Katsumasa Kawahara

Human hair follicle pluripotent stem (hfPS) cells promote regeneration of peripheral‐nerve injury: An advantageous alternative to ES and iPS cells

The optimal source of stem cells for regenerative medicine is a major question. Embryonic stem (ES) cells have shown promise for pluripotency but have ethical issues and potential to form teratomas. Pluripotent stem cells have been produced from skin cells by either viral‐, plasmid‐ or transposon‐mediated gene transfer. These stem cells have been termed induced pluripotent stem cells or iPS cells. iPS cells may also have malignant potential and are inefficiently produced. Embryonic stem cells may not be suited for individualized therapy, since they can undergo immunologic rejection. To address these fundamental problems, our group is developing hair follicle pluripotent stem (hfPS) cells. Our previous studies have shown that mouse hfPS cells can differentiate to neurons, glial cells in vitro, and other cell types, and can promote nerve and spinal cord regeneration in vivo. hfPS cells are located above the hair follicle bulge in what we have termed the hfPS cell area (hfPSA) and are nestin positive and keratin 15 (K‐15) negative. Human hfPS cells can also differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In the present study, human hfPS cells were transplanted in the severed sciatic nerve of the mouse where they differentiated into glial fibrillary‐acidic‐protein (GFAP)‐positive Schwann cells and promoted the recovery of pre‐existing axons, leading to nerve generation. The regenerated nerve recovered function and, upon electrical stimulation, contracted the gastrocnemius muscle. The hfPS cells can be readily isolated from the human scalp, thereby providing an accessible, autologous and safe source of stem cells for regenerative medicine that have important advantages over ES or iPS cells. J. Cell. Biochem. 107: 1016–1020, 2009.

Mutational and functional analysis of SLC4A4 in a patient with proximal renal tubular acidosis

Permanent isolated proximal renal tubular acidosis (pRTA) with ocular abnormalities is a systemic disease with isolated pRTA, short stature and ocular abnormalities. We identified a novel homozygous deletion of nucleotide 2,311 adenine in the kidney type Na+/HCO3− cotransporter (kNBC1) cDNA in a patient with permanent isolated pRTA. This mutation is predicted to result in a frame shift at codon 721 forming a stop codon after 29 amino acids anomalously transcribed from the SLC4A4 gene. Cosegregation of this mutation with the disease was supported by heterozygosity in the parents of the affected patient. The absence of this mutation in 156 alleles of 78 normal individuals indicates that this mutation is related to the disease and is not a common DNA sequence polymorphism. When injected into Xenopus oocytes, the mutant cRNA failed to induce electrogenic transport activity. In addition, immunofluorescence and Western blot analysis failed to detect the expression of the full-length protein in mutant-injected oocytes. Our results expand the spectrum of kNBC1 mutations in permanent isolated pRTA with ocular abnormalities and increase our understanding of the renal tubular mechanism that is essential for acid-base homeostasis.

Localization of the ATP-sensitive potassium channel subunit (Kir6.1/uKATP-1) in rat brain

The Kir6.1/uK(ATP)-1, subunit of ATP-sensitive K(+) channels (K(ATP)), was localized in adult rat brain by in situ hybridization and immunohistochemistry. The mRNA of this subunit was ubiquitously expressed in various neurons and nuclei of the adult rat brain. Interestingly, Kir6.1/uK(ATP)-1 mRNA was also expressed in glial cells. Kir6.1/uK(ATP)-1 protein staining gave a dispersed array of fine dots throughout all neurons and glial cells examined. Under electron microscope, the immunoreactive products were specifically restricted to the mitochondria. The present study indicates that this K(ATP) subunit is localized in the mitochondria and may play a fundamental role in vital brain function.

Human and mouse hair follicles contain both multipotent and monopotent stem cells

No abstract.

Localization of pore-forming subunit of the ATP-sensitive K(+)-channel, Kir6.2, in rat brain neurons and glial cells.

Kir6.2, a subunit of the ATP-sensitive K(+) channel (K(ATP)), was localized in adult rat brain by immunohistochemistry and in situ hybridization. The Kir6.2 mRNA was widely expressed in most rat brain neuronal populations and nuclei examined, intensely in the mitral cell layer and tufted cells of the olfactory bulb, pontine nucleus, pontine reticular nucleus, motor and spinal trigeminal nuclei and cuneate nuclei of the brain stem, moderately in the neocortex and cerebellar Purkinje cells, and weakly in the granular cell layer of the olfactory bulb and the granular layer of the cerebellum. In addition, glial cells also expressed the Kir6.2 gene weakly in the corpus callosum and cerebellar white matter. This wide localization of the gene was quite similar to that of Kir6.2 protein. Double stainings with anti-GFAP and anti-Kir6.2 antibodies were performed in this study. Glial cells showing immunoreactivity to both anti-Kir6.2 and anti-GFAP were confirmed to be astrocytes, and those showing only immunoreactivity to anti-Kir6.2 but not to anti-GFAP were presumed to be oligodendrocytes and confirmed by immunoelectron microscopy. Thus, it may be concluded that both oligodendrocytes and astrocytes contain Kir6.2. Under the electron microscope, we showed in vivo for the first time that the immunoreactive products were localized in the endoplasmic reticulum and Golgi apparatus as well as the plasma membranes of neurons and glial cells.

