Risk factor analysis for cisplatin-induced nephrotoxicity with the short hydration method in diabetic patients.

The occurrence of cisplatin (CDDP)-induced nephrotoxicity (CIN) has decreased with advancements in supportive care. In contrast, we reported that baseline diabetes mellitus (DM) complications significantly worsen CIN. This study aimed to determine further risk factors associated with CIN development in DM patients. Patients with thoracic cancer requiring DM pharmacotherapy, who received CDDP (≥ 60 mg/m2)-containing regimens using the short hydration method (n = 140), were enrolled in this retrospective multicenter observational study. The primary endpoint of the present study was the elucidation of risk factors (patient factors, DM medication influence, and treatment-related factors) associated with CIN development in patients with DM. Cisplatin-induced nephrotoxicity occurred in 22.1% of patients with DM. The median worst variation of serum creatinine levels and creatinine clearance (worst level - baseline level) was 0.16 mg/dL (range: - 0.12-1.41 mg/dL) and - 15.9 mL/min (- 85.5-24.3 mL/min), respectively. Multivariate logistic regression analyses identified female sex as the singular risk factor for CIN development in the DM population (adjusted odds ratio; 2.87, 95% confidence interval; 1.08-7.67, P = 0.04). Diabetes mellitus medication and treatment-related factors did not affect CIN development. In conclusion, our study revealed that female sex is significantly associated with CIN development in patients with DM and thoracic cancer.

In Vivo Modeling of Skeletal Muscle Diseases Using the CRISPR/Cas9 System in Rats.

The CRISPR/Cas9 system is a powerful gene editing tool that can be used to modify a target gene in almost all species. It unlocks the possibility of generating knockout or knock-in genes in laboratory animals other than mice. The Dystrophin gene is implicated in human Duchenne muscular dystrophy; however, Dystrophin gene mutant mice do not show severe muscle degenerating phenotypes when compared to humans. On the other hand, Dystrophin gene mutant rats made with the CRISPR/Cas9 system show more severe phenotypes than those seen in mice. The phenotypes seen in dystrophin mutant rats are more representative of the features of human DMD. This implies that rats are better models of human skeletal muscle diseases than mice. In this chapter, we present a detailed protocol for the generation of gene-modified rats by microinjection into embryos using the CRISPR/Cas9 system.

Suppression of MyoD induces spontaneous adipogenesis in skeletal muscle progenitor cell culture.

The degree of intramuscular adipose tissue accumulation is one of the factors affecting meat quality. Accumulation of adipocytes is also observed under the pathological condition of skeletal muscle such as muscular dystrophy and sarcopenia. The origin of adipocytes seen in skeletal muscle is mesenchymal progenitor cells that can give rise to both adipocytes and fibroblasts. In the present study, we demonstrated that siRNA-mediated suppression of MyoD expression in rat skeletal muscle progenitor cell culture, which comprises both myogenic satellite cells and mesenchymal progenitor cells, resulted in diminished myotube formation and an unexpected spontaneous appearance of white adipocytes. Suppressing myomaker expression also resulted in complete absence of myotube formation without reducing MyoD expression, but no adipogenesis was seen in this scenario, indicating that decline in MyoD expression rather than decreased myotube formation is necessary to induce adipogenesis. In addition, spontaneous adipogenesis induced by suppressing MyoD expression in culture was inhibited by the conditioned medium from control culture, indicating that anti-adipogenic factor(s) are secreted from MyoD-positive myogenic cells. These results indicate the presence of regulatory mechanism on adipogenesis by myogenic cells.

Evaluation of Proteinuria Using Urine Protein : Creatine Ratio in Treatment with Molecular Targeted Agents for Advanced Renal Cell Carcinoma.

The usefulness of the urine protein : creatine ratio (UPCR) in management of molecular targeted therapy and immunotherapy has not been studied, although urine protein dipstick testing (uPr) is widely used in the clinical setting. The aim of this study was to investigate the usefulness of UPCR as compared to uPr in patients undergoing molecular targeted therapy for advanced renal cell carcinoma (RCC). A total of 25 patients (median age 68 years) with advanced RCC were included. Sunitinib, pazopanib, axitinib, sorefenib, everolimus, and nivolumab were administered to 15, 9, 16, 3, 7, and 13 patients, respectively, with duplication. Proteinuria was managed according to the grade determined by UPCR. Data at every treatment visit were retrospectively collected and uPr and UPCR were compared. The overall incidences of any grade of proteinuria associated with sunitinib, pazopanib, axitinib, sorafenib and everolimus were 86.7, 88.9, 93.8, 100, and 85.7%, respectively. There were discordances between the uPr-based grade and UPCR-based grade. UPCR did not meet the criteria of Grade 3 in 70.6, 100, 83.3, and 83.3% at visits in cases with uPr 3+ for sunitinib, pazopanib, sorafenib, and everolimus, respectively. In axitinib treatment, UPCR did not meet the criteria for withholding in 46.2% of the cases of uPr 2+ and more. Our study suggests that UPCR may be useful tool in management of adverse events associated with tyrosine kinase inhibitors, everolimus and can provide patients with optimal opportunities for receiving treatment.

