scholarly journals Mouse β6 Integrin Sequence, Pattern of Expression, and Role in Kidney Development

2000 ◽  
Vol 11 (12) ◽  
pp. 2297-2305
Author(s):  
LOIS J. AREND ◽  
ANN M. SMART ◽  
JOSIE P. BRIGGS
Keyword(s):  
Mrna Expression ◽  
Reverse Transcription ◽  
Kidney Development ◽  
Mouse Kidney ◽  
Thick Ascending Limb ◽  
Integrin Expression ◽  
Coding Region ◽  
Embryonic Mouse ◽  
Nephron Segments ◽  
Reverse Transcription Pcr

Abstract. Integrins mediate cell-cell and cell-extracellular matrix interactions and play key roles in development. β6 integrin expression has been demonstrated in human fetal kidney at a higher level than in the adult, making β6 integrin a marker of interest for the study of development of the nephron. The aims of this study were to determine the cDNA sequence for the mouse β6 integrin and to characterize β6 integrin expression in the developing mouse kidney. Two embryonic mouse kidney cDNA libraries were screened, and the coding region was sequenced. The mouse β6 nucleotide coding region sequence shows 82% nucleotide identity to the human sequence. The putative amino acid sequence has 89.5% identity to human β6 integrin and contains many conserved domains. By reverse transcription-PCR, β6 integrin mRNA expression is very low at 11 d of gestation in the mouse, increases dramatically by E14 and E17 (20-fold, normalized for increases in β actin), and plateaus by 2 wk of age. β6 integrin expression is induced 15- to 20-fold after 5 d in metanephric explant culture. Reverse transcription-PCR of adult rat microdissected nephron segments demonstrates β6 integrin mRNA expression in proximal tubule, cortical thick ascending limb, distal nephron segments (inner and outer medullary collecting ducts), and macula densa—containing segments. Lectin-peroxidase and in situ colocalization studies demonstrated expression of β6 integrin mRNA in developing proximal tubules and thick ascending limb. Culture of mouse metanephric kidneys with antisense oligonucleotides to β6 integrin resulted in inhibition of ureteric bud branching and complete lack of mesenchyme condensation. These studies demonstrate a high homology between the human and mouse β6 integrin sequence, a different pattern of expression in the developing mouse kidney compared with the primate kidney, and abnormal metanephric development in culture in the absence of β6 integrin. These findings suggest an important role for β6 integrin in normal development of the mouse kidney.

Intrarenal and Cellular Localization of CLC-K2 Protein in the Mouse Kidney

2001 ◽  
Vol 12 (7) ◽  
pp. 1327-1334 ◽  
Author(s):  
KATSUKI KOBAYASHI ◽  
SHINICHI UCHIDA ◽  
SHUKI MIZUTANI ◽  
SEI SASAKI ◽  
FUMIAKI MARUMO
Keyword(s):  
Cellular Localization ◽  
Collecting Duct ◽  
Type A ◽  
Mouse Kidney ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Basolateral Membranes ◽  
Nephron Segments ◽  
Henle's Loop ◽  
Distal Tubules

Abstract. CLC-K2, a kidney-specific member of the CLC chloride channel family, is thought to play an important role in the transepithelial Cl- transport in the kidney. This consensus was first reached shortly after it was demonstrated that the mutations of the human CLCNKB gene resulted in Bartter's syndrome type III. To clarify the pathogenesis, the exact intrarenal and cellular localization of CLC-K2 by immunohistochemistry of the Clcnk1-/- mouse kidney were investigated by use of an anti-CLC-K antibody that recognized both CLC-K1 and CLC-K2. CLC-K2 is expressed in the thick ascending limb of Henle's loop and distal tubules, where it is localized to the basolateral membranes. The localization of CLC-K2 to these nephron segments strongly implies that CLC-K2 confers the basolateral chloride conductance in the thick ascending limb of Henle's loop and distal tubules, where Cl- is taken up by the bumetanide-sensitive Na-K-2Cl cotransporter or the thiazide-sensitive Na-Cl cotransporter at the apical membranes. CLC-K2 expression was also shown to extend into the connecting tubule in the basolateral membrane. CLC-K2 was found in basolateral membranes of the type A intercalated cells residing along the collecting duct. This localization strongly suggests that CLC-K2 confers the basolateral conductance in the type A intercalated cells where Cl- is taken up by the anion exchanger in exchange for HCO3- at the basolateral membranes. These aspects of CLC-K2 localization suggest that CLC-K2 is important in Cl- transport in the distal nephron segments.

