Sumiyo Watanabe

Association between activating mutations of calcium-sensing receptor and Bartter's syndrome

Bartters syndrome is a heterogeneous disorder characterised by deficient renal reabsorption of sodium and chloride, and hypokalaemic metabolic alkalosis with hyper-reninaemia and hyperaldosteronaemia. Mutations in several ion transporters and channels have been associated with the pathogenesis of Bartters syndrome. We describe two hypocalcaemic patients with deficient parathyroid hormone secretion who also showed characteristics of Bartters syndrome. We found activating mutations of the gene for the calcium-sensing receptor (CASR) in both patients. Activation of this calcium-sensing receptor inhibits the activity of a renal outer-medullary potassium channel that is mutated in type 2 Bartters syndrome. We therefore suggest that some activating mutations of CASR could provide new mechanisms for the development of Bartters syndrome.

Smart multilayered assembly for biocompatible siRNA delivery featuring dissolvable silica, endosome-disrupting polycation, and detachable PEG.

Multifunctional delivery systems of small interfering RNA (siRNA) are needed to overcome the intrinsic biological barriers toward efficient gene silencing in the cell cytoplasm. In this report, a smart multilayered assembly (SMA) was fabricated by a layer-by-layer method with polyionic materials. The SMA was designed to feature a siRNA-loaded core, a transiently core-stabilizing silica interlayer, an endosome-disrupting polycation interlayer, and a biocompatible poly(ethylene glycol) (PEG) shell with reductive environment-responsive detachability. The SMA was confirmed to be approximately 160 nm in size with narrow distribution and spherical morphology by DLS and TEM analyses. The PEG detachability of the SMA based on disulfide cleavage was also confirmed by the increase in both ζ-potential and size due to the exposure of the polycation interlayer and the compromised colloidal stability. The silica interlayer rendered the SMA highly tolerant to dissociation induced by anionic lipids, while after 24 h dialysis siRNA release from the SMA was clearly observed, presumably due to gradual dissolution of the silica interlayer based on the equilibrium shift to silicate ions. The entrapment ratio of siRNA delivered by the SMA within the endosome was significantly lower than that by nondisulfide control (NDC) without PEG detachability, suggesting the improved endosomal escape of SMA with the exposed, endosome-disrupting interlayer after PEG detachment. SMAs induced significantly higher gene silencing efficiency in various cultured cells, compared to NDC, without associated cytotoxicity. The systemic administration of SMAs for subcutaneous tumor-bearing mice achieved significant endogenous gene silencing in tumor tissue without hematological toxicity.

Systemic siRNA delivery to a spontaneous pancreatic tumor model in transgenic mice by PEGylated calcium phosphate hybrid micelles

Efficient systems for delivery of small interfering RNA (siRNA) are required for clinical application of RNA interference (RNAi) in cancer therapy. Herein, we developed a safe and efficient nanocarrier comprising poly(ethylene glycol)-block-charge-conversional polymer (PEG-CCP)/calcium phosphate (CaP) hybrid micelles for systemic delivery of siRNA and studied their efficacy in spontaneous bioluminescent pancreatic tumors from transgenic mice. PEG-CCP was engineered to provide the siRNA-loaded hybrid micelles with enhanced colloidal stability and biocompatibility due to the PEG capsule and with endosome-disrupting functionality due to the acidic pH-responsive CCP segment where the polyanionic structure could be converted to polycationic structure at acidic pH through cis-aconitic amide cleavage. The resulting hybrid micelles were confirmed to have a diameter of <50nm, with a narrow size distribution. Intravenously injected hybrid micelles significantly reduced the luciferase-based luminescent signal from the spontaneous pancreatic tumors in an siRNA sequence-specific manner. The gene silencing activity of the hybrid micelles correlated with their preferential tumor accumulation, as indicated by fluorescence imaging and histological analysis. Moreover, there were no significant changes in hematological parameters in mice treated with the hybrid micelles. These results demonstrate the great potential of the hybrid micelles as siRNA carriers for RNAi-based cancer therapy.

Decrease in serum leptin by troglitazone is associated with preventing bone loss in type 2 diabetic patients.

