淺沼 克彦

We recently demonstrated that cilnidipine, an L/N-type calcium channel blocker, elicits protective effects against glomerular podocyte injury, in particular, in obese hypertensive rats that express the N-type calcium channel (N-CC). Since the N-CC is known to be expressed in sympathetic nerve endings, we evaluated the reno-protective effects of cilnidipine in innervated and denervated spontaneously hypertensive rats (SHR). Male SHR were uninephrectomized and fed 4% high-salt diet (HS-UNX-SHR). Animals were divided into groups, as follows, and observed from 9 to 27 weeks of age: 1) vehicle (n = 14), 2) vehicle plus renal-denervation (n = 15), 3) cilnidipine (50 mg/kg per day, p.o.; n = 10), and 4) cilnidipine plus renal-denervation (n = 15). Renal denervation attenuated elevations in blood pressure, but failed to suppress urinary protein excretion and podocyte injury in HS-UNX-SHR. Cilnidipine in both innervated and denervated HS-UNX-SHR similarly induced significant antihypertensive effects, as well as suppressing the urinary protein excretion and podocyte injury, compared to vehicle-treated HS-UNX-SHR. These data indicate that renal nerves have a limited contribution to the cilnidipine-induced reno-protective effects in HS-UNX-SHR.

Background: In various animal studies, vitamin D has been shown to have a significant effect on reduction of proteinuria and the progression of kidney disease. However, little is known on its renoprotective effect in adriamycin (ADR)-induced nephrosis mice. The present study was intended to determine the therapeutic benefit of 22-oxa-calcitriol (OCT), a vitamin D analog, in reducing proteinuria and its renoprotective effect, i.e. preventing podocyte injury on ADR-induced nephrosis mice. Methods: Three experimental groups were used as follows: (1) nephrosis mice, established by a single intravenous injection of ADR (2) ADR+OCT mice, nephrosis mice treated with OCT, and (3) mice treated only with OCT as the control group. Podocyte injury was assessed by podocyte apoptosis using the TUNEL assay, podocyte counting, podocyte-specific expressed protein by immunofluorescence and Western blot analysis, and foot process effacement using electron microscopy. Results: Lower proteinuria was observed in ADR+OCT mice. Improvement in glomerulosclerosis and interstitial fibrosis, and prevention of glomerular hyperfiltration were observed in ADR+OCT mice. Immunofluorescence and Western blot analyses showed restoration of downregulated expression of nephrin, CD2AP and podocin. Nevertheless, dendrin expression was not restored. An insignificant reduction in podocyte numbers was found in ADR+OCT mice. Complete foot process effacement was partially prevented in ADR+OCT mice. Conclusions: The results indicate that OCT reduces podocyte injury and has renoprotective effects in ADR nephrosis mice. Copyright © 2011 S. Karger AG, Basel.

Background: The long-term antiproteinuric effects of benidipine, a calcium channel blocker (CCB), have not been evaluated in detail in hypertensive patients with chronic kidney disease (CKD). Methods: Benidipine (4 mg/day) was administered to previously untreated hypertensive patients with CKD, or hypertensive patients with CKD not achieving target blood pressure (BP) despite taking an angiotensin II receptor blocker (ARB). The patients were followed up for 1 year. If target BP was not achieved by 2 weeks after the start of benidipine treatment, the dosage was increased to 8 mg/day. The urinary protein to creatinine (UP/cre) ratio was evaluated before and after benidipine treatment. Results: This study evaluated 65 hypertensive patients with CKD. BP (systolic/diastolic) decreased from 154 +/- 19 / 91 +/- 12 mm Hg before treatment to 134 +/- 16 / 78 +/- 10 mm Hg at 1 year after treatment (p<0.001). The UP/cre ratio decreased significantly from 2.21 +/- 2.47 g/g creatinine (g/g cre) before treatment to 1.43 +/- 2.21 g/g cre after treatment (p<0.001). In both the untreated and ARB-treated groups, the BP and UP/cre ratio decreased significantly at 1 year after treatment. The percentage change in the UP/cre ratio was signifi-cantly greater in patients aged 65 years or older than in those less than 65 years (79.1% vs. 48.7%, p=0.038). Conclusions: Benidipine treatment reduced the UP/cre ratio in hypertensive patients with CKD, and the percentage decrease of the UP/cre ratio was greater in elderly patients, suggesting that benidipine may have more potent antiproteinuric effects in elderly hypertensive patients with CKD.

