumulation induced in experimental C.B-17/Inc-scid/scid (SCID) lupus model mice by injection of IgG(3)-producing hybridoma clones obtained from MRL/lpr mice. Glomerular Fkn expression and accumulation of CD16(+) Mos were semiquantitatively evaluated using laser capture microdissection and real-time PCR. Injection of the 2B11.3 and 7B6.8 clones induced formation of glomerular proliferative and wire-loop lesions, respectively. Immunohistological analysis of the localization of Fkn and CD16(+) Mos revealed that Fkn expression and CD16(+) Mo accumulation were markedly elevated in glomerular lesions induced by 2B11.3, whereas no elevation was detected in those induced by 7B6.8. In addition, to examine the contribution of glomerular Fkn to the development of proliferative lesions, L cells producing an Fkn antagonist (Fkn-AT) were transplanted into SCID mice exhibiting proliferative lupus nephritis (DPLN) induced by 2B11.3. Notably, transplantation of the Fkn-AT-producing cells was functionally and histologically protective against this DPLN. Taken together, our findings suggest that Fkn and CD16(+) Mo accumulation are partially associated with the severity and diversity of histology of lupus nephritis.

Suppressed soluble Fms–like tyrosine kinase-1 production aggravates atherosclerosis in chronic kidney disease

Patients with chronic kidney disease (CKD) die of cardiovascular diseases for unknown reasons. Blood vessel formation in plaques and its relationship with plaque stability could be involved with signaling through the Flt-1 receptor and its ligands, vascular endothelial growth factor, and the closely related placental growth factor (PlGF). Flt-1 also exists as a circulating regulatory splice variant short-inhibitory form (sFlt-1) that serves as a decoy receptor, thereby inactivating PlGF. Heparin releases sFlt-1 by displacing the sFlt-1 heparin-binding site from heparin sulfate proteoglycans. Heparin could provide diagnostic inference or could also induce an antiangiogenic state. In the present study, postheparin sFlt-1 levels were lower in CKD patients than in control subjects. More importantly, sFlt-1 levels were inversely related to atherosclerosis in CKD patients, and this correlation was more robust after heparin injection, as verified by subsequent cardiovascular events. Knockout of apolipoprotein E (ApoE) and/or sFlt-1 showed that the absence of sFlt-1 worsened atherogenesis in ApoE-deficient mice. Thus, the relationship between atherosclerosis and PlGF signaling, as regulated by sFlt-1, underscores the underappreciated role of heparin in sFlt-1 release. These clinical and experimental data suggest that novel avenues into CKD-dependent atherosclerosis and its detection are warranted.

In vitro effects of Habu snake venom on cultured mesangial cells.

Background: Habu snake venom (HSV)-induced glomerulonephritis is a unique model showing a progressive course of mesangial proliferation. To elucidate the in vitro effects of HSV, we examined whether HSV itself could have direct effects on the cultured mesangial cells, such as cell proliferation and activation of chemokine gene expression. Methods: The incorporation of 5-[125I]iodo-2’-deoxyuridine was measured with a γ-counter, and gene expressions of growth factors, chemokines and cytokines were evaluated by a real time quantitative PCR. Results: We demonstrated that excessive or continuous HSV stimulation decreased a mesangial cell viability. However, adequate and temporary HSV stimulation induced proliferation of mesangial cells in vitro along with a significant elevation of monocyte chemoattractant protein-1 (MCP-1) mRNA levels. In addition to these in vitro results, we showed that MCP-1 mRNA levels increased in renal cortices of glomerulonephritis induced by HSV. Immunohistochemistry also showed a positive staining for MCP-1 in the marginal area of glomerulus with mesangiolysis. Conclusions: These data suggest that HSV itself may elicit direct biological effects on mesangial cells which may participate in pathophysiology of glomerulonephritis induced by HSV.

