P0045GENETIC ABLATION OF NFAT5 IN THE PRINCIPAL CELLS OF THE COLLECTING DUCT LEADS TO A WATER HANDLING DEFECT

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Federica Petrillo ◽  
Annika Fischer ◽  
Dmitry Chernyakov ◽  
Cristina Esteva Font ◽  
Søren B Poulsen ◽  
...  
Keyword(s):  
Mouse Model ◽  
Function Analysis ◽  
Collecting Duct ◽  
Distal Tubule ◽  
Dominant Negative ◽  
Urinary Concentration ◽  
Thick Ascending Limb ◽  
Water Restriction ◽  
Activated T Cells ◽  
Principal Cells

Abstract Background and Aims Nuclear Factor of Activated T-Cells 5 (NFAT5), also called TonEBP/OREBP, helps protecting kidney cells from the stress of extracellular hypertonic challenge. In response to hypertonicity, NFAT5 rapidly translocates into the nucleus and enhances the transcription of osmoprotective genes. Mice lacking NFAT5 have embryonic lethality, with those surviving having severe renal atrophy and hydronephrosis. Overexpression of a dominant-negative NFAT5 in all epithelial cells of the thick ascending limb, distal tubule and collecting duct (CD) leads to an impairment in the urine concentration, with reduced expression of AQP2 and urea transporters UT-A1 and UT-A2. Here, we generated a novel mouse model to assess the role of NFAT5 in the water transporting principal cells of the CD. Method The mouse model was generated by breeding floxed NFAT5 mice with mice specifically expressing Cre recombinase in AQP2 expressing cells (NFAT5f/f-AQP2cre+/-). AQP2cre+/- mice served as controls. For renal function analysis, mice were kept in metabolic cages for 5 days and water intake, urinary volume, osmolality, and serum and urine electrolytes were evaluated. Mice were euthanized and the kidneys were used for western blot analysis. Results NFAT5f/f-AQP2cre+/- mice show a significant increase of 24 hour urine output compared to the control (KO: 6.267 ± 0.9367 µl/h/g BW vs WT: 0.9593 ± 0.0796 µl/h/g BW), a decrease in the urine osmolality (KO: 505.6 ± 107.6 mOsm/Kg H2O vs WT: 6205 ± 752.7 mOsm/Kg H2O) and urea concentration. After 3h and half intraperitoneal injection of dDAVP (1ug/kg) the osmolality was still significantly low in NFAT5f/f-AQP2cre+/- mice (KO: 743.7 ± 81.77 vs WT: 3342 ± 80.14 mOsm/Kg H2O), as well as after 8h water restriction (KO: 732.8 ± 24.90 vs WT: 3784 ± 450.9 mOsm/Kg H2O). Immunoblotting demonstrated a significant decrease in AQP2, AQP3 and AQP4 abundance in cortical kidney fractions and inner medulla. There were no differences in vasopressin V2 receptor (V2R) and AVP-regulated transporters NKCC2, ROMK and alpha-ENaC. Conclusion We demonstrate that NFAT5 ablation from AQP2-expressing cells results in a urinary concentration defect. NFAT5f/f-AQP2cre+/- mice have a hypoosmotic polyuria, without major effects on the AVP-V2R axis, suggesting Nephrogenic Diabetes Insipidus.

Structural-functional relationships along the distal nephron

1986 ◽  
Vol 250 (1) ◽  
pp. F1-F15 ◽  
Author(s):  
K. M. Madsen ◽  
C. C. Tisher
Keyword(s):  
Atpase Activity ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Distal Tubule ◽  
Structural Heterogeneity ◽  
Apical Plasma Membrane ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Principal Cells ◽  
Functional Relationships

