Ken Tsuchiya

Cloning of inv, a gene that controls left/right asymmetry and kidney development

Most vertebrate internal organs show a distinctive left/right asymmetry. The inv (inversion of embryonic turning) mutation in mice was created previously by random insertional mutagenesis; it produces both a constant reversal of left/right polarity (situs inversus) and cyst formation in the kidneys. Asymmetric expression patterns of the genes nodal and lefty are reversed in the inv mutant, indicating that inv may act early in left/right determination. Here we identify a new gene located at the inv locus. The encoded protein contains 15 consecutive repeats of an Ank/Swi6 motif, at its amino terminus. Expression of the gene is the highest in the kidneys and liver among adult tissues, and is seen in presomite-stage embryos. Analysis of the transgenic genome and the structure of the candidate gene indicate that the candidate gene is the only gene that is disrupted in inv mutants. Transgenic introduction of a minigene encoding the candidate protein restores normal left/right asymmetry and kidney development in the inv mutant, confirming the identity of the candidate gene.

Reduced klotho expression level in kidney aggravates renal interstitial fibrosis

Renal expression of the klotho gene is markedly suppressed in chronic kidney disease (CKD). Since renal fibrosis is the final common pathology of CKD, we tested whether decreased Klotho expression is a cause and/or a result of renal fibrosis in mice and cultured renal cell lines. We induced renal fibrosis by unilateral ureteral obstruction (UUO) in mice with reduced Klotho expression (kl/+ mice) and compared them with wild-type mice. The UUO kidneys from kl/+ mice expressed significantly higher levels of fibrosis markers such as α-smooth muscle actin (α-SMA), fibronectin, and transforming growth factor-β(1) (TGF-β(1)) than those from wild-type mice. In addition, in cultured renal fibroblast cells (NRK49F), the levels of α-SMA and PAI1 expression were significantly suppressed by addition of recombinant Klotho protein to the medium. The similar effects were observed by a TGF-β(1) receptor inhibitor (ALK5 inhibitor). These observations suggest that low renal Klotho expression enhances TGF-β(1) activity and is a cause of renal fibrosis. On the other hand, TGF-β(1) reduced Klotho expression in renal cultured epithelial cells (inner medullary collecting duct and human renal proximal tubular epithelium), suggesting that low renal Klotho expression is a result of renal fibrosis. Taken together, renal fibrosis can trigger a deterioration spiral of Klotho expression, which may be involved in the pathophysiology of CKD progression.

Prevalence of A-to-G mutation at nucleotide 3243 of the mitochondrial tRNA Leu(UUR) gene in Japanese patients with diabetes mellitus and end stage renal disease

AbstractThe A-to-G mutation at nucleotide 3243 of the mitochondrial tRNALeu(UUR) gene (mt.3243A>G) is associated with both diabetes mellitus and myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Recently, this mutation was found in three diabetic subjects with progressive kidney disease, suggesting that it may be a contributing factor in the development of kidney disease in patients with diabetes. The aim of this study was to evaluate the contribution of this mutation to the development of end stage renal disease (ESRD) in patients with diabetes. The study group consisted of 135 patients with diabetes and ESRD. The control group consisted of 92 non-diabetic subjects with ESRD who were receiving hemodialysis. The mt.3243A>G mutation was detected by polymerase chain reaction-restriction frag-ment length polymorphism (PCR-RFLP). We found the mt.3243A>G mutation in eight patients (8/135; 5.9%), all of whom were initially diagnosed with type II diabetes. Five of the eight patients were subsequently also diagnosed with MELAS. We did not find the mutation in any of the 92 non-diabetic subjects with ESRD. The prevalence of this mutation was 6.5-fold higher in patients with diabetes and ESRD than in those with diabetes alone (8/135 vs 5/550, respectively; χ2 = 13.704; P = 0.0002). The mt.3243A>G mutation may be a contributing genetic factor in the development of ESRD in Japanese patients with diabetes.

Sphingosine‐1‐phosphate acts as a key molecule in the direct mediation of renal fibrosis

The major sphingolipid metabolite, sphingosine‐1‐phosphate (S1P), has important biological functions. S1P serves as a ligand for a family of five G‐protein‐coupled receptors with distinct signaling pathways regulating important biological pathways. S1P induces renal fibrosis through an inflammatory pathway. However, its direct fibrosis‐inducing effect on the kidney has not been shown. The role of S1P as a direct mediator of renal fibrosis was investigated in normal rat kidney interstitial fibroblast (NRK‐49F) cells (in vitro) and kidneys of a unilateral ureteral obstruction (UUO) mouse model (in vivo). To clarify the role of S1P in renal fibrosis, we adopted nude UUO mice with immune response deficits. NRK‐49F cells were stimulated with various concentrations of exogenous S1P and FTY720 (a S1P receptor agonist) or N,N‐dimethylsphingosine (DMS; a sphingosine kinase inhibitor). C57BL6 and nude UUO mice were pretreated with FTY720, DMS, or saline. Expression levels of alpha‐smooth muscle actin (a‐SMA), E‐cadherin, collagen type 1 (COL1), collagen type 4 (COL4), tissue inhibitor of matrix metalloproteinase‐1 (TIMP1), and plasminogen activator inhibitor‐1 (PAI1) were examined. S1P stimulated fibrosis in NRK‐49F cells and UUO mice. Increased a‐SMA, COL1, COL4, TIMP1, and PAI1 and decreased E‐cadherin expression levels were observed in both the S1P‐stimulated cells and UUO mice. Nude UUO mouse kidneys expressed fibrotic markers. Fibrotic changes were successfully induced in both UUO and nude UUO mice, evident through prominent fibronectin and COL1 staining. These S1P‐induced fibrotic changes were suppressed by FTY720 and DMS both in vitro and in vivo. Thus, S1P essentially and directly mediates renal fibrosis.

