Ichiro Ohkido

A nonclassical vitamin D receptor pathway suppresses renal fibrosis

The TGF-β superfamily comprises pleiotropic cytokines that regulate SMAD and non-SMAD signaling. TGF-β-SMAD signal transduction is known to be involved in tissue fibrosis, including renal fibrosis. Here, we found that 1,25-dihydroxyvitamin D3-bound [1,25(OH)2D3-bound] vitamin D receptor (VDR) specifically inhibits TGF-β-SMAD signal transduction through direct interaction with SMAD3. In mouse models of tissue fibrosis, 1,25(OH)2D3 treatment prevented renal fibrosis through the suppression of TGF-β-SMAD signal transduction. Based on the structure of the VDR-ligand complex, we generated 2 synthetic ligands. These ligands selectively inhibited TGF-β-SMAD signal transduction without activating VDR-mediated transcription and significantly attenuated renal fibrosis in mice. These results indicate that 1,25(OH)2D3-dependent suppression of TGF-β-SMAD signal transduction is independent of VDR-mediated transcriptional activity. In addition, these ligands did not cause hypercalcemia resulting from stimulation of the transcriptional activity of the VDR. Thus, our study provides a new strategy for generating chemical compounds that specifically inhibit TGF-β-SMAD signal transduction. Since TGF-β-SMAD signal transduction is reportedly involved in several disorders, our results will aid in the development of new drugs that do not cause detectable adverse effects, such as hypercalcemia.

Interaction of a farnesylated protein with renal type IIa Na/Pi co-transporter in response to parathyroid hormone and dietary phosphate.

Treatment with PTH (parathyroid hormone) or a high-P(i) diet causes internalization of the type IIa sodium-dependent phosphate (Na/P(i) IIa) co-transporter from the apical membrane and its degradation in the lysosome. A dibasic amino acid motif (KR) in the third intracellular loop of the co-transporter is essential for proteins PTH-induced retrieval. To elucidate the mechanism of internalization of Na/P(i) IIa, we identified the interacting protein for the endocytic motif by yeast two-hybrid screening. We found a strong interaction of the Na/P(i) IIa co-transporter with a small protein known as the PEX19 (human peroxisomal farnesylated protein; PxF, Pex19p). PEX19 can bind to the KR motif, but not to a mutant with this motif replaced with NI residues. PEX19 is highly expressed in mouse and rat kidney. Western blot analysis indicates that PEX19 is located in the cytosolic and brush-border membrane fractions (microvilli and the subapical component). Overexpression of PEX19 stimulated the endocytosis of the Na/P(i) IIa co-transporter in opossum kidney cells in the absence of PTH. In conclusion, the present study indicates that PEX19 may be actively involved in controlling the internalization and trafficking of the Na/P(i) IIa co-transporter.

Serum soluble α-klotho in hemodialysis patients.

BACKGROUND The extracellular domain of klotho is cleaved and released into various extracellular fluids, such as blood, urine, and cerebrospinal fluid, as soluble α-klotho (sαKl). METHODS We measured sαKl in 53 hemodialysis patients and 20 healthy controls to examine its role in mineral metabolism. RESULTS The sαKl level of the hemodialysis patients was 430 pg/ml (386 - 540 pg/ml, which was lower than that of healthy controls 740 pg/ml (550 - 913 pg/ml, p < 0.01). The serum sαKl level showed a positive correlation with serum phosphorus (P) (σ = 0.33, p = 0.014), while serum corrected Ca tended to be negatively correlated with sαKl (σ = -0.26, p = 0.06). Both parameters showed the same trends in healthy subjects. However, there was no significant association between serum sαKl and age (σ= 0.21, p = 0.14), intact PTH (σ = -0.08, p= 0.55), whole PTH (σ = -0.08, p = 0.59), or FGF23 (σ = -0.07, p = 0.62). Multiple regression analysis showed that P was independently associated with the serum sαKl levels (total adjusted R2 = 0.18, p = 0.02). CONCLUSIONS The sαKl level was lower in hemodialysis patients than in healthy persons. Serum P level was independently associated with the serum sαKl level.