The distal tubule, which includes the thick ascending limb (TAL), the macula densa, and the distal convoluted tubule (DCT), and the collecting duct are structurally heterogeneous, thus reflecting the functional heterogeneity that is also present. As the TAL ascends from medulla to cortex, the surface area of the apical plasma membrane increases while that of the basolateral membrane decreases. The structure of the DCT resembles that of the medullary TAL. An excellent correlation exists between structure, Na-K-ATPase activity, and NaCl reabsorptive capacity in the distal tubule. The collecting duct is subdivided into the initial collecting tubule (ICT), and cortical (CCD), outer medullary (OMCD), and inner medullary (IMCD) collecting ducts. Between the distal tubule and the collecting duct is a transition region termed the connecting segment or connecting tubule (CNT). Considerable structural heterogeneity exists along the collecting duct within the two major cell populations, the intercalated cells and the principal cells. In the CNT, the ICT, and the CCD, potassium loading and mineralocorticoids stimulate Na-K-ATPase activity and cause proliferation of the basolateral membrane of CNT cells and principal cells, thus identifying the cells responsible for mineralocorticoid-stimulated potassium secretion in these regions. Finally, at least two morphologically distinct populations of intercalated cells exist, types A and B. In the rat, type A predominates in the CNT and the OMCD and is believed to be responsible for H+ secretion, at least in the OMCD. Type B predominates in the CCD, where it may be involved in bicarbonate secretion.

scholarly journals Demonstration of the functional impact of vasopressin signaling in the thick ascending limb by a targeted transgenic rat approach

2016 ◽  
Vol 311 (2) ◽  
pp. F411-F423 ◽  
Author(s):  
Kerim Mutig ◽  
Tordis Borowski ◽  
Christin Boldt ◽  
Aljona Borschewski ◽  
Alexander Paliege ◽  
...  
Keyword(s):  
Epithelial Transport ◽  
Collecting Duct ◽  
Surface Expression ◽  
Urine Concentration ◽  
Dominant Negative ◽  
Transgenic Rat ◽  
Thick Ascending Limb ◽  
Water Reabsorption ◽  
Mediated Effects ◽  
Response To Water

The antidiuretic hormone vasopressin (AVP) regulates renal salt and water reabsorption along the distal nephron and collecting duct system. These effects are mediated by vasopressin 2 receptors (V2R) and release of intracellular Gs-mediated cAMP to activate epithelial transport proteins. Inactivating mutations in the V2R gene lead to the X-linked form of nephrogenic diabetes insipidus (NDI), which has chiefly been related with impaired aquaporin 2-mediated water reabsorption in the collecting ducts. Previous work also suggested the AVP-V2R-mediated activation of Na+-K+-2Cl−-cotransporters (NKCC2) along the thick ascending limb (TAL) in the context of urine concentration, but its individual contribution to NDI or, more generally, to overall renal function was unclear. We hypothesized that V2R-mediated effects in TAL essentially determine its reabsorptive function. To test this, we reevaluated V2R expression. Basolateral membranes of medullary and cortical TAL were clearly stained, whereas cells of the macula densa were unreactive. A dominant-negative, NDI-causing truncated V2R mutant (Ni3-Glu242stop) was then introduced into the rat genome under control of the Tamm-Horsfall protein promoter to cause a tissue-specific AVP-signaling defect exclusively in TAL. Resulting Ni3-V2R transgenic rats revealed decreased basolateral but increased intracellular V2R signal in TAL epithelia, suggesting impaired trafficking of the receptor. Rats displayed significant baseline polyuria, failure to concentrate the urine in response to water deprivation, and hypercalciuria. NKCC2 abundance, phosphorylation, and surface expression were markedly decreased. In summary, these data indicate that suppression of AVP-V2R signaling in TAL causes major impairment in renal fluid and electrolyte handling. Our results may have clinical implications.

Abnormal postpartum renal development and cystogenesis in the bcl-2 (-/-) mouse

1996 ◽  
Vol 271 (1) ◽  
pp. F184-F193 ◽  
Author(s):  
C. M. Sorenson ◽  
B. J. Padanilam ◽  
M. R. Hammerman
Keyword(s):  
Cellular Proliferation ◽  
Kidney Development ◽  
Apoptotic Cell ◽  
Collecting Duct ◽  
Distal Tubule ◽  
Renal Development ◽  
Thick Ascending Limb ◽  
Renal Hypoplasia ◽  
Cellular Origin ◽  
Bax Protein