Cellular senescence-mediated exacerbation of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive disease characterised by chronic muscle degeneration and inflammation. Our previously established DMD model rats (DMD rats) have a more severe disease phenotype than the broadly used mouse model. We aimed to investigate the role of senescence in DMD using DMD rats and patients. Senescence was induced in satellite cells and mesenchymal progenitor cells, owing to the increased expression of CDKN2A, p16- and p19-encoding gene. Genetic ablation of p16 in DMD rats dramatically restored body weight and muscle strength. Histological analysis showed a reduction of fibrotic and adipose tissues invading skeletal muscle, with increased muscle regeneration. Senolytic drug ABT263 prevented loss of body weight and muscle strength, and increased muscle regeneration in rats even at 8 months-the late stage of DMD. Moreover, senescence markers were highly expressed in the skeletal muscle of DMD patients. In situ hybridization of CDKN2A confirmed the expression of it in satellite cells and mesenchymal progenitor cells in patients with DMD. Collectively, these data provide new insights into the integral role of senescence in DMD progression.

Pathological evaluation of rats carrying in-frame mutations in the dystrophin gene: a new model of Becker muscular dystrophy.

Dystrophin, encoded by the DMD gene on the X chromosome, stabilizes the sarcolemma by linking the actin cytoskeleton with the dystrophin-glycoprotein complex (DGC). In-frame mutations in DMD cause a milder form of X-linked muscular dystrophy, called Becker muscular dystrophy (BMD), characterized by the reduced expression of truncated dystrophin. So far, no animal model with in-frame mutations in Dmd has been established. As a result, the effect of in-frame mutations on the dystrophin expression profile and disease progression of BMD remains unclear. In this study, we established a novel rat model carrying in-frame Dmd gene mutations (IF rats) and evaluated the pathology. We found that IF rats exhibited reduced expression of truncated dystrophin in a proteasome-independent manner. This abnormal dystrophin expression caused dystrophic changes in muscle tissues but did not lead to functional deficiency. We also found that the expression of additional dystrophin named dpX, which forms the DGC in the sarcolemma, was associated with the appearance of truncated dystrophin. In conclusion, the outcomes of this study contribute to the further understanding of BMD pathology and help elucidate the efficiency of dystrophin recovery treatments in Duchenne muscular dystrophy, a more severe form of X-linked muscular dystrophy.

Disruption of insulin receptor substrate-2 impairs growth but not insulin function in rats.

Insulin receptor substrate (IRS)-2, along with IRS-1, is a key signaling molecule that mediates the action of insulin and insulin-like growth factor (IGF)-I. The activated insulin and IGF-I receptors phosphorylate IRSs on tyrosine residues, leading to the activation of downstream signaling pathways and the induction of various physiological functions of insulin and IGF-I. Studies using IRS-2 knockout (KO) mice showed that the deletion of IRS-2 causes type 2 diabetes due to peripheral insulin resistance and impaired ß-cell function. However, little is known about the roles of IRS-2 in other animal models. Here, we created IRS-2 KO rats to elucidate the physiological functions of IRS-2 in rats. The body weights of IRS-2 KO rats at birth were lower compared with those of their WT littermates. The postnatal growth of both male and female IRS-2 KO rats was also suppressed. Compared with male WT rats, the glucose and insulin tolerance of male IRS-2 KO rats were slightly enhanced, whereas a similar difference was not observed between female WT and IRS-2 KO rats. Besides the modestly increased insulin sensitivity, male IRS-2 KO rats displayed the enhanced insulin-induced activation of the mTOR complex 1 pathway in the liver compared with WT rats. Taken together, these results indicate that in rats, IRS-2 plays important roles in the regulation of growth but is not essential for the glucose-lowering effects of insulin.

Reduced fibrillar collagen accumulation in skeletal muscle of secreted protein acidic and rich in cysteine (SPARC)-null mice.