Although podocyte damage is known to be responsible for the development of minimal-change disease (MCD), the underlying mechanism remains to be elucidated. Previously, using a rat MCD model, we showed that endoplasmic reticulum (ER) stress in the podocytes was associated with the heavy proteinuric state and another group reported that a mammalian target of rapamycin complex 1 (mTORC1) inhibitor protected against proteinuria. In this study, which utilized a rat MCD model, a combination of immunohistochemistry, dual immunofluorescence and confocal microscopy, western blot analysis, and quantitative real-time RT-PCR revealed co-activation of the unfolded protein response (UPR), which was induced by ER stress, and mTORC1 in glomerular podocytes before the onset of proteinuria and downregulation of nephrin at the post-translational level at the onset of proteinuria. Podocyte culture experiments revealed that mTORC1 activation preceded the UPR that was associated with a marked decrease in the energy charge. The mTORC1 inhibitor everolimus completely inhibited proteinuria through a reduction in both mTORC1 and UPR activity and preserved nephrin expression in the glomerular podocytes. In conclusion, mTORC1 activation may perturb the regulatory system of energy metabolism primarily by promoting energy consumption and inducing the UPR, which underlie proteinuria in MCD. © 2007 USCAP, Inc All rights reserved.

Background: Adriamycin (ADR) nephrosis in mice has been extensively studied and has enabled a greater understanding of the processes underlying the progression of renal injury. Dendrin is a novel component of the slit diaphragm with proapoptotic signaling properties, and it accumulates in the podocyte nucleus in response to glomerular injury in mice. The present study re-evaluated chronic progressive nephropathy in ADR mice and the localization of dendrin in mice and in human glomerulopathy. Methods: To investigate the localization of dendrin, a mouse model of nephrosis and glomerulosclerosis was used, in which ADR was injected once. WT-1-positive cells and apoptotic cells were counted in vivo and in vitro. To check the expression of dendrin in ADR mice, immunostaining and Western blot were performed. A survey of dendrin staining was performed on human kidney biopsy specimens. Results: The injection of ADR induced proteinuria, podocyte loss and glomerulosclerosis. It also caused the relocation of dendrin from the slit diaphragm to the podocyte nucleus. We demonstrated the location of dendrin to podocyte nuclei in several cases of human glomerulopathy. The mean occurrence of dendrin-positive nucleus per glomerulus increased in several cases of human glomerulopathy. Conclusions: These findings suggest that the relocation of dendrin to the podocyte nuclei is useful as a novel marker of podocyte injury in human glomerulopathy. Copyright © 2011 S. Karger AG, Basel.

Background: Adriamycin (ADR) nephrosis in mice has been extensively studied and has enabled a greater understanding of the processes underlying the progression of renal injury. Dendrin is a novel component of the slit diaphragm with proapoptotic signaling properties, and it accumulates in the podocyte nucleus in response to glomerular injury in mice. The present study re-evaluated chronic progressive nephropathy in ADR mice and the localization of dendrin in mice and in human glomerulopathy. Methods: To investigate the localization of dendrin, a mouse model of nephrosis and glomerulosclerosis was used, in which ADR was injected once. WT-1-positive cells and apoptotic cells were counted in vivo and in vitro. To check the expression of dendrin in ADR mice, immunostaining and Western blot were performed. A survey of dendrin staining was performed on human kidney biopsy specimens. Results: The injection of ADR induced proteinuria, podocyte loss and glomerulosclerosis. It also caused the relocation of dendrin from the slit diaphragm to the podocyte nucleus. We demonstrated the location of dendrin to podocyte nuclei in several cases of human glomerulopathy. The mean occurrence of dendrin-positive nucleus per glomerulus increased in several cases of human glomerulopathy. Conclusions: These findings suggest that the relocation of dendrin to the podocyte nuclei is useful as a novel marker of podocyte injury in human glomerulopathy. Copyright (C) 2011 S. Karger AG, Basel