scholarly journals Atlas of gene expression in the mouse kidney: new features of glomerular parietal cells

2011 ◽  
Vol 43 (3) ◽  
pp. 161-173 ◽  
Author(s):  
Lydie Cheval ◽  
Fabien Pierrat ◽  
Carole Dossat ◽  
Mathieu Genete ◽  
Martine Imbert-Teboul ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Kidney ◽  
Thick Ascending Limb ◽  
Proliferating Cells ◽  
Quantitative Expression ◽  
Nephron Segments ◽  
Nephron Segment ◽  
Mrna Markers ◽  
Insight Into

To gain molecular insight into kidney function, we performed a high-resolution quantitative analysis of gene expression in glomeruli and nine different nephron segments dissected from mouse kidney using Serial Analysis of Gene Expression (SAGE). We also developed dedicated bioinformatics tools and databases to annotate mRNA tags as transcripts. Over 800,000 mRNA SAGE tags were sequenced corresponding to >20,000 different mRNA tags present at least twice in at least one library. Hierarchical clustering analysis of tags demonstrated similarities between the three anatomical subsegments of the proximal tubule, between the cortical and medullary segments of the thick ascending limb of Henle's loop, and between the three segments constituting the aldosterone-sensitive distal nephron segments, whereas the glomerulus and distal convoluted tubule clusterized independently. We also identified highly specific mRNA markers of each subgroup of nephron segments and of most nephron segments. Tag annotation also identified numbers of putative antisense mRNAs. This database constitutes a reference resource in which the quantitative expression of a given gene can be compared with that of other genes in the same nephron segment, or between different segments of the nephron. To illustrate possible applications of this database, we performed a deeper analysis of the glomerulus transcriptome that unexpectedly revealed expression of several ion and water carriers; within the glomerulus, they were found to be preferentially expressed in the parietal sheet. It also revealed the major role of the zinc finger transcription factor Wt1 in the specificity of gene expression in the glomerulus. Finally, functional annotation of glomerulus-specific transcripts suggested a high proliferation activity of glomerular cells. Immunolabeling for PCNA confirmed a high percentage of proliferating cells in the glomerulus parietal sheet.

scholarly journals Development and Evaluation of a Real-Time Reverse Transcription-PCR Assay for Quantification of Gamma Interferon mRNA To Diagnose Tuberculosis in Multiple Animal Species

2007 ◽  
Vol 14 (12) ◽  
pp. 1563-1571 ◽  
Author(s):  
Noel P. Harrington ◽  
Om P. Surujballi ◽  
W. Ray Waters ◽  
John F. Prescott
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Reverse Transcription ◽  
Skin Testing ◽  
Gamma Interferon ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sequence Information ◽  
Tuberculin Skin Testing ◽  
Reverse Transcription Pcr ◽  
Ifn Γ