We have previously shown that secreted protein acidic and rich in cysteine (SPARC) promotes myogenic differentiation of rat skeletal muscle progenitor cells in vitro, and in vivo small interfering RNA (siRNA)-mediated transient suppression of SPARC expression in skeletal muscle of mice causes atrophic changes of myofibers, suggesting that SPARC plays a role in the maintenance of skeletal muscle function. In order to know the effect of long-term deficiency of SPARC on skeletal muscle, we performed phenotypic analyses of skeletal muscle of SPARC-null mice. Age-associated changes of myofiber diameters were comparable between wild type (WT) and SPARC-null mice at all ages examined, indicating that the growth of myofibers is unaffected by the absence of SPARC. On the other hand, accumulation of fibrillar collagen was significantly reduced in SPARC-null mice compared to WT mice after 5 months of age without significant changes of collagen I gene expression. The results obtained in the present study suggest that SPARC plays a role to maintain the stiffness of skeletal muscle by regulating collagen accumulation.

Pneumothorax as an Adverse Event in Patients with Lung Metastasis of Soft Tissue Sarcoma under Eribulin Treatment.

Pneumothorax has been reported as a pazopanib-associated adverse event in patients with lung metastases of soft tissue sarcoma (STS). However, pneumothorax triggered by eribulin treatment has never been reported. We herein report two cases of spontaneous pneumothorax in patients with STS treated with eribulin. Both patients experienced pneumothorax accompanied by sudden dyspnea on day 9 or 10 of eribulin treatment. These two cases suggest that spontaneous pneumothorax may occur as an adverse event of eribulin treatment in such patients. We should therefore be alert for the potential development of pneumothorax during eribulin treatment of patients with STS and lung metastases.

Attributes' Importance for Zero-Shot Pose-Classification Based on Wearable Sensors.

This paper presents a simple yet effective method for improving the performance of zero-shot learning (ZSL). ZSL classifies instances of unseen classes, from which no training data is available, by utilizing the attributes of the classes. Conventional ZSL methods have equally dealt with all the available attributes, but this sometimes causes misclassification. This is because an attribute that is effective for classifying instances of one class is not always effective for another class. In this case, a metric of classifying the latter class can be undesirably influenced by the irrelevant attribute. This paper solves this problem by taking the importance of each attribute for each class into account when calculating the metric. In addition to the proposal of this new method, this paper also contributes by providing a dataset for pose classification based on wearable sensors, named HDPoseDS. It contains 22 classes of poses performed by 10 subjects with 31 IMU sensors across full body. To the best of our knowledge, it is the richest wearable-sensor dataset especially in terms of sensor density, and thus it is suitable for studying zero-shot pose/action recognition. The presented method was evaluated on HDPoseDS and outperformed relative improvement of 5.9% in comparison to the best baseline method.

Roles of chondroitin sulfate proteoglycan 4 in fibrogenic/adipogenic differentiation in skeletal muscle tissues.

Intramuscular adipose tissue and fibrous tissue are observed in some skeletal muscle pathologies such as Duchenne muscular dystrophy and sarcopenia, and affect muscle strength and myogenesis. They originate from common fibrogenic/adipogenic cells in the skeletal muscle. Thus, elucidating the regulatory mechanisms underlying fibrogenic/adipogenic cell differentiation is an important step toward the mediation of these disorders. Previously, we established a highly adipogenic progenitor clone, 2G11, from rat skeletal muscle and showed that basic fibroblast growth factor (bFGF) is pro-adipogenic in these cells. Here, we demonstrated that 2G11 cells give rise to fibroblasts upon transforming growth factor (TGF)-ß1 stimulation, indicating that they possess mesenchymal progenitor cells (MPC)-like characteristics. The previously reported MPC marker PDGFRα is expressed in other cell populations. Accordingly, we produced monoclonal antibodies that specifically bind to 2G11 cell surface antigens and identified chondroitin sulfate proteoglycan 4 (CSPG4) as a potential MPC marker. Based on an RNA interference analysis, we found that CSPG4 is involved in both the pro-adipogenic effect of bFGF and in TGF-ß-induced alpha smooth muscle actin expression and stress fiber formation. By establishing an additional marker for MPC detection and characterizing its role in fibrogenic/adipogenic differentiation, these results will facilitate the development of effective treatments for skeletal muscle pathologies.

Basic fibroblast growth factor is pro-adipogenic in rat skeletal muscle progenitor clone, 2G11 cells.