Difference in coronary artery intima and media calcification in autopsied patients with chronic kidney disease.

BACKGROUND In patients with chronic kidney disease (CKD), coronary artery calcification occurs at two distinct sites in the vessel wall: the intima and the media. Arterial media calcification (AMC), a nonocclusive condition, affects hemodynamics differently compared to arterial intima calcification (AIC), which occurs in atherosclerotic plaques. Arterial calcification is considered a cell-regulated process resembling intramembranous bone formation. The purpose of this retrospective observational study was to clarify the morphological differences between AIC and AMC and to evaluate the role of vascular smooth muscle cells (VSMCs) and macrophages in AIC and AMC formation. METHODS We histologically analyzed 14 tissue specimens from 14 autopsies of patients with CKD Stage 5D who underwent hemodialysis and 5 specimens from 5 patients with CKD Stage 2 - 3 (90 ml/min/1.73 m2 > estimated GFR >= 30 ml/min/1.73 m2). We performed immunohistochemical staining of osteopontin (OPN) as a marker for bone matrix protein, alpha-smooth muscle actin (alphaSMA) for VSMCs, Cbfa1/Runx2 as a marker for osteoblastic differentiation of VSMCs, and CD68 for macrophages. RESULTS In the CKD 2/3 group, we also found AIC and AMC. OPN and CD68 expression in the CKD 2/3 group was similar to that in the CKD 5D group. Although we did not find Cbfa1/Runx2 positive cell expression in the CKD 2/3 group, we did find it in the CKD 5D group. We found CD68-positive cells predominantly in AIC and absent in AMC in both groups. CONCLUSIONS These findings suggest that the influence of Cbfa1/Runx2 pathway in coronary artery calcification depends on the CKD Stage. Expression of CD68-positive cells depends on the location of the coronary artery calcification.

The Negative Ca2+ Balance Is Involved in the Stimulation of PTH Secretion

The low calcium (Ca2+) dialysate have been developed to diminish the risk of hypercalcemia with the administration of active vitamin D and Ca2+ carbonate as phosphate binder. Today, increasing numbers of hemodialysis (HD) patients have been on the low Ca2+ dialysate (Ca2+ = 2.5 mEq/l). However, the clinical consequences of a negative calcium net-balance which may be induced by the use of low Ca dialysate are not well evaluated. In the present study, we explored the effects of low Ca2+ dialysate on the calcium balance and the PTH secretion. Eighty one chronic HD patients (male/female: 47/34; mean age: 60.2 ± 1.5 years; mean HD periods: 11.1 ± 0.8 years) who had been dialyzed with 3.0 mEq/l Ca2+ dialysate were studied. All patients were transferred to the low Ca dialysate, which actually brought about a negative net-balance in Ca (mean: –94.5 mg) and an increase in serum intact PTH levels (mean: +23.7%: p = 0.03) during a single HD session. However, no changes in serum ionized Ca2+ were found in spite of negative Ca2+ balance. One month after change to the low Ca2+ dialysate (total 12 sessions in each case), serum intact PTH levels increased significantly (186.7 ± 19.5 vs. 216.2 ± 21.9 pg/ml: p = 0.01) in spite of the fact that no changes were found in serum ionized Ca2+, Pi and Mg. This result indicates that the negative Ca2+ balance during low-Ca2+ hemodialysis-stimulated PTH secretion, which offset the decrease of serum Ca2+; a trade-off phenomenon between negative Ca balance and PTH. This suggests that low Ca2+ dialysate may exaggerate the progression of secondary hyperparathyroidism.

