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

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.

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Claudins 6, 9, and 13 are developmentally expressed renal tight junction proteins

AJP Renal Physiology ◽

10.1152/ajprenal.00063.2006 ◽

2006 ◽

Vol 291 (6) ◽

pp. F1132-F1141 ◽

Cited By ~ 80

Author(s):

Ghazala Abuazza ◽

Amy Becker ◽

Scott S. Williams ◽

Sumana Chakravarty ◽

Hoang-Trang Truong ◽

...

Keyword(s):

Proximal Tubule ◽

Real Time ◽

Adult Mouse ◽

Mouse Kidney ◽

Proximal Tubules ◽

Paracellular Pathway ◽

Rt Pcr ◽

Adult Kidney ◽

Convoluted Tubules ◽

Proximal Convoluted Tubules

The adult proximal tubule is a low-resistance epithelium where there are high rates of both active transcellular and passive paracellular NaCl transport. We have previously demonstrated that the neonatal rabbit and rat proximal tubule have substantively different passive paracellular transport properties than the adult proximal tubule, which results in a maturational change in the paracellular passive flux of ions. Neonatal proximal tubules have a higher PNa/PCl ratio and lower chloride and bicarbonate permeabilities than adult proximal tubules. Claudins are a large family of proteins which are the gate keepers of the paracellular pathway, and claudin isoform expression determines the permeability characteristics of the paracellular pathway. Previous studies have shown that claudins 1, 2, 3, 4, 5, 7, 8, 10, 11, 12, 15, and 16 are expressed in the adult mouse kidney. To determine whether there are developmental claudin isoforms, we compared the claudin isoforms present in the neonatal and adult kidney using RT-PCR to detect mRNA of claudin isoforms. Claudin 6, claudin 9, and claudin 13 were either not expressed or barely detectable in the adult mouse kidney using traditional PCR, but were expressed in the neonatal mouse kidney. Using real-time RT-PCR, we were able to detect a low level of claudin 6 mRNA expression in the adult kidney compared with the neonate, but claudin 9 and claudin 13 were only detected in the neonatal kidney. There was the same maturational decrease in these claudin proteins with Western blot analysis. Immunohistochemistry showed high levels of expression of claudin 6 in neonatal proximal tubules, thick ascending limb, distal convoluted tubules, and collecting ducts in a paracellular distribution but there was no expression of claudin 6 in the adult kidney. Using real-time RT-PCR claudin 6 and 9 mRNA were present in 1-day-old proximal convoluted tubules and were virtually undetectable in proximal convoluted tubules from adults. Claudin 13 was not detectable in neonatal or adult proximal convoluted tubules. In summary, we have identified developmentally expressed claudin isoforms, claudin 6, claudin 9, and claudin 13. These paracellular proteins may play a role in the maturational changes in paracellular permeability.

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

Journal of the American Society of Nephrology ◽

10.1681/asn.v1271327 ◽

2001 ◽

Vol 12 (7) ◽

pp. 1327-1334 ◽

Cited By ~ 1

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.

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Differential Expression Patterns of Claudins, Tight Junction Membrane Proteins, in Mouse Nephron Segments

Journal of the American Society of Nephrology ◽

10.1681/asn.v134875 ◽

2002 ◽

Vol 13 (4) ◽

pp. 875-886 ◽

Cited By ~ 5

Author(s):

Yumiko Kiuchi-Saishin ◽

Shimpei Gotoh ◽

Mikio Furuse ◽

Akiko Takasuga ◽

Yasuo Tano ◽

...

Keyword(s):

Membrane Proteins ◽

Tight Junction ◽

Polyclonal Antibodies ◽

Collecting Duct ◽

Expression Patterns ◽

Distal Tubule ◽

Northern Blotting ◽

Thick Ascending Limb ◽

Specific Expression ◽

Nephron Segments

ABSTRACT. As the first step in understanding the physiologic functions of claudins (tight junction integral membrane proteins) in nephrons, the expression of claudin-1 to -16 in mouse kidneys was examined by Northern blotting. Among these claudins, only claudin-6, -9, -13, and -14 were not detectable. Claudin-5 and -15 were detected only in endothelial cells. Polyclonal antibodies specific for claudin-7 and -12 were not available. Therefore, the distributions of claudin-1, -2, -3, -4, -8, -10, -11, and -16 in nephron segments were examined with immunofluorescence microscopy. For identification of individual segments, antibodies specific for segment markers were used. Immunofluorescence microscopic analyses of serial frozen sections of mouse kidneys with polyclonal antibodies for claudins and segment markers revealed that claudins demonstrated very complicated, segment-specific, expression patterns in nephrons, i.e., claudin-1 and -2 in Bowman’s capsule, claudin-2, -10, and -11 in the proximal tubule, claudin-2 in the thin descending limb of Henle, claudin-3, -4, and -8 in the thin ascending limb of Henle, claudin-3, -10, -11, and -16 in the thick ascending limb of Henle, claudin-3 and -8 in the distal tubule, and claudin-3, -4, and -8 in the collecting duct. These segment-specific expression patterns of claudins are discussed, with special reference to the physiologic functions of tight junctions in nephrons.

