“Avian-type” renal medullary tubule organization causes immaturity of urine-concentrating ability in neonates

“Avian-type” renal medullary tubule organization causes immaturity of urine-concentrating ability in neonates.BackgroundWhile neonatal kidneys are not powerful in concentrating urine, they already dilute urine as efficiently as adult kidneys. To elucidate the basis for this paradoxical immaturity in urine-concentrating ability, we investigated the function of Henle's loop and collecting ducts (IMCDs) in the inner medulla of neonatal rat kidneys.MethodsAnalyses of individual renal tubules in the inner medulla of neonatal and adult rat kidneys were performed by measuring mRNA expression of membrane transporters, transepithelial voltages, and isotopic water and ion fluxes. Immunofluorescent identification of the rCCC2 and rCLC-K1 using polyclonal antibodies was also performed in neonatal and adult kidney slices.ResultsOn day 1, the transepithelial voltages (VTs) in the thin ascending limbs (tALs) and IMCDs were 14.6 ± 1.1mV (N = 27) and -42.7 ± 6.1mV (N = 14), respectively. The VTs in the thin descending limbs (tDLs) were zero on day 1. The VTs in the tALs were strongly inhibited by luminal bumetanide or basolateral ouabain, suggesting the presence of a NaCl reabsorption mechanism similar to that in the thick ascending limb (TAL). The diffusional voltage (VD) of the tAL due to transepithelial NaCl gradient was almost insensitive to a chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The VTs in the IMCDs were strongly inhibited by luminal amiloride.On day 1, both the tDL and tAL were impermeable to water, indicating the water impermeability of the entire loop. Diffusional water permeability (Pdw) and urea permeabilities (Purea) in the IMCDs indicated virtual impermeability to water and urea on day 1. Stimulation by vasopressin (1nmol/L) revealed that only Pdw was sensitive to vasopressin by day 14. A partial isoosmolar replacement of luminal urea by NaCl evoked negligible water flux across the neonatal IMCDs, indicating the absence of urea-dependent volume flux in the neonatal IMCD. These transport characteristics in each neonatal tubule are similar to those in quail kidneys. Identification of mRNAs and immunofluorescent studies for specific transporters, including rAQP-1, rCCC2, rCLC-K1, rENaC β subunit, rAQP-2, and rUT-A1, supported these findings.ConclusionWe hypothesize that the renal medullary tubule organization of neonatal rats shares a tremendous similarity with avian renal medulla. The qualitative changes in the organization of medullary tubules may be primarily responsible for the immature urine-concentrating ability in mammalian neonates

Melanin pigments in the melanocytic nevus regress spontaneously after inactivation by high hydrostatic pressure

We report a novel treatment for giant congenital melanocytic nevi (GCMN) that involves the reuse of resected nevus tissue after high hydrostatic pressurization (HHP). However, the remaining melanin pigments in the inactivated nevus tissue pose a problem; therefore, we performed a long-term observation of the color change of inactivated nevus tissue after HHP. Pressurized nevus specimens (200 MPa group, n = 9) and non-pressurized nevus tissues (control group, n = 9) were subcutaneously implanted into nude mice (BALB/c-nu) and then harvested 3, 6, and 12 months later. Color changes of the nevus specimens were evaluated. In the 200 MPa group, the specimen color gradually regressed and turned white, and brightness values were significantly higher in the 200 MPa group than in the control group after 6 months. This indicated that melanin pigments in the pressurized nevus tissue had spontaneously degraded and regressed. Therefore, it is not necessary to remove melanin pigments in HHP-treated nevus tissue

The Alteration of the Epidermal Basement Membrane Complex of Human Nevus Tissue and Keratinocyte Attachment after High Hydrostatic Pressurization

We previously reported that human nevus tissue was inactivated after high hydrostatic pressure (HHP) higher than 200 MPa and that human cultured epidermis (hCE) engrafted on the pressurized nevus at 200 MPa but not at 1000 MPa. In this study, we explore the changes to the epidermal basement membrane in detail and elucidate the cause of the difference in hCE engraftment. Nevus specimens of 8 mm in diameter were divided into five groups (control and 100, 200, 500, and 1000 MPa). Immediately after HHP, immunohistochemical staining was performed to detect the presence of laminin-332 and type VII collagen, and the specimens were observed by transmission electron microscopy (TEM). hCE was placed on the pressurized nevus specimens in the 200, 500, and 1000 MPa groups and implanted into the subcutis of nude mice; the specimens were harvested at 14 days after implantation. Then, human keratinocytes were seeded on the pressurized nevus and the attachment was evaluated. The immunohistochemical staining results revealed that the control and 100 MPa, 200 MPa, and 500 MPa groups were positive for type VII collagen and laminin-332 immediately after HHP. TEM showed that, in all of the groups, the lamina densa existed; however, anchoring fibrils were not clearly observed in the 500 or 1000 MPa groups. Although the hCE took in the 200 and 500 MPa groups, keratinocyte attachment was only confirmed in the 200 MPa group. This result indicates that HHP at 200 MPa is preferable for inactivating nevus tissue to allow its reuse for skin reconstruction in the clinical setting