Abstract. The thiazolidinedione (TZD) class of antidiabetic drugs has been shown to inhibit the formation of bone-resorbing osteoclasts in vitro and to decrease bone resorption markers in vivo. These drugs also inhibit the expression of leptin in adipocytes. Less leptin can be associated with higher bone mass, based on analyses of mice deficient in leptin action. Effects of 1-year treatment with troglitazone, a member of the TZDs, on bone mineral density (BMD) and bone metabolism were examined in 25 Japanese type 2 diabetic patients. Glucose metabolism was improved, whereas body mass index and percent body fat did not change throughout the study. The percent change of BMD was negatively correlated with that of serum leptin, whereas it was not associated with changes of bone metabolic markers, type I collagen N-telopeptide (NTx), bone alkaline phosphatase (ALP), body mass index, or HbA1c. Serum leptin decreased in 68% of subjects (responders) after 1-month treatment and was consistently lower than the basal level throughout the treatment. Percent changes of BMD were significantly higher in the responders than in the nonresponders and in nondiabetic subjects at 6 and 12 months. NTx and bone ALP decreased at 1 month but increased thereafter in either group of patients. Thus, it is suggested that the decrease in serum leptin with no reduction in body fat mass by troglitazone is associated with preventing bone loss in type 2 diabetic patients. Hence, TZDs may have an advantage for diabetic patients who have risk factors for osteoporosis.

Pancreatic cancer therapy by systemic administration of VEGF siRNA contained in calcium phosphate/charge-conversional polymer hybrid nanoparticles

Development of an efficient in vivo delivery vehicle of small interfering RNA (siRNA) is the key challenge for successful siRNA-based therapies. In this study, toward systemic delivery of siRNA to solid tumors, a smart polymer/calcium phosphate (CaP)/siRNA hybrid nanoparticle was prepared to feature biocompatibility, reversible stability and endosomal escape functionality using a pH sensitive block copolymer of poly(ethylene glycol) and charge-conversional polymer (PEG-CCP), of which anionic functional groups could be converted to cationic groups in an endosomal acidic condition for facilitated endosomal escape. Nanoparticles were confirmed to be approximately 100nm in size, narrowly dispersed and spherical. Also, the nanoparticle was highly tolerable in medium containing serum, while releasing the entrapped siRNA in a cytoplasm-mimicking ionic condition, presumably based on the equilibrium between CaP complexes and calcium ions. Further, the nanoparticle showed high gene silencing efficiency in cultured pancreatic cancer cells (BxPC3) without associated cytotoxicity. Ultimately, systemic administration of the nanoparticles carrying vascular endothelium growth factor (VEGF) siRNA led to the significant reduction in the subcutaneous BxPC3 tumor growth, well consistent with the enhanced accumulation of siRNA and the significant VEGF gene silencing (~68%) in the tumor. Thus, the hybrid nanoparticle was demonstrated to be a promising formulation toward siRNA-based cancer therapies.

Direct and instantaneous observation of intravenously injected substances using intravital confocal micro-videography.

We describe the development and application of intravital confocal micro-videography to visualize entrance, distribution, and clearance of drugs within various tissues and organs. We use a Nikon A1R confocal laser scanning microscope system attached to an upright ECLIPSE FN1. The Nikon A1R allows simultaneous four channel acquisition and speed of 30 frames per second while maintaining high resolution of 512 × 512 scanned points. The key techniques of our intravital imaging are (1) to present a flat and perpendicular surface to the objective lens, and (2) to expose the subject with little or no bleeding to facilitate optical access to multiple tissues and organs, and (3) to isolate the subject from the body movement without compressing the blood vessels, and (4) to insert a tail vein catheter for timed injection without moving the subject. Ear lobe dermis tissue was accessible without surgery. Liver, kidney, and subcutaneous tumor were accessed following exteriorization through skin incision. In order to image initial extravasations of compounds into tissue following intravenous injection, movie acquisition was initialized prior to drug administration. Our technique can serve as a powerful tool for investigating biological mechanisms and functions of intravenously injected drugs, with both spatial and temporal resolution.