Neutrophil gelatinase-associated lipocalin is a sensitive biomarker for the early diagnosis of acute rejection after living-donor kidney transplantation

BackgroundEarly diagnosis of kidney allograft dysfunction is crucial for the management and long-term survival of transplanted kidneys. We investigated whether neutrophil gelatinase-associated lipocalin (NGAL), interleukin 18 (IL-18), and liver-type fatty acid-binding protein (L-FABP) are capable of being used as novel biomarkers of acute kidney allograft dysfunction.MethodsWe measured serum and urine NGAL, urine IL-18, and urine L-FABP levels on the first 3xa0days after transplantation. To assess the diagnostic sensitivity of these biomarkers, a receiver-operating characteristic curve (ROC) was plotted, and the area under the curve (AUC) was calculated to quantify the accuracy of the parameter. Sections from paraffin-embedded biopsy specimens were examined by immunohistochemistry for NGAL expression.ResultsTwelve cases were clinically diagnosed as acute rejection (AR) by renal biopsy. Urine NGAL was the most sensitive of these markers for detection of acute kidney allograft dysfunction. The cutoff value of urine NGAL was 66.0xa0ng/ml, with an AUC of 0.79 (95xa0% CI 0.68–0.88). Sensitivity of serum NGAL was about the same as urine NGAL with an AUC of 0.75 (0.64–0.85). IL-18 and L-FABP were 0.584 (95xa0% CI 0.433–0.725) and 0.612 (95xa0% CI 0.460–0.749), respectively. NGAL was more useful than other biomarkers to detect AR of kidney allograft dysfunction. NGAL staining intensity was significantly increased in the proximal tubules of the transplants with AR than in transplants that were not acutely rejected.ConclusionUrine NGAL level was found to be the most sensitive biomarker of acute kidney allograft dysfunction after living-donor kidney transplantation.

Metabolic syndrome and risk of progression of chronic kidney disease: a single-center cohort study in Japan

Metabolic syndrome (MetS) is a risk factor for the development of diabetes and cardiovascular disease, and recently was linked to incident chronic kidney disease (CKD). The purpose of this study is to examine whether MetS is associated with CKD progression in Japanese at a single center. Outcome variables were a decrease in estimated glomerular filtration rate (eGFR) of 50 % or 25 ml/min/1.73 m2, end-stage renal disease (ESRD), death, or a composite outcome of all three. There were 213 subjects in the analysis, 40.4 % of whom met the criteria for MetS. The group of subjects with MetS had higher urinary albumin-to-creatinine (UACR) levels. Survival curves stratified by MetS status showed early separation of the curves and a significantly higher survival rate in the group without MetS (P = 0.0086). Comparisons with normoalbuminuria and microalbuminuria showed that macroalbuminuria was equally associated with predicted composite outcome (GFR, ESRD, or death) both in the presence and absence of MetS. Multivariate analyses for all covariates showed that eGFR (hazard ratio (HR) 8.286, 95 % confidence interval (CI) 2.360–28.044, P = 0.0012) and the UACR (HR 2.338, 95 % CI 1.442–3.861, P = 0.0005) at baseline were independently associated with the composite outcomes. The results show that MetS was associated with albuminuria in a cohort of Japanese CKD patients, and both MetS and albuminuria were independently associated with CKD progression.

Inhibitory effects of the transcription factor Ets-1 on the expression of type I collagen in TGF-β1-stimulated renal epithelial cells.

Extracellular matrix (ECM) production and epithelial-mesenchymal transition (EMT) are important for phenotypic conversion in normal development and disease states such as tissue fibrosis. Transforming growth factor-β1 (TGFβ1) is one of the most potent inducers of ECM proteins, and its role in the pathogenesis of fibrosis is well established. Ets family is involved in a diverse array of biologic functions including cellular growth, migration, and differentiation. In the present study, we investigated whether Ets-1 has a role in ECM production and EMT in human renal tubuloepithelial cells (HKC cells). TGFβ1 treatment increases Ets-1 expression and nuclear translocation in the HKC cells. Overexpression of recombinant Ets-1 suppressed transcription of α2(I) collagen (COL1A2) and type I collagen production in the TGFβ1-activated HKC cells. From the experiments using specific inhibitors against Smad3 or mitogen-activated protein (MAP) kinase pathways, Ets-1 has an inhibitory role for COL1A2 transcription and the p38 MAPK pathway participates in the negative contribution of Ets-1 in TGFβ1/Smad3-activated renal cells.

A new classification of Diabetic Nephropathy 2014: a report from Joint Committee on Diabetic Nephropathy

The Joint Committee on Diabetic Nephropathy has revised its Classification of Diabetic Nephropathy (Classification of Diabetic Nephropathy 2014) in line with the widespread use of key concepts, such as the estimated glomerular filtration rate (eGFR) and chronic kidney disease. In revising the Classification, the Committee carefully evaluated, as relevant to current revision, the report of a study conducted by the Research Group of Diabetic Nephropathy, Ministry of Health, Labour and Welfare of Japan. Major revisions to the Classification are summarized as follows: (1) eGFR is substituted for GFR in the Classification; (2) the subdivisions A and B in stage 3 (overt nephropathy) have been reintegrated; (3) stage 4 (kidney failure) has been redefined as a GFR less than 30xa0mL/min/1.73xa0m2, regardless of the extent of albuminuria; and (4) stress has been placed on the differential diagnosis of diabetic nephropathy versus non-diabetic kidney disease as being crucial in all stages of diabetic nephropathy.