ABSTRACT Tuberculosis of free-ranging and captive wildlife, including species implicated in the maintenance and transmission of Mycobacterium bovis, is a difficult disease to diagnose and control. Historically, diagnosis of tuberculosis has relied largely upon assays of cell-mediated immunity (CMI), such as tuberculin skin testing. This approach, however, is problematic or impractical for use with many wildlife species. Increasingly, in vitro diagnostic tests, including gamma interferon (IFN-γ)-based assays, are replacing or complementing skin testing of cattle and humans. Analogous assays are unavailable for most wildlife because of a lack of species-specific immunological reagents. This report describes the development and validation of a whole-blood assay to quantify antigen-specific IFN-γ mRNA expression by quantitative real-time reverse transcription-PCR. Oligonucleotide primers and probes were designed and tested for reactivity towards several susceptible species of interest with respect to tuberculosis infection. The assay was subsequently optimized to quantify the IFN-γ mRNA expression in elk and red deer (Cervus elaphus) and was evaluated for its ability to detect mycobacterial antigen-specific responses of experimentally tuberculosis-infected animals. The assay was a simple, rapid, and sensitive measure of antigen-specific CMI. The IFN-γ mRNA responses correlated well with IFN-γ protein production and showed performance in determining an animal's infection status superior to that of either lymphocyte proliferation or IFN-γ protein enzyme-linked immunosorbent assay methods. An additional advantage is the ease with which the assay can be modified to reliably quantify IFN-γ expression by using consensus sequences of closely related species or of other species for which IFN-γ sequence information is available.

Identification and localization of BK-β subunits in the distal nephron of the mouse kidney

2007 ◽  
Vol 293 (1) ◽  
pp. F350-F359 ◽  
Author(s):  
P. Richard Grimm ◽  
Ruth M. Foutz ◽  
Robert Brenner ◽  
Steven C. Sansom
Keyword(s):  
Immunohistochemical Staining ◽  
Bk Channels ◽  
Mouse Kidney ◽  
Thick Ascending Limb ◽  
Distal Nephron ◽  
Rt Pcr ◽  
Western Blots ◽  
Nephron Segments ◽  
High K ◽  
Specific Localization

Large-conductance, Ca2+-activated K+ channels (BK), comprised of pore-forming α- and accessory β-subunits, secrete K+ in the distal nephron under high-flow and high-K+ diet conditions. BK channels are detected by electrophysiology in many nephron segments; however, the accessory β-subunit associated with these channels has not been determined. We performed RT-PCR, Western blotting, and immunohistochemical staining to determine whether BK-β1 is localized to the connecting tubule's principal-like cells (CNT) or intercalated cells (ICs), and whether BK-β2-4 are present in other distal nephron segments. RT-PCR and Western blots revealed that the mouse kidney expresses BK-β1, BK-β2, and BK-β4. Available antibodies in conjunction with BK-β1−/− and BK-β4−/− mice allowed the specific localization of BK-β1 and BK-β4 in distal nephron segments. Immunohistochemical staining showed that BK-β1 is localized in the CNT but not ICs of the connecting tubule. The localization of BK-β4 was discerned using an anti-BK-β4 antibody on wild-type tissue and anti-GFP on GFP-replaced BK-β4 mouse (BK-β4−/−) tissue. Both antibodies (anti-BK-β4 and anti-GFP) localized BK-β4 to the thick ascending limb (TAL), distal convoluted tubule (DCT), and ICs of the distal nephron. It is concluded that BK-β1 is narrowly confined to the apical membrane of CNTs in the mouse, whereas BK-β4 is expressed in the TAL, DCT, and ICs.

scholarly journals β-catenin regulates the formation of multiple nephron segments in the mouse kidney

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Patrick Deacon ◽  
Charles W. Concodora ◽  
Eunah Chung ◽  
Joo-Seop Park
Keyword(s):  
Kidney Development ◽  
Critical Role ◽  
Mouse Kidney ◽  
Proximal Tubules ◽  
Stable Form ◽  
Loss Of Function ◽  
Nephron Segments ◽  
Mesenchymal To Epithelial Transition ◽  
Nephron Progenitors ◽  
Nephron Segmentation

