Ruchira Singh

Kansas State University, Manhattan, KS, United States

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Publications (5)19.02 Total impact

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    ABSTRACT: The low luminal Ca2+ concentration of mammalian endolymph in the inner ear is required for normal hearing and balance. We recently reported the expression of mRNA for a Ca2+-absorptive transport system in primary cultures of semicircular canal duct (SCCD) epithelium. We now identify this system in native vestibular and cochlear tissues by qRT-PCR, immunoblots and confocal immunolocalization. Transcripts were found and quantified for several isoforms of epithelial calcium channels (TRPV5, TRPV6), calcium buffer proteins (calbindin-D9K, calbindin-D28K), sodium-calcium exchangers (NCX1, NCX2, NCX3) and plasma membrane Ca2+-ATPase (PMCA1, PMCA2, PMCA3, and PMCA4) in native SCCD, cochlear lateral wall (LW) and stria vascularis (SV) of adult rat as well as Ca2+ channels in neonatal SCCD. All components were expressed except TRPV6 in SV and PMCA2 in SCCD. 1,25-(OH)2vitamin D3 (VitD) significantly up-regulated transcripts of TRPV5 in SCCD, calbindin-D9K in SCCD and LW, NCX2 in LW, while PMCA4 in SCCD and PMCA3 in LW were down-regulated. The expression of TRPV5 relative to TRPV6 was in the sequence SV > Neonatal SCCD > Adult SCCD > LW > primary culture SCCD. Expression of TRPV5 protein from primary culture of SCCD did not increase significantly when cells were incubated with VitD (1.2 times control; P > 0.05). Immunolocalization showed the distribution of TRPV5 and TRPV6. TRPV5 was found near the apical membrane of strial marginal cells and both TRPV5 and TRPV6 in outer and inner sulcus cells of the cochlea and in the SCCD of the vestibular system. These findings demonstrate for the first time the expression of a complete Ca2+ absorptive system in native cochlear and vestibular tissues. Regulation by vitamin D remains equivocal since the results support the regulation of this system at the transcript level but evidence for control of the TRPV5 channel protein was lacking.
    BMC Physiology 01/2010; 10:1.
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    ABSTRACT: Mutations of SLC26A4 cause an enlarged vestibular aqueduct, nonsyndromic deafness, and deafness as part of Pendred syndrome. SLC26A4 encodes pendrin, an anion exchanger located in the cochlea, thyroid, and kidney. The goal of the present study was to determine whether developmental delays, possibly mediated by systemic or local hypothyroidism, contribute to the failure to develop hearing in mice lacking Slc26a4 (Slc26a4(-/-)). We evaluated thyroid function by voltage and pH measurements, by array-assisted gene expression analysis, and by determination of plasma thyroxine levels. Cochlear development was evaluated for signs of hypothyroidism by microscopy, in situ hybridization, and quantitative RT-PCR. No differences in plasma thyroxine levels were found in Slc26a4(-/-) and sex-matched Slc26a4(+/-) littermates between postnatal day 5 (P5) and P90. In adult Slc26a4(-/-) mice, the transepithelial potential and the pH of thyroid follicles were reduced. No differences in the expression of genes that participate in thyroid hormone synthesis or ion transport were observed at P15, when plasma thyroxine levels peaked. Scala media of the cochlea was 10-fold enlarged, bulging into and thereby displacing fibrocytes, which express Dio2 to generate a cochlear thyroid hormone peak at P7. Cochlear development, including tunnel opening, arrival of efferent innervation at outer hair cells, endochondral and intramembraneous ossification, and developmental changes in the expression of Dio2, Dio3, and Tectb were delayed by 1-4 days. These data suggest that pendrin functions as a HCO3- transporter in the thyroid, that Slc26a4(-/-) mice are systemically euthyroid, and that delays in cochlear development, possibly due to local hypothyroidism, lead to the failure to develop hearing.
    AJP Renal Physiology 09/2009; 297(5):F1435-47. · 4.42 Impact Factor
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    Ruchira Singh, Philine Wangemann
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    ABSTRACT: Pendred syndrome is due to loss-of-function mutations of Slc26a4, which codes for the HCO(3)(-) transporter pendrin. Loss of pendrin causes deafness via a loss of the K(+) channel Kcnj10 in stria vascularis and consequent loss of the endocochlear potential. Pendrin and Kcnj10 are expressed in different cell types. Here, we report that free radical stress provides a link between the loss of Kcnj10 and the loss of pendrin. Studies were performed using native and cultured stria vascularis from Slc26a4(+/-) and Slc26a4(-/-) mice as well as Chinese hamster ovary (CHO)-K1 cells. Kcnj10, oxidized proteins, and proteins involved in iron metabolism were quantified by Western blotting. Nitrated proteins were quantified by ELISA. Total iron was measured by ferrozine spectrophotometry and gene expression was quantified by qRT-PCR. At postnatal day 10 (P10), stria vascularis from Slc26a4(+/-) and Slc26a4(-/-) mice expressed similar amounts of Kcnj10. Slc26a4(-/-) mice lost Kcnj10 expression during the next 5 days of development. In contrast, stria vascularis, obtained from P10 Slc26a4(-/-) mice and kept in culture for 5 days, maintained Kcnj10 expression. Stria vascularis from Slc26a4(-/-) mice was found to suffer from free radical stress evident by elevated amounts of oxidized and nitrated proteins and other changes in protein and gene expression. Free radical stress induced by 3-morpholinosydnonimine-N-ethylcarbamide was found to be sufficient to reduce Kcnj10 expression in CHO-K1 cells. These data demonstrate that free radical stress provides a link between loss of pendrin and loss of Kcnj10 in Slc26a4(-/-) mice and possibly in human patients suffering from Pendred syndrome.
