Genetic reprogramming with stem cells regenerates glomerular epithelial podocytes in Alport syndrome.

Glomerular filtration relies on the type IV collagen (ColIV) network of the glomerular basement membrane, namely, in the triple helical molecules containing the α3, α4, and α5 chains of ColIV. Loss of function mutations in the genes encoding these chains (Col4a3, Col4a4, and Col4a5) is associated with the loss of renal function observed in Alport syndrome (AS). Precise understanding of the cellular basis for the patho-mechanism remains unknown and a specific therapy for this disease does not currently exist. Here, we generated a novel allele for the conditional deletion of Col4a3 in different glomerular cell types in mice. We found that podocytes specifically generate α3 chains in the developing glomerular basement membrane, and that its absence is sufficient to impair glomerular filtration as seen in AS. Next, we show that horizontal gene transfer, enhanced by TGFß1 and using allogenic bone marrow-derived mesenchymal stem cells and induced pluripotent stem cells, rescues Col4a3 expression and revive kidney function in Col4a3-deficient AS mice. Our proof-of-concept study supports that horizontal gene transfer such as cell fusion enables cell-based therapy in Alport syndrome.

Transcriptome Analysis by RNA Sequencing of Mouse Embryonic Stem Cells Stocked on International Space Station for 1584 Days in Frozen State after Culture on the Ground.

As a space project, in "Stem Cells" by the Japan Aerospace Exploration Agency (JAXA), frozen mouse ES cells were stored on the International Space Station (ISS) in the Minus Eighty Degree Laboratory Freezer for ISS (MELFI) for 1584 days. After taking these cells back to the ground, the cells were thawed and cultured, and their gene expressions were comprehensively analyzed using RNA sequencing in order to elucidate the early response of the cells to long-time exposure to space radiation consisting of various ionized particles. The comparisons of gene expression involved in double-stranded break (DSB) repair were examined. The expressions of most of the genes that were involved in homologous recombination (HR) and non-homologous end joining (NHEJ) were not significantly changed between the ISS-stocked cells and ground-stocked control cells. However, the transcription of Trp53inp1 (tumor protein 53 induced nuclear protein-1), Cdkn1a (p21), and Mdm2 genes increased in ISS-stocked cells as well as Fe ion-irradiated cells compared to control cells. This suggests that accumulated DNA damage caused by space radiation exposure would activate these genes, which are involved in cell cycle arrest for repair and apoptosis in a p53-dependent or -independent manner, in order to prevent cells with damaged genomes from proliferating and forming tumors.

Comparison of biological measurement and physical estimates of space radiation in the International Space Station.

Nowadays, ordinary people can travel in space, and the possibility of extended durations in an environment such as moon of the Earth and Mars with higher space radiation exposures compared to past missions, is increasing. Until now, the physical doses of space radiation have been measured, but measurement of direct biological effects has been hampered by its low dose and low dose-rate effect. To assess the biological effects of space radiation, we launched and kept frozen mouse embryonic stem (ES) cells in minus eighty degree Celsius freezer in ISS (MELFI) on the International Space Station (ISS) for a maximum of 1,584 days. The passive dosimeter for life science experiments in space (PADLES) was attached on the surface of the sample case of the ES cells. The physical dosimeter measured the absorbed dose in water. After return, the frozen cells were thawed and cultured and their chromosome aberrations were analyzed. Comparative experiments with proton and iron ion irradiation were performed at particle accelerators on Earth. The wild-type ES cells showed no differences in chromosomal aberrations between the ground control and ISS exposures. However, we detected an increase of chromosome aberrations in radio-sensitized histone H2AX heterozygous-deficient mouse ES cells and found that the rate of increase against the absorbed dose was 1.54-fold of proton irradiation at an accelerator. On the other hand, we estimated the quality factor of space radiation as 1.48 ± 0.2. using formulas of International Commission of Radiation Protection (ICRP) 60. The relative biological effectiveness (RBE) observed from our experiments (1.54-fold of proton) was almost equal (1.04-fold) to the physical estimation (1.48 ± 0.2). It should be important to clarify the relation between biological effect and physical estimates of space radiation. This comparative study paves a way to reveal the complex radiation environments to reduce the uncertainty for risk assessment of human stay in space.