Objective : Objective of the present study is to evaluate the stable potency of PS-Ca Jelly in CKD patients with hyperkalemia. Materials and Methods : We examined 55 hyperkalemic patients (serum potassium concentration &gt 5.0 mEq/L) caused by CKD. Jelly type formulation of PS-Ca (PS-Ca 5 g/25 g Jelly=one cup) was used in this study. One to three cups which contained 5-15 g PS-Ca were administered depending on the patients' condition for more than 6 months. Serum potassium level, electrolytes and indexes of renal function were measured every two months during the study period. Results : Serum creatinine, SUN, proteinurea, blood pressure and HbA1c in these patients did not change and patients' clinical conditions were stable during this study period. Serum potassium levels were 5.56±0.05 mEq/L at baseline, 4.69±0.05 mEq/L after two months, 4.79±0.06 mEq/L after 4 months, 4.72±0.06 mEq/L after 6 months, 4.76±0.07 mEq/L after 8 months and 4.79±0.09 mEq/L after 10 months, respectively. Serum potassium reduction after the treatment (average observation period : 8.36±0.24 months) was 0.82±0.06 mEq/L. Different daily dose of PS-Ca Jelly reduced serum potassium level in a dose dependent manner [one cup/day : 0.65±0.05 mEq/L (p&lt 0.001), two cups/day : 0.84±0.15 mEq/L (p&lt 0.001), three cups/day : 1.00±0.11 mEq/L (p&lt 0.001)]. Conclusion : It appears that PS-Ca Jelly was effective for the treatment of hyperkalemia in CKD patients and its potency was stable during 10 months study period. This effect also showed a dose dependence.

OBJECTIVES: Clinical studies have indicated the beneficial effect of an L/N-type calcium channel blocker (CCB), cilnidipine, on the progression of proteinuria in hypertensive patients compared with an L-type CCB, amlodipine. In the present study, we examined the effects of cilnidipine and amlodipine on the renal injury in spontaneously hypertensive rat/ND mcr-cp (SHR/ND) and their underlying mechanism. METHODS AND RESULTS: SHR/ND were treated with vehicle (nU10), cilnidipine [33 mg/kg per day, orally (p.o.); nU11] or amlodipine (20 mg/kg per day, p.o.; nU9) for 20 weeks. SHR/ND developed proteinuria in an age-dependent manner. Cilnidipine suppressed the proteinuria greater than amlodipine did. The immunohistochemical analysis showed that N-type calcium channel and Wilm's tumor factor, a marker of podocyte, were co-expressed. SHR/ND had significantly greater desmin staining, an indicator of podocyte injury, with lower podocin and nephrin expression in the glomeruli than Wistar-Kyoto rat or SHR. Cilnidipine significantly prevented the increase in desmin staining and restored the glomerular podocin and nephrin expression compared with amlodipine. Cilnidipine also prevented the increase in renal angiotensin II content, the expression and membrane translocation of NADPH oxidase subunits and dihydroethidium staining in SHR/ND. In contrast, amlodipine failed to change these renal parameters. CONCLUSION: These data suggest that cilnidipine suppressed the development of proteinuria greater than amlodipine possibly through inhibiting N-type calcium channel-dependent podocyte injury in SHR/ND.

LAT3 is a Na+-independent neutral L-amino acid transporter recently isolated from a human hepatocellular carcinoma cell line. Although liver, skeletal muscle, and pancreas are known to express LAT3, the tissue distribution and physiologic function of this transporter are not completely understood. Here, we observed that glomeruli express LAT3. Immunofluorescence, confocal microscopy, and immunoelectron microscopy revealed that LAT3 localizes to the apical plasma membrane of podocyte foot processes. In mice, starvation upregulated glomerular LAT3, phosphorylated AKT1, reconstituted the actin network, and elongated foot processes. In the fetal kidney, we observed intense LAT3 expression at the capillary loops stage of renal development. Finally, zebrafish morphants lacking lat3 function showed collapsed glomeruli with thickened glomerular basement membranes. Permeability studies of the glomerular filtration barrier in these zebrafish morphants demonstrated a disruption of selective glomerular permeability. Our data suggest that LAT3 may play a crucial role in the development and maintenance of podocyte structure and function by regulating protein synthesis and the actin cytoskeleton. Copyright © 2009 by the American Society of Nephrology.