Mice deficient for B cell leukemia/lymphoma gene 2 [bcl-2(-/-) mice] manifest congenital renal hypoplasia and develop multicystic kidney disease and renal failure postnatally. To characterize postpartum renal development, to identify the cellular origin of the cysts, and to provide insight into the role that bcl-2 deficiency plays in the cystogenic process, we examined the morphology of kidneys from bcl-2 (-/-) mice and wild-type littermates [bcl-2 (+/+)] from birth (P0) to postpartum day 28 (P28), determined whether abnormalities of cellular proliferation and apoptosis accompany cyst development, and characterized expression of the bcl-2-related protein, bax. Between P0 and P7, kidneys from bcl-2 (-/-) and bcl-2 (+/+) mice undergo a comparable increase in weight and have similar histological appearances. However, during the next 2 wk of life, weight gain in kidneys from bcl-2 (-/-) mice is reduced compared with that in kidneys from bcl-2 (+/+) animals, and cysts develop in tubules with staining characteristics of proximal tubule, distal tubule/medullary thick ascending limb of Henle's loop, and collecting duct. Unaffected glomeruli and proximal tubules in kidneys of bcl-2 (-/-) mice undergo compensatory growth. Cystogenesis is accompanied by enhanced incorporation of 5-bromo-2'-deoxyuridine in cells within cortex and medulla and apoptosis of cells within cysts and in the renal interstitium. Bax protein is expressed in the distal tubule in kidneys of bcl-2 (+/+) and bcl-2 (-/-) mice and in some, but not all cysts. We conclude that abnormal regulation of DNA synthesis and apoptosis accompany cystogenesis in bcl-2 (-/-) mice during postpartum kidney development. Continued expression of bax could enhance apoptotic cell death.0

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

2002 ◽  
Vol 13 (4) ◽  
pp. 875-886 ◽  
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.

Immunocytochemical localization of Na+ channels in rat kidney medulla

1989 ◽  
Vol 256 (2) ◽  
pp. F366-F369 ◽  
Author(s):  
D. Brown ◽  
E. J. Sorscher ◽  
D. A. Ausiello ◽  
D. J. Benos
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Na Channel ◽  
Thick Ascending Limb ◽  
Na Channels ◽  
Kidney Medulla ◽  
Principal Cells

Amiloride-sensitive Na+ channels were localized in semithin frozen sections of rat renal medullary collecting ducts, using polyclonal antibodies directed against purified bovine kidney Na+ channel protein. The apical plasma membrane of collecting duct principal cells was heavily stained by indirect immunofluorescence, whereas intercalated cells were negative. Basolateral plasma membranes of both cell types were unstained, as were subapical vesicles in the cytoplasm of these cells. In the thick ascending limb of Henle, some scattered granular fluorescence was seen in the cytoplasm and close to the apical pole of epithelial cells, suggesting the presence of antigenic sites associated with some membrane domains in these cells. No staining was detected in thin limbs of Henle, or in proximal tubules in the outer medulla. These results show that amiloride-sensitive sodium channels are located predominantly on the apical plasma membrane of medullary collecting duct principal cells, the cells that are involved in Na+ homeostasis in this region of the kidney.

scholarly journals Renal phenotype in Bardet-Biedl syndrome: a combined defect of urinary concentration and dilution is associated with defective urinary AQP2 and UMOD excretion

2016 ◽  
Vol 311 (4) ◽  
pp. F686-F694 ◽  
Author(s):  
Miriam Zacchia ◽  
Enza Zacchia ◽  
Enrica Zona ◽  
Giovanna Capolongo ◽  
Ilaria Raiola ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Urine Osmolality ◽  
Normal Function ◽  
Urinary Concentration ◽  
Thick Ascending Limb ◽  
Renal Anomalies ◽  
Bardet Biedl Syndrome ◽  
Urine Albumin ◽  
Plasma Factor ◽  
Renal Phenotype

The renal phenotype in Bardet-Biedl syndrome (BBS) is highly variable. The present study describes renal findings in 41 BBS patients and analyzes the pathogenesis of hyposthenuria, the most common renal dysfunction. Five of 41 patients (12%) showed an estimated glomerular filtration rate < 60 ml·min−1·1.73 m−2. Urine protein and urine albumin-to-creatinine ratio were over 200 and 30 mg/g in 9/24 and 7/23 patients, respectively. Four of 41 patients showed no renal anomalies on ultrasound. Twenty of 34 patients had hyposthenuria in the absence of renal insufficiency. In all 8 of the hyposthenuric patients studied, dDAVP failed to elevate urine osmolality (Uosm), suggesting a nephrogenic origin. Interestingly, water loading (WL) did not result in a significant reduction of Uosm, indicating combined concentrating and diluting defects. dDAVP infusion induced a significant increase of plasma Factor VIII and von Willebrand Factor levels, supporting normal function of the type 2 vasopressin receptor at least in endothelial cells. While urinary aquaporin 2 (u-AQP2) abundance was not different between patients and controls at baseline, the dDAVP-induced increased u-AQP2 and the WL-induced reduction of u-AQP2 were blunted in patients with a combined concentrating and diluting defect, suggesting a potential role of AQP2 in the defective regulation of water absorption. Urine Uromodulin excretion was reduced in all hyposthenuric patients, suggesting a thick ascending limb defect. Interestingly, renal Na, Cl, Ca, but not K handling was impaired after acute WL but not at basal. In summary, BBS patients show combined urinary concentration and dilution defects; a thick ascending limb and collecting duct tubulopathy may underlie impaired water handling.