Nutritional Status Association With Sarcopenia in Patients Undergoing Maintenance Hemodialysis Assessed by Nutritional Risk Index.

Background: Malnutrition and sarcopenia are frequently observed in patients undergoing maintenance hemodialysis (MHD). To elucidate whether malnutrition is associated with sarcopenia in those cases, the relationship of nutritional status with sarcopenia was investigated. Methods: Nutritional status was assessed using a nutritional risk index (NRI) developed for patients undergoing MHD. This retrospective cross-sectional study included 315 MHD patients (199 males, 116 females), who were divided into low-risk (score 0-7) and medium-/high-risk (score 8-13) groups. Sarcopenia and severe sarcopenia, along with low muscle mass, low muscle strength, and low physical performance were defined using the Asian Working Group for Sarcopenia 2019 criteria. Results: The median NRI score was 5.0, while the prevalence of medium-/high-risk cases among the patients was 31.1%. Additionally, the rates of those with low muscle mass, low muscle strength, and low physical performance were 55.9, 60.6, and 31.4%, respectively, while those of sarcopenia and severe sarcopenia were 44.1 and 20.0%, respectively. Multivariable logistic regression analyses revealed a significant (P < 0.001) association of NRI score with sarcopenia [odds ratio (OR) 1.255, 95% confidence interval (CI) 1.143-1.377] and severe sarcopenia (OR 1.257, 95% CI 1.122-1.407), as well as low muscle mass (OR 1.260, 95% CI 1.157-1.374), low muscle strength (OR 1.310, 95% CI 1.178-1.457), and low physical performance (OR 1.216, 95% CI 1.104-1.339). Furthermore, medium-/high-risk status showed a significant (P < 0.05) association with sarcopenia (OR 2.960, 95% CI 1.623-5.401) and severe sarcopenia (OR 2.241, 95% CI 1.151-4.362), as well as low muscle mass (OR 2.141, 95% CI 1.219-3.760), low muscle strength (OR 7.665, 95% CI 3.438-17.091), and low physical performance (OR 2.570, 95% CI 1.401-4.716). Conclusions: These results suggest that malnutrition contributes to sarcopenia/severe sarcopenia in MHD patients by reducing muscle mass and strength, and physical performance.

Xanthine Oxidoreductase Inhibitor Use Associated With Reduced Risk of Sarcopenia and Severe Sarcopenia in Patients Undergoing Hemodialysis.

BACKGROUND: Xanthine oxidoreductase (XOR) inhibition reduces reactive oxygen species (ROS) production and enhances adenosine triphosphate (ATP) synthesis. We investigated the protective effects of XOR inhibitor treatment on sarcopenia, frequently observed in patients undergoing hemodialysis (HD), in which increased ROS and ATP shortage are known to be involved. METHODS: This retrospective cross-sectional study included 296 HD patient (203 males, 93 females). Muscle mass, physical performance, and muscle strength were assessed using dual-energy X-ray absorptiometry, five-time chair stand testing, and handgrip strength, respectively. The Asian Working Group for Sarcopenia 2019 criteria were used to define low muscle mass, low physical performance, and low muscle strength, as well as sarcopenia and severe sarcopenia. RESULTS: Sarcopenia and severe sarcopenia prevalence rates were 42.2 and 20.9%, respectively. XOR inhibitor users (n = 119) showed a significantly (p < 0.05) lower prevalence of sarcopenia and severe sarcopenia, as well as reduced muscle mass, physical performance, and muscle strength than non-users (n = 177). Multivariate logistic regression analyses also revealed XOR inhibitor use to be significantly associated with low muscle mass [odds ratio (OR), 0.384; 95% confidence interval (CI), 0.183-0.806; p = 0.011] and low physical performance (OR, 0.286; 95% CI, 0.142-0.578; p < 0.001), while significance with low muscle strength was borderline. Furthermore, XOR inhibitor use was significantly associated with sarcopenia (OR, 0.462; 95% CI, 0.226-0.947; p = 0.035) and severe sarcopenia (OR, 0.236; 95% CI, 0.091-0.614; p = 0.003). CONCLUSIONS: XOR inhibitor use was significantly associated with reduced risk of sarcopenia/severe sarcopenia in HD patients, suggesting that XOR inhibitor treatment has protective effects on sarcopenia in HD patients.