Synaptopodin is the founding member of a novel class of proline-rich actin-associated proteins highly expressed in telencephalic dendrites and renal podocytes. Synaptopodin-deficient (synpo(-/-)) mice lack the dendritic spine apparatus and display impaired activity-dependent long-term synaptic plasticity. In contrast, the ultrastructure of podocytes in synpo(-/-) mice is normal. Here we show that synpo(-/-) mice display impaired recovery from protamine sulfate-induced podocyte foot process (FP) effacement and LPS-induced nephrotic syndrome. Similarly, synpo(-/-) podocytes show impaired actin filament reformation in vitro. We further demonstrate that synaptopodin exists in 3 isoforms, neuronal Synpo-short (685 AA), renal Synpo-long (903 AA), and Synpo-T (181 AA). The C terminus of Synpo-long is identical to that of Synpo-T. All 3 isoforms specifically interact with a-actinin and elongate alpha-actinin-induced actin filaments. synpo(-/-) mice lack Synpo-short and Synpo-long expression but show an upregulation of Synpo-T protein expression in podocytes, though not in the brain. Gene silencing of Synpo-T abrogates stress-fiber formation in synpo(-/-) podocytes, demonstrating that Synpo-T serves as a backup for Synpo-long in synpo(-/-) podocytes. In concert, synaptopodin regulates the actin-bundling activity of a-actinin in highly dynamic cell compartments, such as podocyte FPs and the dendritic spine apparatus.

Kidney podocytes and their slit diaphragms form the final barrier to urinary protein loss. This explains why podocyte injury is typically associated with nephrotic syndrome. The present study uncovered an unanticipated novel role for costimulatory molecule B7-1 in podocytes as an inducible modifier of glomerular permselectivity. B7-1 in podocytes was found in genetic, drug-induced, immune-mediated, and bacterial toxin-induced experimental kidney diseases with nephrotic syndrome. The clinical significance of our results is underscored by the observation that podocyte expression of B7-1 correlated with the severity of human lupus nephritis. In vivo, exposure to low-dose LPS rapidly upregulates B7-1 in podocytes of WT and SCID mice, leading to nephrotic-range proteinuria. Mice lacking B7-1 are protected from LPS-induced nephrotic syndrome, suggesting a link between podocyte B7-1 expression and proteinuria. LPS signaling through toll-like receptor-4 reorganized the podocyte actin cytoskeleton in vitro, and activation of B7-1 in cultured podocytes led to reorganization of vital slit diaphragm proteins. In summary, upregulation of B7-1 in podocytes may contribute to the pathogenesis of proteinuria by disrupting the glomerular filter and provides a novel molecular target to tackle proteinuric kidney diseases. Our findings suggest a novel function for B7-1 in danger signaling by nonimmune cells.

Parietal epithelial cells (PEC) of Bowman's capsules cover the inner aspect of Bowman's capsules and are believed to contribute to extracapillary lesions of glomerulonephritis such as crescent formation. In glomerular research including cell culture experiments and pathology, differentiation between PEC and podocytes has frequently been a major problem. Immunohistochemistry of the adult rat kidney for protein gene product 9.5 (PGP 9.5), a neuron-specific ubiquitin C-terminal hydrolase, demonstrated selective localization of the immunoreactivity in PEG. At the urinary pole of the glomerulus, immunoreactive PEC were clearly differentiated from proximal tubular cells that were negative for PGP 9.5. In the subcapsular nephrogenic zone of newborn rat kidney, immunoreactivity was observed in almost all cells in the comma-shaped body and early S-shaped body and selectively in PEC in the late S-shaped body and capillary-stage glomerulus. In rat glomerular disease models (Masugi-nephritis and puromycin aminonucleoside nephrosis), cells that consisted of cellular crescents or adhered to glomerular tufts were positive for PGP 9.5. The selective localization of PGP 9.5 in PEC in rat kidney provides a new cytochemical marker for identifying the cells. Developmental expression of the protein suggests that PGP 9.5 is involved in the processes of nephrogenesis of rat kidney.

Hydrogen peroxide (H2O2) contributes to the development of reperfusion injury. The aim of this study was to characterize the effects of H2O2 on cardiac function, autacoid release and myocardial damage. Rat hearts were Langendorff-perfused with Krebs solution containing 70, 200 or 700 μM H2O2 for 30 min, then washed out with normal Krebs solution. The left ventricular pressure, heart rate and coronary perfusion pressure were monitored, released prostanoid and NO was quantified and the activity of released glutamic oxaloacetic transaminase (GOT) was estimated. H2O2 (70 μM) significantly increased prostacyclin release with no evidence of myocardial damage. H2O2 (200 μM) stimulated the release of all the prostanoids quantified markedly without affecting NO release. GOT release did not increase during H2O2 perfusion, but did increase after washout. Inhibition of prostanoid release with indomethacin did not affect the washout-induced increase in GOT release. These results indicate that a low concentration of H2O2 stimulates prostanoid release without affecting myocardial contraction, while higher concentrations damage the heart indirectly. The mechanism of the washout- induced myocardial damage is not clear yet, but the H2O2-induced change in prostanoid release did not seem to be responsible for the damage.