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

Physiological Genomics ◽

10.1152/physiolgenomics.00093.2010 ◽

2011 ◽

Vol 43 (3) ◽

pp. 161-173 ◽

Cited By ~ 40

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.

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Cell-specific expression of amiloride-sensitive, Na(+)-conducting ion channels in the kidney

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.4.c1303 ◽

1996 ◽

Vol 271 (4) ◽

pp. C1303-C1315 ◽

Cited By ~ 40

Author(s):

F. Ciampolillo ◽

D. E. McCoy ◽

R. B. Green ◽

K. H. Karlson ◽

A. Dagenais ◽

...

Keyword(s):

Cell Lines ◽

Collecting Duct ◽

Extracellular Fluid ◽

Cation Channel ◽

Thick Ascending Limb ◽

Distal Nephron ◽

Rt Pcr ◽

Specific Expression ◽

Nephron Segments ◽

Medullary Collecting Duct

Amiloride-sensitive, electrogenic Na+ absorption across the distal nephron plays a vital role in regulating extracellular fluid volume and blood pressure. Recently, two amiloride-sensitive, Na(+)-conducting ion channel cDNAs were cloned. One, an epithelial Na(+)-selective channel (ENaC), is responsible for Na+ absorption throughout the distal nephron. The second, a guanosine 3',5'-cyclic monophosphate (cGMP)-inhibitable cation channel, is conductive to Na+ and Ca2+ and contributes to Na+ absorption across the inner medullary collecting duct (IMCD). As a first step toward understanding the segment-specific contributions(s) of cGMP-gated cation channels and ENaC to Na+ and Ca2+ uptake along the nephron, we used in situ reverse transcription-polymerase chain reaction (RT-PCR) hybridization, solution-phase RT-PCR, and Western blot analysis to examine the nephron and cell-specific expression of these channels in mouse kidney cell lines and/or dissected nephron segments. cGMP-gated cation channel mRNA was detected in proximal tubule, medullary thick ascending limb (mTAL), distal convoluted tubule (DCT), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and IMCD. cGMP-gated cation channel protein was detected in DCT, CCD, and IMCD cell lines. These observations suggest that hormones that modulate intracellular cGMP levels may regulate Na+, and perhaps Ca2+, uptake throughout the nephron. mRNA for alpha-mENaC, a subunit of the mouse ENaC, was detected in mTAL, DCT, CCD, OMCD, and IMCD. Coexpression of alpha-mENaC and cGMP-gated cation channel mRNAs in mTAL, DCT, CCD, OMCD, and IMCD suggests that both channels may contribute to Na+ absorption in these nephron segments.

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

AJP Renal Physiology ◽

10.1152/ajprenal.00018.2007 ◽

2007 ◽

Vol 293 (1) ◽

pp. F350-F359 ◽

Cited By ~ 58

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.

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

Scientific Reports ◽

10.1038/s41598-019-52255-w ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

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.

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Phosphate-dependent glutaminase activity in rat renal cortical and medullary tubule segments

AJP Renal Physiology ◽

10.1152/ajprenal.1990.259.6.f961 ◽

1990 ◽

Vol 259 (6) ◽

pp. F961-F970 ◽

Cited By ~ 14

Author(s):

P. A. Wright ◽

M. A. Knepper

Keyword(s):

Collecting Duct ◽

Proximal Tubules ◽

Thick Ascending Limb ◽

Loop Of Henle ◽

Ammonium Accumulation ◽

Ammonium Production ◽

Medullary Collecting Duct ◽

Acid Loading ◽

Glutaminase Activity ◽

Convoluted Tubules

To determine whether local production of ammonium by medullary renal tubule segments may contribute to medullary ammonium accumulation, we measured activities of phosphate-dependent glutaminase (PDG) in microdissected tubule segments from rat medulla and cortex. PDG activities were very low in medullary loop of Henle segments but surprisingly high in inner medullary collecting duct (IMCD). In cortex, PDG levels were highest in distal convoluted tubule and cortical thick ascending limb, but substantial levels were also found in proximal segments, as reported previously. To determine effects of acid loading and alkali loading on PDG activity, 0.28 M NH4Cl (acid) or 0.28 M NaHCO3 (alkali) was added to rats' drinking water for 7 days. PDG activities in medullary segments were not affected by acid or alkali intake. Acid intake by rats increased PDG activity in S1 and S2 proximal convoluted tubules severalfold but did not affect the other cortical segments. We conclude that medullary loop of Henle segments probably contribute relatively little to medullary ammonium accumulation because of their low activities. The high PDG activity in IMCD suggests that ammonium could be produced and secreted by this segment. However, because total tubule length of IMCD is very low compared with proximal tubules, it appears unlikely that IMCD contributes substantially to overall renal ammonium production. PDG activity is regulated only in S1 and S2 proximal tubules, consistent with the view that the proximal tubule is the major site of regulation of renal ammonium production.