Heart Failure Associated with Metastatic Myocardial Calcification in a Hemodialysis Patient with Progressive Calcification of the Hand

Metastatic myocardial calcification is a frequent cause of heart failure in hemodialysis patients. However, early detection is difficult, often resulting in a poor prognosis. A 47-year-old man with hemodialysis-dependent end-stage renal disease presented with progressive dyspnea. Levels of serum phosphate, calcium, and intact parathyroid hormone were poorly controlled. He developed pain in his right thumb 1 year before presentation, and the pain gradually increased and extended to the entire right hand. Hand radiography 1 month earlier had revealed significant progressive calcification. Echocardiography showed severe, diffuse hypokinesis and pericardial effusion as well as possible anterior myocardial calcification with high echogenicity. Chest computed tomography revealed a severely dilated heart with anterior massive myocardial calcification and a large amount of pericardial effusion, which was not detected on computed tomography performed 20 months earlier. The patient was diagnosed with heart failure associated with metastatic myocardial calcification and died suddenly 2 weeks later. This experience suggests that progressive metastatic calcification of the skin and subcutaneous tissue is useful for predicting myocardial calcification.

Clinical Significance of Fibroblast-Specific Protein-1 Expression on Podocytes in Patients with Focal Segmental Glomerulosclerosis

Backgrounds/Aims: We previously reported that fibroblast-specific protein 1 (FSP1) is a marker of epithelial-mesenchymal transition (EMT) in tubulointerstitial fibrosis. The EMT-like changes observed in podocytes are reportedly associated with podocyte detachment which may cause focal glomerulosclerosis. Methods: In cross-sectional studies, we analyzed podocyte expression of FSP1 immunohistochemically using renal biopsy specimens from 31 patients with focal segmental glomerulosclerosis (FSGS) and 39 patients with minimal change disease (MCD). We also semiquantitatively analyzed glomerular expression of FSP1 mRNA using laser capture microdissection and real-time PCR. Results: We found that FSP1 was localized to podocytes in both FSGS and MCD patients; however, the number of FSP1+ podocytes per glomerular profile was significantly higher in patients with FSGS than in those with MCD, and there was a corresponding difference in the levels of FSP1 mRNA. FSP1+ podocyte counts per glomerular profile in FSGS patients correlated significantly with the prevalence of glomerulosclerosis and the extent of interstitial type-I collagen-positive areas. Conclusion: Taken together, these data suggest that podocyte expression of FSP1 could shed light on the potential linkage between EMT-like changes, detachment of podocytes from the glomerular basal membrane and the pathophysiology underlying FSGS.

Levels of MCP-1 and GM-CSF mRNA Correlated with Inflammatory Cytokines mRNA Levels in Experimental Autoimmune Myocarditis in Rats

Infiltration of monocytes and T cells is known to be an essential trigger for the progression of experimental autoimmune myocarditis (EAM) in rats. Monocyte chemotactic protein-1 (MCP-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were shown to mediate the migration of monocytes and T cells into inflammatory sites and to proliferate monocytes. Thus, we evaluated levels of MCP-1 and GM-CSF mRNA in the myocardium of EAM in rats using a real time quantitative PCR method. We also examined the correlation of MCP-1 or GM-CSF mRNA levels with those of inflammatory cytokines such as tumor necrosis factor- f (TNF- f ), interleukin-1 g (IL-1 g ) and interleukin-6 (IL-6) in the same lesion. Levels of MCP-1, GM-CSF, TNF- f, IL-1 g and IL-6 mRNA increased with the progression of myocarditis which was accompanied by the accumulation of ED-1 positive cells. The MCP-1 and GM-CSF mRNA levels were positively correlated with TNF- f, IL-1 g and IL-6 mRNA levels in the same lesion of EAM. We also demonstrated that serum MCP-1 concentrations were increased during the active stage of EAM, and were correlated with MCP-1 mRNA levels in the myocardium of each rat. These findings suggest that elevated MCP-1 and GM-CSF may associate with the migration and proliferation of monocytes/macrophages in EAM. Thus, MCP-1 and GM-CSF may play an important role in the progression of EAM.

Therapeutic Benefits of Tocilizumab Vary in Different Organs of a Patient with AA Amyloidosis.