Functional Activities of Mutant Calcium-Sensing Receptors Determine Clinical Presentations in Patients With Autosomal Dominant Hypocalcemia

OBJECTIVE Autosomal dominant hypocalcemia (ADH) is a congenital isolated hypoparathyroidism caused by activating mutations in the calcium-sensing receptor (CASR) gene. The clinical features of ADH are heterogeneous; some patients are asymptomatic, and others show severe hypocalcemia with Bartters syndrome. We therefore recruited 12 patients with ADH to clarify the determinants of their clinical presentation. DESIGN AND METHODS We studied two sporadic and 10 familial cases of ADH. Serum concentrations of calcium, intact PTH, and magnesium (Mg(2+)) were measured in each patient. Fractional excretion of Mg (FE(Mg)) was calculated in spot urine samples. A nuclear factor of activated T cells luciferase assay was used to analyze the responsiveness of each mutant CaSR to extracellular Ca(2+). RESULTS Genomic analysis revealed five known activating mutations and a novel mutation, E481K, in the CASR. Patients with the A843E, C131W, or F788C mutation showed hypomagnesemia with elevated FE(Mg). Intact PTH in these patients was consistently near the detection limit. In contrast, patients with the P221L, K47N, or E481K mutation exhibited normal Mg(2+) levels. In these patients, intact PTH increased in response to low calcium, and their maximum intact PTH exceeded the lower limit of the reference range. Functional analysis showed an association between the disease severity and the in vitro activity of the mutant CaSR. CONCLUSIONS The functional activity of mutant CaSR determines the serum Mg(2+) level, renal Mg(2+) handling, and intact PTH in patients with ADH. The presence of hypomagnesemia with elevated FE(Mg) may indicate the diagnosis of ADH among patients with PTH-deficient hypoparathyroidism.

Influence of RNA Strand Rigidity on Polyion Complex Formation with Block Catiomers.

Polyion complexes (b-PICs) are prepared by mixing single- or double-stranded oligo RNA (aniomer) with poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) (block catiomer) to clarify the effect of aniomer chain rigidity on association behaviors at varying concentrations. Here, a 21-mer single-stranded RNA (ssRNA) (persistence length: 1.0 nm) and a 21-mer double-stranded RNA (small interfering RNA, siRNA) (persistence length: 62 nm) are compared. Both oligo RNAs form a minimal charge-neutralized ionomer pair with a single PEG-PLL chain, termed unit b-PIC (uPIC), at low concentrations (<≈ 0.01 mg mL(-1)). Above the critical association concentration (≈ 0.01 mg mL(-1)), ssRNA b-PICs form secondary associates, PIC micelles, with sizes up to 30-70 nm, while no such multimolecular assembly is observed for siRNA b-PICs. The entropy gain associated with the formation of a segregated PIC phase in the multimolecular PIC micelles may not be large enough for rigid siRNA strands to compensate with appreciably high steric repulsion derived from PEG chains. Chain rigidity appears to be a critical parameter in polyion complex association.

Targeting gene expression to specific cells of kidney tubules in vivo, using adenoviral promoter fragments

Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. “adenovirus driven NPT2a-EGFP and endogenous NHE3 protein”, “adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein” and “adenovirus driven AQP2-EGFP and endogenous AQP2 protein”. Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and effective gene therapy in the kidney tubule diseases.

Proteomics Approach Identifies Factors Associated With the Response to Low-Density Lipoprotein Apheresis Therapy in Patients With Steroid-Resistant Nephrotic Syndrome.

Low‐density lipoprotein apheresis (LDL‐A) has been shown to reduce proteinuria in a subgroup of nephrotic syndrome patients refractory to immunosuppressive therapy. Factors influencing the efficacy of LDL‐A in nephrotic syndrome are completely unknown. Using a proteomics approach, we aimed to identify biological markers that predict the response to LDL‐A in patients with steroid‐resistant nephrotic syndrome (SRNS). Identification of plasma proteins bound to the dextran‐sulfate column at the first session of LDL‐A was determined by mass spectrometry. To investigate biological factors associated with the response to LDL‐A, we compared profiles of column‐bound proteins between responders (defined by more than 50% reduction of proteinuria after the treatment) and non‐responders by 2‐dimensional gel electrophoresis (2‐DE) coupled to mass spectrometry in seven patients with SRNS. Evaluation of proteins adsorbed to LDL‐A column in patients with SRNS revealed the identity of 62 proteins, which included apolipoproteins, complement components, and serum amyloid P‐component (SAP). Comparative analysis of the column‐bound proteins between responders and non‐responders by 2‐DE demonstrated that apolipoprotein E (APOE) and SAP levels were increased in non‐responders as compared with responders. These results were confirmed by western blotting. Moreover, serum levels of APOE and SAP were significantly higher in the non‐responder group than in the responder group by ELISA. Our data provide comprehensive analysis of proteins adsorbed by LDL‐A in SRNS, and demonstrate that the serum levels of APOE and SAP may be used to predict the response to LDL‐A in these patients.