BMP7 Signaling in TGFBR2-Deficient Stromal Cells Provokes Epithelial Carcinogenesis.

Deregulated transforming growth factor-ß (TGFß) signaling is a common feature of many epithelial cancers. Deletion of TGFß receptor type 2 (TGFBR2) in fibroblast specific protein-1 (FSP1)-positive stromal cells induces squamous cell carcinoma in the murine forestomach, implicating fibroblast-derived hepatocyte growth factor (HGF) as the major driver of the epithelium carcinogenesis. Prior to cancer development, hyperproliferative FSP1+ fibroblasts lacking TGFBR2 accumulate in the forestomach, disrupting the regulatory signaling cross-talk with the forestomach epithelium. Here, concurrent loss in TGFBR2 and SMAD4 completely abrogates the development of forestomach cancer. Bone morphogenic protein-7 (BMP7) was highly upregulated in forestomach cancer tissue, activating Smad1/5/8 signaling, cell proliferation, and HGF production in TGFBR2-deficient FSP1+ fibroblasts. This stimulation by BMP7 was lost in the combined TGFBR2 and SMAD4 double knockout fibroblasts, which included a profound decrease in HGF expression. Thus, Smad4-mediated signaling is required to initiate epithelial carcinogenesis subsequent to TGFBR2 deletion in FSP1+ fibroblasts.Implications: These findings reveal a complex cross-talk between epithelial cells and the stroma, wherein Smad4 is required to elicit squamous cell carcinomas in the forestomach of mice with TGFBR2-deficient stromal cells. Mol Cancer Res; 16(10); 1568-78. ©2018 AACR.

Low-dose irradiation of mouse embryos increases Smad-p21 pathway activity and preserves pluripotency.

PURPOSE: To study the outcomes of mouse preimplantation embryos irradiated with low doses of X-rays (≤ 1 Gy) and investigate apoptosis and pluripotency of the irradiated embryos. METHODS: Mouse embryos at the 2-cell stage were collected for in vitro culture. After reaching the 8-cell stage, embryos were irradiated with various low doses of X-rays (0-1 Gy). Blastocysts with a normal appearance were transferred into a pseudopregnant uterus. The developmental rate to blastocysts and the survival rate following embryo transfer were examined. Expression levels of p21, Smad2, Foxo1, Cdx2, Oct4, and Nanog genes were measured by RT-PCR. Apoptotic cells in mouse blastocysts were examined immunofluorescently by staining for cleaved caspase-3. RESULTS: More than 90% of non-irradiated and low-dose X-ray-irradiated preimplantation embryos developed to morphologically normal blastocysts that could be implanted and survive in the uterus. However, embryos irradiated with X-rays had more apoptotic cells in a dose-dependent manner. Expression of p21, Smad2, and Foxo1 genes in X-ray-irradiated embryos was increased significantly, while expression of Cdx2, Oct4, and Nanog genes was maintained in comparison with non-irradiated embryos. CONCLUSIONS: Although irradiated embryos contained apoptotic cells, the low doses of irradiation did not disturb development of 8-cell stage embryos to blastocysts or their survival in utero. The underlying mechanisms might involve anti-apoptotic systems, including the Smad-p21 pathway, and preservation of pluripotency.

Gene-modified embryonic stem cell test to characterize chemical risks.

A high-throughput test of cell growth inhibition was performed using mouse embryonic stem (ES) cells to assess chemical toxicities. We herein demonstrated using a 96-well culture plate approach and the MTT assay that this method was suitable for prioritization of chemicals for their cytotoxic properties. In order to categorize chemicals, we used p53 gene-modified mouse ES cells as well as wild-type ES cells. The p53 gene is a well-known tumor suppressor and controls programmed cell death (apoptosis) and cellular senescence that is triggered by DNA-damaging agents such as alkylating agents and radiation. In the present study, p53-deficient ES cells were found to be more resistant to a tumor initiator, diethylnitrosamine (DEN), than wild-type ES cells, suggesting the inhibition of apoptosis or senescence by a dysfunction in p53. Chromosome aberrations were more frequently detected in p53-deficient ES cells than in wild-type cells, indicating genomic instability due to the deletion of p53. Other tumor initiators, methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (NMU), did not reveal apparent differences in cytotoxicity between wild-type and p53-deficient ES cells. Thus, ES test system using gene-modified ES cells may be used to categorize chemicals by detecting their characteristic effects on apoptosis, genotoxic potentials as well as general cytotoxicity.