Metanephros Transplantation Inhibits the Progression of Vascular Calcification in Rats with Adenine-Induced Renal Failure

Background/Aim: Recent research has shown that transplanted metanephroi form primitive vascularized kidneys with histologically recognizable renal features. The aim of the present study was to determine the metabolic function of transplanted metanephroi in rats with chronic renal failure (CRF), with particular reference to secondary hyperparathyroidism and vascular calcification. Methods: CRF was induced in 11-week-old male Wistar rats by maintaining them on a 0.75% adenine diet for 4 weeks, followed by normal diet for an additional 2 weeks. At the end of adenine loading, whole metanephroi from embryonic day 15 rats were transplanted into the omentum and epididymis of the transplantation group. Vascular calcification was evaluated 2 weeks after metanephroi transplantation. Results: Metanephros transplantation significantly reduced vascular calcium and phosphorus content and suppressed the progression of vascular calcification as indicated by von Kossa staining of the media of the thoracic aorta. However, no significant differences between the adenine-fed control and transplantation groups were found regarding the serum levels of 1,25(OH)2D3, calcium or phosphorus or the calcium × phosphorus product. Conclusion: The present study has shown that transplantation of metanephroi suppresses the progression of vascular calcification via a mechanism that is independent of calcium-phosphorus dynamics.

Proton Pump Inhibitor Use and Magnesium Concentrations in Hemodialysis Patients: A Cross-Sectional Study

Magnesium concentration is a proven predictor of mortality in hemodialysis patients. Recent reports have indicated that proton pump inhibitor (PPI) use affects serum magnesium levels, however few studies have investigated the relationship between PPI use and magnesium levels in hemodialysis patients. This study aimed to clarify the association between PPI use and serum magnesium levels in hemodialysis patients. We designed this cross sectional study and included 1189 hemodialysis patients in stable condition. Associations between PPI and magnesium-related factors, as well as other possible confounders, were evaluated using a multiple regression model. We defined hypomagnesemia as a value < 2.0 mg/dL, and created comparable logistic regression models to assess the association between PPI use and hypomagnesemia. PPI use is associated with a significantly lower mean serum magnesium level than histamine 2 (H2) receptor antagonists or no acid-suppressive medications (mean [SD] PPI: 2.52 [0.45] mg/dL; H2 receptor antagonist: 2.68 [0.41] mg/dL; no acid suppressive medications: 2.68 [0.46] mg/dL; P = 0.001). Hypomagnesemia remained significantly associated with PPI (adjusted OR, OR: 2.05; 95% CI: 1.14–3.69; P = 0.017). PPI use is associated with an increased risk of hypomagnesemia in hemodialysis patients. Future prospective studies are needed to explore magnesium replacement in PPI users on hemodialysis.

Interactions between Serum Vitamin D Levels and Vitamin D Receptor Gene FokI Polymorphisms for Renal Function in Patients with Type 2 Diabetes

Background We aimed to examine associations among serum 25-hydroxyvitamin D (25OHD) levels, 1,25-dihyroxyvitamin D (1,25OHD) levels, vitamin D receptor (VDR) polymorphisms, and renal function based on estimated glomerular filtration rate (eGFR) in patients with type 2 diabetes. Methods In a cross-sectional study of 410 patients, chronic kidney disease (CKD) stage assessed by eGFR was compared with 25OHD, 1,25OHD, and VDR FokI (rs10735810) polymorphisms by an ordered logistic regression model adjusted for the following confounders: disease duration, calendar month, use of angiotensin converting enzyme inhibitors/angiotensin receptor blockers or statins, and serum calcium, phosphate, and intact parathyroid hormone levels. Results 1,25OHD levels, rather than 25OHD levels, showed seasonal oscillations; peak levels were seen from May to October and the lowest levels were seen from December to February. These findings were evident in patients with CKD stage 3∼5 but not stage 1∼2. eGFR was in direct proportion to both 25OHD and 1,25OHD levels (P<0.0001), but it had stronger linearity with 1,25OHD (r = 0.73) than 25OHD (r = 0.22) levels. Using multivariate analysis, 1,25OHD levels (P<0.001), but not 25OHD levels, were negatively associated with CKD stage. Although FokI polymorphisms by themselves showed no significant associations with CKD stage, a significant interaction between 1,25OHD and FokITT was observed (P = 0.008). The positive association between 1,25OHD and eGFR was steeper in FokICT and CC polymorphisms (r = 0.74) than FokITT polymorphisms (r = 0.65). Conclusions These results suggest that higher 1,25OHD levels may be associated with better CKD stages in patients with type 2 diabetes and that this association was modified by FokI polymorphisms.