Abstract The nephron is composed of distinct segments that perform unique physiological functions. Little is known about how multipotent nephron progenitor cells differentiate into different nephron segments. It is well known that β-catenin signaling regulates the maintenance and commitment of mesenchymal nephron progenitors during kidney development. However, it is not fully understood how it regulates nephron segmentation after nephron progenitors undergo mesenchymal-to-epithelial transition. To address this, we performed β-catenin loss-of-function and gain-of-function studies in epithelial nephron progenitors in the mouse kidney. Consistent with a previous report, the formation of the renal corpuscle was defective in the absence of β-catenin. Interestingly, we found that epithelial nephron progenitors lacking β-catenin were able to form presumptive proximal tubules but that they failed to further develop into differentiated proximal tubules, suggesting that β-catenin signaling plays a critical role in proximal tubule development. We also found that epithelial nephron progenitors lacking β-catenin failed to form the distal tubules. Expression of a stable form of β-catenin in epithelial nephron progenitors blocked the proper formation of all nephron segments, suggesting tight regulation of β-catenin signaling during nephron segmentation. This work shows that β-catenin regulates the formation of multiple nephron segments along the proximo-distal axis of the mammalian nephron.

scholarly journals Membrane Adsorption with Direct Cell Culture Combined with Reverse Transcription-PCR as a Fast Method for Identifying Enteroviruses from Sewage

2005 ◽  
Vol 71 (1) ◽  
pp. 72-79 ◽  
Author(s):  
D. Papaventsis ◽  
N. Siafakas ◽  
P. Markoulatos ◽  
G. T. Papageorgiou ◽  
C. Kourtis ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Sewage Treatment ◽  
Rflp Analysis ◽  
Fast Method ◽  
Coding Region ◽  
Protein Coding ◽  
Membrane Filters ◽  
Reverse Transcription Pcr ◽  
Detection And Identification ◽  
Study Sites

ABSTRACT We present a new approach for the detection and identification of enteroviruses concentrated and isolated from sewage. Samples were collected from two study sites located at Nicosia and Limassol sewage treatment plants in Cyprus. Viruses were adsorbed to cellulose nitrate membrane filters, cultured directly from the membrane filters by using the VIRADEN method, and identified by reverse transcription-PCR, followed by 5′ untranslated region (5′-UTR) restriction fragment length polymorphism (RFLP) analysis and partial sequencing of the VP1 protein coding region. Initial subgrouping based on the HpaII restriction profile showed that all of the isolates except one belonged to the same genetic subcluster. Partial VP1 sequencing revealed that most isolates belonged to serotypes coxsackie B4 (42.5%) and coxsackie Α9 (30%), whereas coxsackie B2 (17.5%) and coxsackie B1 (3%) isolates were less frequently observed. One poliovirus type 2 isolate (2.5%) of vaccine origin was also found. The HpaII digests predicted the genetic subcluster for all isolates. They also accurately differentiated the isolates as nonpolio or polio isolates. This approach seems to be very promising for environmental surveillance of enterovirus circulation and epidemiology, with all of the significant effects that this entails for public health. Partial VP1 sequencing is efficient for molecular serotyping of enteroviruses, while 5′-UTR RFLP analysis with HpaII can also be considered an asset for the initial subclassification of enterovirus isolates.

scholarly journals Nephron-specific expression of components of the renin–angiotensin–aldosterone system in the mouse kidney

2012 ◽  
Vol 13 (1) ◽  
pp. 46-55 ◽  
Author(s):  
Stephan W Reinhold ◽  
Bernd Krüger ◽  
Caroline Barner ◽  
Flavius Zoicas ◽  
Martin C Kammerl ◽  
...  
Keyword(s):  
Mouse Kidney ◽  
Proximal Tubules ◽  
Thick Ascending Limb ◽  
Specific Expression ◽  
Renin Angiotensin Aldosterone System ◽  
Renin Angiotensin ◽  
Nephron Segments ◽  
Specific Distribution ◽  
Convoluted Tubules ◽  
Proximal Convoluted Tubules