High Hydrostatic Pressure Therapy Annihilates Squamous Cell Carcinoma in a Murine Model

Cutaneous squamous cell carcinoma (cSCC) is one of the most common skin cancers. In the treatment of cSCC, it is necessary to remove it completely, and reconstructive surgery, such as a skin graft or a local or free flap, will be required, depending on the size, with donor-site morbidity posing a burden to the patient. The high hydrostatic pressure (HHP) technique has been developed as a physical method of decellularizing various tissues. We previously reported that HHP at 200 MPa for 10 min could inactivate all cells in the giant congenital melanocytic nevus, and we have already started a clinical trial using this technique. In the present study, we explored the critical pressurization condition for annihilating cSCC cells in vitro and confirmed that this condition could also annihilate cSCC in vivo. We prepared 5 pressurization conditions in this study (150, 160, 170, 180, and 190 MPa for 10 min) and confirmed that cSCC cells were killed by pressurization at ≥160 MPa for 10 min. In the in vivo study, the cSCC cells inactivated by HHP at 200 MPa for 10 min were unable to proliferate after injection into the intradermal space of mice, and transplanted cSCC tissues that had been inactivated by HHP showed a decreased weight at 5 weeks after implantation. These results suggested that HHP at 200 MPa for 10 min was able to annihilate SCC, so HHP technology may be a novel treatment of skin cancer

Exploration of the Pressurization Condition for Killing Human Skin Cells and Skin Tumor Cells by High Hydrostatic Pressure

High hydrostatic pressure (HHP) is a physical method for inactivating cells or tissues without using chemicals such as detergents. We previously reported that HHP at 200 MPa for 10 min was able to inactivate all cells in skin and giant congenital melanocytic nevus (GCMN) without damaging the extracellular matrix. We also reported that HHP at 150 MPa for 10 min was not sufficient to inactivate them completely, while HHP at 200 MPa for 10 min was able to inactivate them completely. We intend to apply HHP to treat malignant skin tumor as the next step; however, the conditions necessary to kill each kind of cell have not been explored. In this work, we have performed a detailed experimental study on the critical pressure and pressurization time using five kinds of human skin cells and skin tumor cells, including keratinocytes (HEKas), dermal fibroblasts (HDFas), adipose tissue-derived stem cells (ASCs), epidermal melanocytes (HEMa-LPs), and malignant melanoma cells (MMs), using pressures between 150 and 200 MPa. We pressurized cells at 150, 160, 170, 180, or 190 MPa for 1 s, 2 min, and 10 min and evaluated the cellular activity using live/dead staining and proliferation assays. The proliferation assay revealed that HEKas were inactivated at a pressure higher than 150 MPa and a time period longer than 2 min, HDFas and MMs were inactivated at a pressure higher than 160 MPa and for 10 min, and ASCs and HEMa-LPs were inactivated at a pressure higher than 150 MPa and for 10 min. However, some HEMa-LPs were observed alive after HHP at 170 MPa for 10 min, so we concluded that HHP at a pressure higher than 180 MPa for 10 min was able to inactivate five kinds of cells completely

Calcium Channel Blockers, More than Diuretics, Enhance Vascular Protective Effects of Angiotensin Receptor Blockers in Salt-Loaded Hypertensive Rats

The combination therapy of an angiotensin receptor blocker (ARB) with a calcium channel blocker (CCB) or with a diuretic is favorably recommended for the treatment of hypertension. However, the difference between these two combination therapies is unclear. The present work was undertaken to examine the possible difference between the two combination therapies in vascular protection. Salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP) were divided into 6 groups, and they were orally administered (1) vehicle, (2) olmesartan, an ARB, (3) azelnidipine, a CCB, (4) hydrochlorothiazide, a diuretic, (5) olmesartan combined with azelnidipine, or (6) olmesartan combined with hydrochlorothiazide. Olmesartan combined with either azelnidipine or hydrochlorothiazide ameliorated vascular endothelial dysfunction and remodeling in SHRSP more than did monotherapy with either agent. However, despite a comparable blood pressure lowering effect between the two treatments, azelnidipine enhanced the amelioration of vascular endothelial dysfunction and remodeling by olmesartan to a greater extent than did hydrochlorothiazide in salt-loaded SHRSP. The increased enhancement by azelnidipine of olmesartan-induced vascular protection than by hydrochlorothiazide was associated with a greater amelioration of vascular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, superoxide, mitogen-activated protein kinase activation, and with a greater activation of the Akt/endothelial nitric oxide synthase (eNOS) pathway. These results provided the first evidence that a CCB potentiates the vascular protective effects of an ARB in salt-sensitive hypertension, compared with a diuretic, and provided a novel rationale explaining the benefit of the combination therapy with an ARB and a CCB