    American journal of physiology. Renal physiology 02/2008; 294(1):F139-48. · 3.61 Impact Factor
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    ABSTRACT: Pendred syndrome, an autosomal-recessive disorder characterized by deafness and goiter, is caused by a mutation of SLC26A4, which codes for the anion exchanger pendrin. We investigated the relationship between pendrin expression and deafness using mice that have (Slc26a4+/+ or Slc26a4+/-) or lack (Slc26a4-/-) a complete Slc26a4 gene. Previously, we reported that stria vascularis of adult Slc26a4-/- mice is hyperpigmented and that marginal cells appear disorganized. Here we determine the time course of hyperpigmentation and marginal cell disorganization, and test the hypothesis that inflammation contributes to this tissue degeneration. Slc26a4-/- and age-matched control (Slc26a4+/+ or Slc26a4+/-) mice were studied at four postnatal (P) developmental stages: before and after the age that marks the onset of hearing (P10 and P15, respectively), after weaning (P28-41) and adult (P74-170). Degeneration and hyperpigmentation stria vascularis was evaluated by confocal microscopy. Gene expression in stria vascularis was analyzed by microarray and quantitative RT-PCR. In addition, the expression of a select group of genes was quantified in spiral ligament, spleen and liver to evaluate whether expression changes seen in stria vascularis are specific for stria vascularis or systemic in nature. Degeneration of stria vascularis defined as hyperpigmentation and marginal cells disorganization was not seen at P10 or P15, but occurred after weaning and was associated with staining for CD68, a marker for macrophages. Marginal cells in Slc26a4-/-, however, had a larger apical surface area at P10 and P15. No difference in the expression of Lyzs, C3 and Cd45 was found in stria vascularis of P15 Slc26a4+/- and Slc26a4-/- mice. However, differences in expression were found after weaning and in adult mice. No difference in the expression of markers for acute inflammation, including Il1a, Il6, Il12a, Nos2 and Nos3 were found at P15, after weaning or in adults. The expression of macrophage markers including Ptprc (= Cd45), Cd68, Cd83, Lyzs, Lgals3 (= Mac2 antigen), Msr2, Cathepsins B, S, and K (Ctsb, Ctss, Ctsk) and complement components C1r, C3 and C4 was significantly increased in stria vascularis of adult Slc26a4-/- mice compared to Slc26a4+/+ mice. Expression of macrophage markers Cd45 and Cd84 and complement components C1r and C3 was increased in stria vascularis but not in spiral ligament, liver or spleen of Slc26a4-/- compared to Slc26a4+/- mice. The expression of Lyzs was increased in stria vascularis and spiral ligament but not in liver or spleen. The data demonstrate that hyperpigmentation of stria vascularis and marginal cell reorganization in Slc26a4-/- mice occur after weaning, coinciding with an invasion of macrophages. The data suggest that macrophage invasion contributes to tissue degeneration in stria vascularis, and that macrophage invasion is restricted to stria vascularis and is not systemic in nature. The delayed onset of degeneration of stria vascularis suggests that a window of opportunity exists to restore/preserve hearing in mice and therefore possibly in humans suffering from Pendred syndrome.
    BMC Medicine 02/2006; 4:37. · 7.28 Impact Factor
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    ABSTRACT: Gene expression profiling using microarrays requires microgram amounts of RNA, which limits its direct application for the study of nanogram RNA samples obtained using microdissection, laser capture microscopy, or needle biopsy. A novel system based on Ribo-SPIA technology (RS, Ovation-Biotin amplification and labeling system) was recently introduced. The utility of the RS system, an optimized prototype system for picogram RNA samples (pRS), and two T7-based systems involving one or two rounds of amplification (One RA, Standard Protocol, or Two RA, Small Sample Prototcol, version II) were evaluated in the present study. Mouse kidney (MK) and mouse universal reference (MUR) RNA samples, 0.3 ng to 10 mug, were analyzed using high-density Affymetrix Mouse Genome 430 2.0 GeneChip arrays. Call concordance between replicates, correlations of signal intensity, signal intensity ratios, and minimal fold increase necessary for significance were determined. All systems amplified partially overlapping sets of genes with similar signal intensity correlations. pRS amplified the highest number of genes from 10-ng RNA samples. We detected 24 of 26 genes verified by RT-PCR in samples prepared using pRS. Two RA yielded somewhat higher call concordances than did RS and pRS (91.8% vs. 89.3% and 88.1%, respectively). Although all target preparation methods were suitable, pRS amplified the highest number of targets and was found to be suitable for amplification of as little as 0.3 ng of total RNA. In addition, RS and pRS were faster and simpler to use than the T7-based methods and resulted in the generation of cDNA, which is more stable than cRNA.
    AJP Cell Physiology 06/2005; 288(5):C1179-89. · 3.71 Impact Factor