Intramuscular adipose tissue (IMAT) formation is a hallmark of marbling in cattle. IMAT is considered to originate from skeletal muscle progenitor cells with adipogenic potential. However, the mechanism involved in IMAT formation from these progenitor cells in vivo remains unclear. In the present study, among the growth factors tested, which were known to be expressed in skeletal muscle, we found only basic fibroblast growth factor (bFGF) has a pro-adipogenic effect on skeletal muscle derived adipogenic progenitor clone, 2G11 cells. Pre-exposure of 2G11 cells to bFGF did not affect initial gene expressions of CCAAT/enhancer-binding protein (C/EBP)ß and C/EBPδ, while resulting in an enhancement of subsequent expressions of C/EBPα and proliferator-activated receptor gamma (PPARγ) during adipogenesis, indicating that bFGF is acting on the transcriptional regulation of C/EBPα and PPARγ. In addition, the effect of bFGF is mediated via two types of FGF receptor (FGFR) isoforms: FGFR1 and FGFR2 IIIc, and both receptors are prerequisite for bFGF to express its pro-adipogenic effect. These results suggest that bFGF plays an important role as a key trigger of IMAT formation in vivo.

Possible involvement of the cerebellum in motor-function impairment in progranulin-deficient mice.

Progranulin (PGRN) is a multifunctional growth factor involved in many physiological and pathological processes in the brain such as sexual differentiation, neurogenesis, neuroinflammation, and neurodegeneration. Previously, we showed that PGRN was expressed broadly in the brain and the Purkinje cells in the cerebellum were one of the regions with the highest expression level of PGRN. Thus, in the present study, we investigated the possible roles of PGRN in the cerebellum by comparing wild-type (WT) and PGRN-deficient (KO) mice with immunohistochemical staining for calbindin, a marker of Purkinje cells. The results showed that the density of Purkinje cell dendrites in the molecular layer of the cerebellum was significantly higher in KO mice than in WT mice, although the number of cell bodies was comparable between the genotypes. Subsequently, as the cerebellum is the center of the motor function, we performed a rotarod test and found that KO mice remained on the rotating rod for significantly shorter periods than WT mice. However, KO and WT mice did not differ significantly with respect to the diameter of myofibers in a skeletal muscle. These results suggest that PGRN is involved in the development and/or maturation of neuronal networks comprising Purkinje cells in the cerebellum, which may be a prerequisite to normal motor function.

[Transdisciplinary Approach for Sarcopenia. Molecular mechanism of sarcopenia : The role of skeletal muscle niche component SPARC in the regulation of myogenesis and adipogenesis and its alteration with age].

Sarcopenia, an age-related decline in skeletal muscle mass and strength, causes the decline of the quality of life in the elderly. The age-related alteration in the differentiation potency of satellite cells, myogenic tissue specific stem cells in skeletal muscle, and preadipocytes in skeletal muscle is possibly involved in the disruption of homeostasis in skeletal muscle. The differentiation of the cells is affected by the microenvironment surrounding the cells, called niche. Here, we focused on SPARC (secreted protein acidic and rich in cysteine) as a secreted glycoprotein existing in the niche. We review the roles of SPARC on the differentiation of satellite cells and preadipocytes in the muscle and their alteration with age.

Generation of muscular dystrophy model rats with a CRISPR/Cas system.

Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder caused by mutations in the Dmd gene encoding Dystrophin. DMD model animals, such as mdx mice and canine X-linked muscular dystrophy dogs, have been widely utilized in the development of a treatment for DMD. Here, we demonstrate the generation of Dmd-mutated rats using a clustered interspaced short palindromic repeats (CRISPR)/Cas system, an RNA-based genome engineering technique that is also adaptive to rats. We simultaneously targeted two exons in the rat Dmd gene, which resulted in the absence of Dystrophin expression in the F0 generation. Dmd-mutated rats exhibited a decline in muscle strength, and the emergence of degenerative/regenerative phenotypes in the skeletal muscle, heart, and diaphragm. These mutations were heritable by the next generation, and F1 male rats exhibited similar phenotypes in their skeletal muscles. These model rats should prove to be useful for developing therapeutic methods to treat DMD.

Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner.

Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies.

Secreted protein acidic and rich in cysteine internalization and its age-related alterations in skeletal muscle progenitor cells.