スピードガン計測におけるボールスピードの信頼性

The purpose of this study is to clarify the reliability of ball speed in speed radar gun measurement. Intraclass correlation coefficient (ICC) was designed to assess consistency between speed radar gun and picture analysis measurements. The ICC was calculated using random-effects model of two-way analysis of variance. One male university baseball pitcher participated in the experiment. The subject was required to pitch the ball five times at the subjective efforts of 60% or 70%. The ball speed (initial velocity) of each trial was measured simultaneously using both speed radar gun and high-speed video camera (250 frames/s). The speed radar gun was placed at distance of 22m from the subject (1.6m height above the ground) on the extension of pitcher\u27s plate and home base. The pitching motion including the ball was filmed using high-speed video camera positioned at 40m on the right hand side of the subject. The ICC for the ball speed between speed radar gun and picture analysis measurements was 0.94 (SEM=0.44). This result indicated that the consistency of the ball speed between two measuring method is very high, and if the ball speed calculated by picture analysis considers as the actual (or approximate actual) value, the ball speed in speed radar gun measurement has high reliability

Tissue-engineered submillimeter-diameter vascular grafts for free flap survival in rat model

Vascular grafts for free flap transfers should be of very small diameter and remain patent for approximately three weeks to supply blood until the revascularization from the surrounding tissue is established, with the autologous vein grafts acting as the gold standard. Artificial submillimeter-diameter vascular grafts with clinically useful size of 0.6 mm inner diameter and 5 cm length were prepared and evaluated by replacing the axial artery of free flap in rats. The rat tail artery, selected as a novel bioscaffold material, was decellularized using ultrahigh-hydrostatic pressure (UHP) method and compared with the detergent-based conventional method. To induce rapid endothelialization, the graft lumen was modified with synthesized peptides, having high affinity to the endothelial progenitor cells. The UHP method and peptide modification at 37 °C were found to preserve the extracellular matrix structure well, leading to the stable immobilization of the peptide at the luminal surface. These grafts showed the neointima formation, even at the center position far from the native vessels, remained patent for three weeks, and resulted in the flap survival in the rat free-flap model. The tissue-engineered vascular grafts with functionalized lumen have great future potential as an alternative to autologous vein grafts in free flap transfers

REDV-modified decellularized microvascular grafts for arterial and venous reconstruction

Recently, a decellularized microvascular graft (inner diameter: 0.6 mm) modified with the integrin α4β1 ligand, REDV, was developed to provide an alternative to autologous-vein grafting in reconstructive microsurgery, showing good early-stage patency under arterial flow in rats. This consecutive study evaluated its potential utility not only as an arterial substitute, but also as a venous substitute, using a rat-tail replantation model. Graft remodeling depending on hemodynamic status was also investigated. ACI rat tail arteries were decellularized via ultra-high-hydrostatic pressure treatment and modified with REDV to induce antithrombogenic interfaces and promote endothelialization after implantation. Grafts were implanted into the tail artery and vein to re-establish blood circulation in amputated Lewis rat tails (n = 12). The primary endpoint was the survival of replants. Secondary endpoints were graft patency, remodeling, and regeneration for 6 months. In all but three cases with technical errors or postoperative self-mutilation, tails survived without any evidence of ischemia or congestion. Six-month Kaplan–Meier patency was 100% for tail-artery implanted grafts and 62% for tail-vein implanted grafts. At 6 months, the neo-tunica media (thickness: 95.0 μm in tail-artery implanted grafts, 9.3 μm in tail-vein implanted grafts) was regenerated inside the neo-intima. In conclusion, the microvascular grafts functioned well both as arterial and venous paths of replanted-rat tails, with different remodeling under arterial and venous conditions

Complete Cell Killing by Applying High Hydrostatic Pressure for Acellular Vascular Graft Preparation

Pressure treatment has been developed in tissue engineering application. Although the tissue scaffold prepared by a ultrahydrostatic pressure treatment has been reported, an excessive pressure has a potential to disrupt a structure of extracellular matrix through protein denaturation. It is important to understand the suitable low-pressure condition and mechanisms for cell killing. In this study, cellular morphology, mitochondria activity, and membrane permeability of mammalian cells with various pressure treatments were investigated with in vitro models. When the cells were treated with a pressure of 100 MPa for 10 min, cell morphology and adherence were the same as an untreated cells. Dehydrogenase activity in mitochondria was almost the same as untreated cells. On the other hand, when the cells were treated with the pressure of more than 200 MPa, the cells did not adhere, and the dehydrogenase activity was completely suppressed. However, green fluorescence was observed in the live/dead staining images, and the cells were completely stained as red after above 500 MPa. That is, membrane permeability was disturbed with the pressure treatment of above 500 MPa. These results indicated that the pressure of 200 MPa for 10 min was enough to induce cell killing through inactivation of mitochondria activity