Two zebrafish (Danio rerio) antizymes with different expression and activitiesThe nucleotide sequence data reported will appear in DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers AB017117 (AZS) and AB017118 (AZL).

Cellular polyamines are regulated by a unique feedback mechanism involving ornithine decarboxylase (ODC) antizyme. The synthesis of mammalian antizyme requires a programmed translational frameshift event induced by polyamines. Antizyme represses ODC, a key enzyme for polyamine synthesis, through accelerating enzyme degradation by the 26 S proteasome. Antizyme also inhibits the cellular uptake of polyamines. In the present study we isolated two distinct zebrafish (Danio rerio) antizyme cDNA clones (AZS and AZL) from an embryonic library. Their sequences revealed that both clones required translational frameshifting for expression. Taking account of +1 frameshifting, AZS and AZL products were 214 and 218 residues long respectively and shared 51.8% amino acid identity. In rabbit reticulocyte lysates, both mRNA species were translated through spermidine-induced frameshifting. The presence of the two antizyme mRNA species in embryos, adult fish and a cultured cell line was confirmed by Northern blot analysis. The ratio of AZS mRNA to AZL mRNA in the adult fish was 1.8-fold higher than in the embryos. Whole-mount hybridization in situ demonstrated that both mRNA species are expressed in every tissue in embryo, but predominantly in the central nervous system and the eyes. Bacterial expression products of both cDNA species inhibited ODC activity, but only the AZS product accelerated ODC degradation in vitro. These results show that both zebrafish antizymes are induced by polyamines but their mRNA species are expressed differently during development. The difference in activities on ODC degradation suggests their functional divergence.

Plasma cell-rich rejection accompanied by acute antibody-mediated rejection in a patient with ABO-incompatible kidney transplantation

We report a case of plasma cell‐rich rejection accompanied by acute antibody‐mediated rejection in a patient with ABO‐incompatible kidney transplantation. A 33‐year‐old man was admitted for an episode biopsy; he had a serum creatinine (S‐Cr) level of 5.7 mg/dL 1 year following primary kidney transplantation. Histological features included two distinct entities: (1) a focal, aggressive tubulointerstitial inflammatory cell (predominantly plasma cells) infiltration with moderate tubulitis; and (2) inflammatory cell infiltration (including neutrophils) in peritubular capillaries. Substantial laboratory examination showed that the patient had donor‐specific antibodies for DQ4 and DQ6. Considering both the histological and laboratory findings, we diagnosed him with plasma cell‐rich rejection accompanied by acute antibody‐mediated rejection. We started 3 days of consecutive steroid pulse therapy three times every 2 weeks for the former and plasma exchange with intravenous immunoglobulin (IVIG) for the latter histological feature. One month after treatment, a second allograft biopsy showed excellent responses to treatment for plasma cell‐rich rejection, but moderate, acute antibody‐mediated rejection remained. Therefore, we added plasma exchange with IVIG again. After treatment, allograft function was stable, with an S‐Cr level of 2.8 mg/dL. This case report demonstrates the difficulty of the diagnosis of, and treatment for, plasma cell‐rich rejection accompanied by acute antibody‐mediated rejection in a patient with ABO‐incompatible kidney transplantation. We also include a review of the related literature.