Systemic reactive AA amyloidosis is a life-threatening complication of chronic inflammatory diseases. Anti-interleukin-6 receptor, tocilizumab (TCZ), has been shown to improve clinical symptoms of patients with AA amyloidosis, accompanied with regression of the amyloid deposition. We report a case of AA amyloidosis evaluated by histology of multiple organs before and after TCZ treatment. A woman in her 60s with rheumatoid arthritis was referred to our hospital because of cardiac and renal dysfunction. A gastric and renal biopsy revealed the deposition of AA amyloid, and echocardiography revealed concentric left ventricular hypertrophy. Her estimated glomerular filtration rate was decreased to 8.6 mL/min/1.73 m2, and B-type natriuretic peptide, C-reactive protein, and serum amyloid A protein were significantly elevated. TCZ treatments markedly decreased her serum amyloid A protein and C-reactive protein levels, but hemodialysis was required 1 year later. Endoscopic gastric rebiopsy 3 years after initiation of TCZ treatments revealed the regression of amyloid deposition and echocardiography revealed improvement of her left ventricular hypertrophy. However, a renal rebiopsy revealed that the amyloid deposition had not regressed. In conclusion, these observations suggest that the therapeutic effects of TCZ can vary among organs in patients with AA amyloidosis.

Placental Growth Factor as a Predictor of Cardiovascular Events in Patients with CKD from the NARA-CKD Study

Placental growth factor (PlGF) contributes to atherogenesis through vascular inflammation and plaque destabilization. High levels of PlGF may be associated with mortality and cardiovascular disease, but the relationship between PlGF level and adverse outcomes in patients with CKD is unclear. We conducted a prospective cohort study of 1351 consecutive participants with CKD enrolled in the Novel Assessment of Risk management for Atherosclerotic diseases in CKD (NARA-CKD) study between April 1, 2004, and December 31, 2011. During a median follow-up of 3 years, 199 participants died and 383 had cardiovascular events, defined as atherosclerotic disease or heart failure requiring hospitalization. In adjusted analyses, mortality and cardiovascular risk increased in each successive quartile of serum PlGF level; hazard ratios (HRs) (95% confidence intervals [95% CIs]) for mortality and cardiovascular risk, respectively, were 1.59 (0.83 to 3.16) and 1.55 (0.92 to 2.66) for the second quartile, 2.97 (1.67 to 5.59) and 3.39 (2.20 to 5.41) for the third quartile, and 3.87 (2.24 to 7.08) and 8.42 (5.54 to 13.3) for the fourth quartile. The composite end point of mortality and cardiovascular events occurred during the study period in 76.4% of patients in both the highest PlGF quartile (≥19.6 pg/ml) and the lowest eGFR tertile (<30 ml/min per 1.73 m(2)). The association between PlGF and mortality or cardiovascular events was not attenuated when participants were stratified by age, sex, traditional risk factors, and eGFR. These data suggest elevated PlGF is an independent risk factor for all-cause mortality and cardiovascular events in patients with CKD.

Prognostic Impact of Placental Growth Factor on Mortality and Cardiovascular Events in Dialysis Patients

Background: Placental growth factor (PlGF), a member of the vascular endothelial growth factor (VEGF) family, has recently emerged as a predictor of survival and cardiovascular risk. Along with others, we have shown an independent association between PlGF and cardiovascular events in CKD patients, but not much is known about patients receiving dialysis. Methods: We studied 205 dialysis patients undergoing cardiac catheterization at the Nara Medical University between April 1, 2004, and December 31, 2012. Serum levels of PlGF and VEGF were measured with ELISA in all the patients. Results: During a median follow-up of 20 months, 121 participants died from any cause or experienced a cardiovascular event. In the fully adjusted analysis, having an above-median PlGF or VEGF level was associated with a hazards ratio for adverse outcomes of 2.55 (1.72-3.83) and 1.39 (0.95-2.04), respectively. Using a multimarker strategy in a model with age, serum albumin, history of coronary artery disease, brain natriuretic peptide and PlGF, patients with 2, 3 and 4 positive markers had a 3.82-, 5.77- and 6.59-fold higher risk of mortality or a cardiovascular event, respectively, compared to those with no positive markers. The model with PlGF had a significantly higher c-statistic, integrated discrimination improvement index and category-free net reclassification improvement index than the model without PlGF. Conclusion: PlGF is independently associated with mortality and cardiovascular events, but the association between VEGF and adverse events was attenuated with covariate adjustment. The addition of PlGF to models with established clinical predictors provides additional useful prognostic information in patients receiving dialysis.