Endothelial-mesenchymal transition as a novel mechanism for generating myofibroblasts during diabetic nephropathy.

This Commentary provides perspective on epithelial-mesenchymal transition in diabetic nephropathy.

Different risk factors for vascular calcification in end-stage renal disease between diabetics and nondiabetics: the respective importance of glycemic and phosphate control.

Vascular calcification is highly prevalent in dialysis patients, and significantly increases cardiovascular mortality. The presence and progression of vascular calcification is significantly associated with chronic inflammation and malnutrition. Disorders of mineral metabolism, particularly hyperphosphatemia, have been emphasized as risk factors for vascular calcification. Although vascular calcification has been reported to be highly prevalent in diabetic patients with end-stage renal disease (ESRD), the risk factors for vascular calcification in these patients have not been fully explored. Through a review of the literature and our recent studies examining vascular calcification in ESRD patients, hyperphosphatemia is significantly associated with vascular calcification in nondiabetic ESRD patients, while it may not be a significant risk factor for vascular calcification in diabetic ESRD patients. In diabetic patients, vascular calcification occurs long before the initiation of dialysis therapy, and the factors associated with vascular calcification in non-uremic diabetics appear to be hyperglycemia and related metabolic disorders, such as increased glycation and oxidative stress. In diabetic ESRD patients, hyperglycemia is also suggested to be a significant factor associated with the progression of vascular calcification. Thus, the importance of glycemic and phosphate control is suggested to be emphasized in diabetic and nondiabetic ESRD patients, respectively, for prevention of the progression of vascular calcification.

Receptor for advanced glycation end products is involved in impaired angiogenic response in diabetes.

Angiogenic response is impaired in diabetes. Here, we examined the involvement of receptor for advanced glycation end products (RAGE) in diabetes-related impairment of angiogenesis in vivo. Angiogenesis was determined in reconstituted basement membrane protein (matrigel) plugs containing vascular endothelial growth factor (VEGF) implanted into nondiabetic or insulin-deficient diabetic wild-type or RAGE(-/-) mice. The total, endothelial, and smooth muscle (or pericytes) cells in the matrigel were significantly decreased in diabetes, with the regulation dependent on RAGE. In the matrigel, proangiogenic VEGF expression was decreased, while antiangiogenic thrombospondin-1 was upregulated in diabetic mice, regardless of the presence of RAGE. In wild-type mice, proliferating cell nuclear antigen (PCNA)-positive cells in the matrigel were significantly less in diabetic than in nondiabetic mice, while the numbers of transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly higher. This alteration in PCNA- and TUNEL-positive cells in diabetes was not observed in RAGE(-/-) mice. Similarly, the percentage of nuclear factor kappaB-activated cells is enhanced in diabetes, with the regulation dependent on the presence of RAGE. Importantly, adenovirus-mediated overexpression of endogenous secretory RAGE, a decoy receptor for RAGE, restores diabetes-associated impairment of angiogenic response in vivo. Thus, RAGE appears to be involved in impairment of angiogenesis in diabetes, and blockade of RAGE might be a potential therapeutic target.

[Mechanism of vascular calcification].

Vascular calcification in dialysis patients is associated with morbidity and mortality risks. Recent evidence suggests that vascular calcification is an active process resembling osteogenesis and chondrogenesis process. In this process, hyperphosphatemia is one of the important regulators. Inorganic phosphates directly regulate vascular calcification in vitro through a sodium-dependent phosphate cotransporter and promote expression of the osteoblastic differentiation markers.

Statins inhibit in vitro calcification of human vascular smooth muscle cells induced by inflammatory mediators.