Introduction: The renin–angiotensin–aldosterone system (RAAS) plays an integral role in the regulation of blood pressure, electrolyte and fluid homeostasis in mammals. The capability of the different nephron segments to form components of the RAAS is only partially known. This study therefore aimed to characterize the nephron-specific expression of RAAS components within the mouse kidney. Materials and methods: Defined nephron segments of adult C57B/16 mice were microdissected after collagenase digestion. The gene expression of renin, angiotensinogen (AGT), angiotensin-converting enzyme (ACE), angiotensin II receptors 1a (AT1a), 1b (AT1b), and 2 (AT2) was assessed by reverse transcriptase polymerase chain reaction (RT-PCR). Results: Renin mRNA was present in glomeruli, in proximal tubules, in distal convoluted tubules (DCT) and cortical collecting ducts (CCD). AGT mRNA was found in proximal tubules, descending thin limb of Henle’s loop (dTL) and in the medullary part of the thick ascending limb (mTAL). ACE mRNA was not detectable in microdissected mouse nephron segments. AT1a, AT1b and AT2 mRNA was detected in glomeruli and proximal convoluted tubules. Conclusions: Our data demonstrate a nephron-specific distribution of RAAS components. All components of the local RAAS – except ACE – are present in proximal convoluted tubules, emphasizing their involvement in sodium and water handling.

scholarly journals Intracellular Calcium Concentration in the Inositol Trisphosphate Receptor Type 1 Knockout Mouse

1999 ◽  
Vol 10 (10) ◽  
pp. 2094-2101
Author(s):  
MATSUHIKO HAYASHI ◽  
TOSHIAKI MONKAWA ◽  
TADASHI YOSHIDA ◽  
HIROYUKI SASAMURA ◽  
MINEO MATSUMOTO ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Reverse Transcription ◽  
Calcium Concentration ◽  
Hormone Receptors ◽  
Receptor Type ◽  
Wild Type ◽  
Ip3 Receptor ◽  
Reverse Transcription Pcr ◽  
Receptor Isoforms

Abstract. Recently, mice with a disrupted inositol trisphosphate (IP3) receptor type 1 allele were produced by gene targeting. To examine the role of IP3 receptor type 1 in the regulation of intracellular calcium concentration ([Ca2+]i) of glomerular cells, [Ca2+]i was measured with fura 2-acetoxymethyl-ester in the superfused glomeruli from homozygous and wild-type mice. [Ca2+]i was determined in calcium-free medium before and after the addition of 10-7 M endothelin-1 (ET-1) and 10-6 M angiotensin II (AngII). The expression of mRNA of IP3 receptor isoforms and hormone receptors in the glomeruli from these animals also was measured by quantitative reverse transcription-PCR with specific primers for IP3 receptor isoforms (types 1, 2, and 3), AngII receptor type 1, and ET receptors (types A and B). In homozygous mutants, the shorter mRNA of IP3 receptor type 1, which lacks the first exon, is transcribed. Basal [Ca2+]i and the responses to ET-1 and AngII in homozygous mutants (ET-1, 55 ± 7 nM to 73 ± 7 nM; AngII, 66 ± 6 to 91 ± 8 nM) were significantly lower than those in the wild-type mice (ET-1, 93 ± 13 nM to 162 ± 13 nM; AngII, 87 ± 7 to 147 ± 9 nM; P < 0.05 for both hormones) without significant changes in mRNA expression of hormone receptors. The results with quantitative reverse transcription-PCR also revealed that mRNA expression of the IP3 receptor gene family was not significantly different between the two groups. The present study clearly shows that IP3 receptor type 1 plays a major role in the regulation of [Ca2+]i in the glomeruli and that lack of an isoform of IP3 receptor in the glomeruli does not induce expression of the other isoforms of the IP3 receptor.

Sign in / Sign up

Export Citation Format

Share Document