Vasopressin regulates the renin-angiotensin-aldosterone system via V1a receptors in macula densa cells

The neuropeptide hormone arginine-vasopressin (AVP) is well known to exert its antidiuretic effect via the vasopressin V2 receptor (V2R), whereas the role of the vasopressin V1a receptor (V1aR) in the kidney remains to be clarified. Previously, we reported decreased plasma volume and blood pressure in V1a receptor-deficient (V1aR-/-) mice (Koshimizu T, Nasa Y, Tanoue A, Oikawa R, Kawahara Y, Kiyono Y, Adachi T, Tanaka T, Kuwaki T, Mori T. Proc Natl Acad Sci USA 103: 7807-7812, 2006). In this study, we investigated the role of V1aR in urine concentration, renal function, and the renin-angiotensin system (RAS) using V1aR-/- mice. Urine volume of V1aR-/- mice was greater than that of wild-type mice, particularly when water was loaded, while the glomerular filtration rate (GFR), urinary NaCl excretion, AVP-dependent cAMP generation, V2R, and aquaporin 2 (AQP2) expression in the kidney were lower, indicating that the diminished GFR and V2R-AQP2 system led to impaired urinary concentration in V1aR-/- mice. Since the GFR and V2R-AQP2 system are regulated by RAS, we analyzed renin and angiotensin II in V1aR-/- mice and found that the plasma renin and angiotensin II were decreased. The expression of renin in granule cells was decreased in V1aR-/- mice, which led to a decreased level of plasma renin. In addition, the expression of renin stimulators such as neuronal nitric oxide synthase and cyclooxygenase-2 in macula densa (MD) cells, where V1aR was specifically expressed, was decreased in V1aR-/- mice. These data indicate that AVP regulates body fluid homeostasis and GFR via the V1aR in MD cells by activating RAS and subsequently the V2R-AQP2 system.

ATP-sensitive K+ -channel Subunits on the Mitochondria and Endoplasmic Reticulum of Rat Cardiomyocytes

ATP-sensitive K+ (KATP) channel subunits on the subcellular structures of rat cardiomyocytes were studied with antibodies against Kir6.1 and Kir6.2. According to the results of Western blot analysis, Kir6.1 was strongly expressed in mitochondrial and microsome fractions, and faintly expressed in cell membrane fraction, whereas Kir6.2 was mainly expressed in the microsome fraction and weakly in cell membrane and mitochondrial fractions. Immunohistochemistry showed that Kir6.1 and Kir6.2 were expressed in the endocardium, atrial and ventricular myocardium, and in vascular smooth muscles. Immunoelectron microscopy revealed that Kir6.1 immunoreactivity was mainly localized in the mitochondria, whereas Kir6.2 immunoreactivity was mainly localized in the endoplasmic reticulum and a few in the mitochondria. Both Kir6.1 and Kir6.2 are candidates of mitochondrial KATP channel subunits. The data obtained in this study will be useful for analyzing the composition of KATP channels of cardiomyocytes and help to understanding the cardioprotective role of KATP channels during heart ischemia.

Water May Cure Patients With Meniere Disease

Objectives/Hypothesis: We examined whether sufficient water intake is effective in the long‐term control of vertigo and hearing activity in patients with Meniere disease (MD) for whom conventional therapy has proven unsuccessful.

From hair to heart: nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells differentiate to beating cardiac muscle cells

We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair follicle stem cells located in the bulge area which are termed hair-follicle-associated pluripotent (HAP) stem cells. HAP stem cells from mouse and human could form spheres in culture, termed hair spheres, which are keratin 15-negative and CD34-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Subsequently, we demonstrated that nestin-expressing stem cells could effect nerve and spinal cord regeneration in mouse models. In the present study, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. We separated the mouse vibrissa hair follicle into 3 parts (upper, middle, and lower), and suspended each part separately in DMEM containing 10% FBS. All three parts of hair follicle differentiated to beating cardiac muscle cells as well as neurons, glial cells, keratinocytes and smooth muscle cells. The differentiation potential to cardiac muscle is greatest in the upper part of the follicle. The beat rate of the cardiac muscle cells was stimulated by isoproterenol and inhibited by propanolol. HAP stem cells have potential for regenerative medicine for heart disease as well as nerve and spinal cord repair.

Aldosterone Requires Vasopressin V1a Receptors on Intercalated Cells to Mediate Acid-Base Homeostasis

Both aldosterone and luminal vasopressin may contribute to the maintenance of acid-base homeostasis, but the functional relationship between these hormones is not well understood. The effects of luminal vasopressin likely result from its interaction with V1a receptors on the luminal membranes of intercalated cells in the collecting duct. Here, we found that mice lacking the V1a receptor exhibit type 4 renal tubular acidosis. The administration of the mineralocorticoid agonist fludrocortisone ameliorated the acidosis by restoring excretion of urinary ammonium via increased expression of Rhcg and H-K-ATPase and decreased expression of H-ATPase. In a cell line of intercalated cells established from transgenic rats expressing the mineralocorticoid and V1a receptors, but not V2 receptors, knockdown of the V1a receptor gene abrogated the effects of aldosterone on H-K-ATPase, Rhcg, and H-ATPase expression. These data suggest that defects in the vasopressin V1a receptor in intercalated cells can cause type 4 renal tubular acidosis and that the tubular effects of aldosterone depend on a functional V1a receptor in the intercalated cells.