Although lipid-lowering therapy with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) decreases the progression of coronary artery and aortic valve calcification, the mechanism of action of these drugs to inhibit the calcification process remains unclear. In this study, we investigated the effect of statins such as cerivastatin and atorvastatin on vascular calcification by utilizing an in vitro model of inflammatory vascular calcification. Cerivastatin and atorvastatin dose-dependently inhibited in vitro calcification of human vascular smooth muscle cells (HVSMCs) induced by the following inflammatory mediators (IM): interferon-gamma, 1alpha,25-dihydroxyvitamin D3, tumor necrosis factor-alpha, and oncostatin M. These statins also depressed expression of alkaline phosphatase (ALP) in HVSMCs induced by these factors. Mevalonate and geranylgeranylpyrophosphate reversed the inhibitory effect of cerivastatin on ALP expression in HVSMCs, while farnesylpyrophosphate showed no effect on the ALP activities inhibited by this drug, suggesting that inhibition of Rho and its downstream target, Rho kinase may mediate the inhibitory effect of cerivastatin. Cerivastatin prevented RhoA activation in HVSMCs induced by the IM. A specific inhibitor of Rho kinase (Y-27632) inhibited in vitro calcification and induction of ALP in HVSMCs. These findings provide a possible mechanism of statins to prevent the progression of calcification in inflammatory vascular diseases such as atherosclerosis and cardiac valvular calcification.

Matrix Gla protein is associated with coronary artery calcification as assessed by electron-beam computed tomography.

Matrix Gla protein (MGP) is an extracellular matrix protein with wide tissue distribution. It has been demonstrated that the expression of MGP is detected not only in the normal blood vessels but also calcified atherosclerotic plaques, and that MGP deficient mice develop extensive arterial calcification. MGP is thought to be a regulator of vascular calcification. A recent clinical study demonstrates the association between polymorphisms of the MGP gene and increased risk of myocardial infarction. However, there are no reports of the relationship between serum MGP levels and coronary artery calcification (CAC). We evaluated the severity of CAC using electron-beam computed tomography (EBCT), and measured serum MGP levels by enzyme-linked immunosorbent assay in 115 subjects with suspected coronary artery disease. CAC scores were correlated with traditional risk factors, such as age, gender, hyper-tension, diabetes, hyperlipidemia and smoking. The serum MGP levels were lower in patients with CAC than in those without CAC (p<0.001). As the severity of CAC increased, there was a significant decrease in serum MGP levels. Serum MGP levels (U/L) were 116.7 +/- 20.3, 104.9 +/- 19.2, 95.2 +/- 15.2 and 82.2 +/- 19.7, (medians 115.5, 105.0, 94.8, and 81.9) for the subjects with normal (CAC score=0), mild (CAC score=1 to 99), moderate (CAC score=100 to 400), and severe (CAC score >400) coronary calcification, respectively. We found that serum MGP levels are inversely correlated with the severity of CAC. These data suggest a possible role for MGP in the development of vascular calcification.

Arterial wall stiffness is associated with peripheral circulation in patients with type 2 diabetes.

The prevalence of peripheral vascular disease (PVD) in diabetic patients is manyfold higher than that of age- and sex-matched nondiabetic subjects. This study was designed to evaluate the relationship between quantitatively determined peripheral circulation in the lower extremities and arterial wall thickness or stiffness in 68 patients with type 2 diabetes. Peripheral circulation during treadmill-exercise was monitored by transcutaneous oxygen tension (TcPO2) and was expressed as percentage of post-exercise TcPO2 adjusted by that of pre-exercise (TcPO2 index). Arterial wall thickness (intima-media thickness; IMT) and stiffness (stiffness beta) were measured by ultrasonography. TcPO2 index was negatively (r=-0.350, P=0.0007) correlated with stiffness beta, not with IMT, of the femoral artery. In patients without insulin therapy (n=52), both fasting plasma insulin concentration (r=-0.323, P=0.0023) and HOMA IR, an insulin resistance index, (r=-0.281, P=0.0084) were negatively correlated with TcPO2 index. Multiple regression analyses showed that association of stiffness beta of the femoral artery or HOMA IR with the TcPO2 index was independent of other factors including age, smoking index, ankle brachial pressure index and IMT of femoral artery. Thus, arterial wall stiffness of femoral artery appears to be a major determinant of peripheral circulation in patients with type 2 diabetes.