Aging causes phenotypic changes in skeletal muscle progenitor cells (Skm-PCs), such as reduced myogenesis and increased adipogenesis due to alterations in their environment or niche. Secreted protein acidic and rich in cysteine (SPARC), which is secreted into the niche of Skm-PCs, inhibits adipogenesis and promotes myogenesis. We have previously reported that Skm-PC responsiveness to SPARC declines with age, although the mechanism underlying this decline is unknown. In this study, we found that SPARC is internalized by Skm-PCs and that this uptake increases with age. Internalization is dependent on integrin-α5, a cell surface SPARC-binding molecule, and clathrin-mediated endocytosis. We also demonstrated that internalized SPARC is transported to Rab7-positive endosomes. Skm-PCs from old rats exhibited increased clathrin expression and decreased Rab7 expression exclusively in MyoD-negative cells. In loss-of-function analyses, clathrin knockdown increased the anti-adipogenic effect of SPARC, whereas Rab7 knockdown reduced it, indicating that alterations in SPARC internalization may mediate the age-related decline in its anti-adipogenic effect. These results provide insights into age-related SPARC resistance in Skm-PCs, which may lead to sarcopenia.

Ex vivo bupivacaine treatment results in increased adipogenesis of skeletal muscle cells in the rat.

Intramuscular adipose tissue (IMAT) is observed in some skeletal muscle pathologies. IMAT is implicated not only in the disorders of muscle contraction, but also of metabolism and insulin sensitivity due to its nature as a secretary organ. Several studies indicate the presence of cells with adipogenic potential in skeletal muscle. However, the mechanism of fate specification that triggers these cells to enter an adipogenic program in vivo remains to be solved. In the present study, we examined whether activation of the adipogenic program of muscle-resident cells precedes their proliferation upon muscle injury. For this purpose, muscle injury was induced by injecting bupivacaine (BPVC) to excised skeletal muscle ex vivo. Cells isolated from ex vivo BPVC-treated muscle exhibited higher adipogenic potential than those from saline-treated muscle. Pre-plating exposure of skeletal muscle cells to basic fibroblast growth factor (bFGF) mimicked the effect of ex vivo BPVC-treatment, suggesting that bFGF released from extracellular matrix in response to muscle injury activates their adipogenic program. Interestingly, the number of myotubes were significantly reduced in the culture from BPVC-treated muscle, suggesting that adipocytes negatively regulate myogenesis.

Loss of SPARC in mouse skeletal muscle causes myofiber atrophy.

INTRODUCTION: The expression of secreted protein acidic and rich in cysteine (SPARC) in skeletal muscle decreases with age. Here, we examined the role of SPARC in skeletal muscle by reducing its expression. METHODS: SPARC expression was suppressed by introducing short interfering RNA (siRNA) into mouse tibialis anterior muscle. Myofiber diameter, atrogin1, and muscle RING-finger protein 1 (MuRF1) expression, and tumor necrosis factor-α (TNFα) and transforming growth factor-ß (TGFß) signaling were then analyzed. RESULTS: Reduced SPARC expression caused decreases in the diameter of myofibers, especially fast-type ones, accompanied by upregulation of atrogin1, but not MuRF1, at 10 days after siRNA transfection. The expression of TNFα and TGFß and the phosphorylation status of p38 were not affected by SPARC knockdown, whereas Smad3 phosphorylation was increased at 2 days after siRNA transfection. CONCLUSIONS: The loss of SPARC not only upregulates atrogin1 expression but also enhances TGFß signaling, which may in turn cause muscle atrophy.

In vivo electroporation induces cell cycle reentry of myonuclei in rat skeletal muscle.

Adult urodele amphibians such as newts are capable of regenerating lost structures including their limbs. In these species, dedifferentiation of myofiber is essential for the regenerative process. Upon terminal differentiation, nuclei of myofiber (myonuclei) are withdrawn from cell cycle, but prior to dedifferentiation, myonuclei reenter the cell cycle. In contrast with urodele amphibians, it is generally accepted that mammalian myofibers are not able to dedifferentiate in response to muscle injury. A recent study has suggested that electroporation can induce dedifferentiation response of skeletal muscle in newt limbs. In the present study, we examined whether myonuclei of skeletal muscle of mammals are capable of reentering the cell cycle by means of electroporation. Electroporation was applied to tibialis anterior muscle of the rat with or without plasmid DNA. Histological analyses revealed that, while electroporation induces degenerative/regenerative responses in skeletal muscle irrespective of the presence of plasmid DNA, the expression of proliferating cell nuclear antigen (PCNA) in myonuclei was observed only in the presence of plasmid DNA. The present results indicate that myonuclei of skeletal muscle are capable of reentering the cell cycle and suggest that in vivo electroporation can induce dedifferentiation of mammalian skeletal muscle.