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Expression profiling of antisense transcripts on DNA arrays

Physiological Genomics ◽

10.1152/physiolgenomics.00127.2006 ◽

2007 ◽

Vol 28 (3) ◽

pp. 294-300 ◽

Cited By ~ 21

Author(s):

Andreas Werner ◽

Gabriele Schmutzler ◽

Mark Carlile ◽

Colin G. Miles ◽

Heiko Peters

Keyword(s):

Large Scale ◽

Expression Patterns ◽

Distribution Patterns ◽

Mouse Kidney ◽

Dna Arrays ◽

Antisense Transcripts ◽

Specific Expression ◽

Tissue Specific ◽

Antisense Rnas ◽

Specific Distribution

The majority of mouse genes are estimated to undergo bidirectional transcription; however, their tissue-specific distribution patterns and physiological significance are largely unknown. This is in part due to the lack of methodology to routinely assess the expression of natural antisense transcripts (NATs) on a large scale. Here we tested whether commercial DNA arrays can be used to monitor antisense transcription in mouse kidney and brain. We took advantage of the reversely annotated oligonucleotides on the U74 mouse genome array from Affymetrix that hybridize to NATs overlapping with the sense transcript in the area of the probe set. In RNA samples from mouse kidney and brain, 11.9% and 10.1%, respectively, of 5,652 potential NATs returned positive and about half of the antisense RNAs were detected in both tissues, which was similar to the fraction of sense transcripts expressed in both tissues. Notably, we found that the majority of NATs are related to the sense transcriptome since corresponding sense transcripts were detected for 92.5% (kidney) and 74.5% (brain) of the detected antisense RNAs. Antisense RNA transcription was confirmed by real-time PCR and included additional RNA samples from heart, thymus, and liver. The randomly selected transcripts showed tissue specific expression patterns and varying sense/antisense ratios. The results indicate that antisense transcriptomes are tissue specific, and although pairing of sense/antisense transcripts are known to result in rapid degradation, our data provide proof of principle that the sensitivity of commercial DNA arrays is sufficient to assess NATs in total RNA of whole organs.

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Expression of 25-Hydroxyvitamin D3-1α-Hydroxylase in the Human Kidney

Journal of the American Society of Nephrology ◽

10.1681/asn.v10122465 ◽

1999 ◽

Vol 10 (12) ◽

pp. 2465-2473

Author(s):

DANIEL ZEHNDER ◽

ROSEMARY BLAND ◽

ELIZABETH A. WALKER ◽

ARTHUR R. BRADWELL ◽

ALEXANDER J. HOWIE ◽

...

Keyword(s):

Vitamin D ◽

Enzyme Activity ◽

Bone Growth ◽

Human Kidney ◽

Polyclonal Antiserum ◽

Cytochrome P450 Enzyme ◽

Thick Ascending Limb ◽

Variable Expression ◽

Convoluted Tubules ◽

Proximal Convoluted Tubules

Abstract. The secosteroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a vital role in calcium metabolism, tissue differentiation, and normal bone growth. Biosynthesis of 1,25(OH)2D3 is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D3 1α-hydroxylase (1α-hydroxylase). Although activity of this enzyme has been described in several tissues, the kidneys are recognized to be the principal site of 1,25(OH)2D3 production. To date, enzyme activity studies using vitamin D-deficient animals have suggested that 1α-hydroxylase is expressed exclusively in proximal convoluted tubules. With the recent cloning of 1α-hydroxylase, specific cRNA probes and in-house polyclonal antiserum have been used to determine the distribution of 1α-hydroxylase along the human nephron. Immunohistochemistry and in situ hybridization studies indicated strong expression of 1α-hydroxylase protein and mRNA in the distal convoluted tubule, the cortical and medullary part of the collecting ducts, and the papillary epithelia. Lower expression was observed along the thick ascending limb of the loop of Henle and Bowman's capsule. Weaker and more variable expression of 1α-hydroxylase protein and mRNA was seen in proximal convoluted tubules, and no expression was observed in glomeruli or vascular structures. These data show for the first time the distribution of 1α-hydroxylase expression in normal human kidney. In contrast to earlier enzyme activity studies conducted in vitamin D-deficient animals, our data indicate that the distal nephron is the predominant site of 1α-hydroxylase expression under conditions of vitamin D sufficiency.

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