Aquaporin-2 abundance in the renal collecting duct: new insights from cultured cell models

2009 ◽  
Vol 297 (1) ◽  
pp. F10-F18 ◽  
Author(s):  
Udo Hasler ◽  
Valérie Leroy ◽  
Pierre-Yves Martin ◽  
Eric Féraille
Keyword(s):  
Gene Transcription ◽  
Collecting Duct ◽  
Mrna Translation ◽  
Osmotic Gradient ◽  
Thick Ascending Limb ◽  
Whole Cell ◽  
Cell Abundance ◽  
Principal Cells ◽  
Aquaporin 2 ◽  
Aqp2 Gene

The renal cortico-papillary osmotic gradient is generated by sodium reabsorption in the thick ascending limb. The antidiuretic hormone arginine vasopressin (AVP) increases collecting duct water permeability by enhancing aquaporin-2 (AQP2) water channel insertion in the apical membrane of principal cells, allowing water to passively flow along the osmotic gradient from the tubule lumen to the interstitium. In addition to short-term AQP2 redistribution between intracellular compartments and the cell surface, AQP2 whole cell abundance is tightly regulated. AVP is a major transcriptional activator of the AQP2 gene, and stimulation of insulin- and calcium-sensing receptors respectively potentiate and reduce its action. Extracellular tonicity is another key factor that determines the levels of AQP2 abundance. Its effect is dependent on activation of the tonicity-responsive enhancer binding protein that reinforces AVP-induced AQP2 transcriptional activation. Conversely, activation of the NF-κB transcriptional factor by proinflammatory factors reduces AQP2 gene transcription. Aldosterone additionally regulates AQP2 whole cell abundance by simultaneously reducing AQP2 gene transcription and stimulating AQP2 mRNA translation. These examples illustrate how cross talk between various stimuli regulates AQP2 abundance in collecting duct principal cells and consequently contributes to maintenance of body water homeostasis.

scholarly journals In vivo nuclear translocation of mineralocorticoid and glucocorticoid receptors in rat kidney: differential effect of corticosteroids along the distal tubule

2010 ◽  
Vol 299 (6) ◽  
pp. F1473-F1485 ◽  
Author(s):  
Daniel Ackermann ◽  
Nikolay Gresko ◽  
Monique Carrel ◽  
Dominique Loffing-Cueni ◽  
Daniel Habermehl ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Glucocorticoid Receptors ◽  
Nuclear Translocation ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Distal Tubule ◽  
Plasma Aldosterone ◽  
Thick Ascending Limb ◽  
Cell Nuclei

Aldosterone and corticosterone bind to mineralocorticoid (MR) and glucocorticoid receptors (GR), which, upon ligand binding, are thought to translocate to the cell nucleus to act as transcription factors. Mineralocorticoid selectivity is achieved by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) that inactivates 11β-hydroxy glucocorticoids. High expression levels of 11β-HSD2 characterize the aldosterone-sensitive distal nephron (ASDN), which comprises the segment-specific cells of late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct (CD). We used MR- and GR-specific antibodies to study localization and regulation of MR and GR in kidneys of rats with altered plasma aldosterone and corticosterone levels. In control rats, MR and GR were found in cell nuclei of thick ascending limb (TAL), DCT, CNT, CD cells, and intercalated cells (IC). GR was also abundant in cell nuclei and the subapical compartment of proximal tubule (PT) cells. Dietary NaCl loading, which lowers plasma aldosterone, caused a selective removal of GR from cell nuclei of 11β-HSD2-positive ASDN. The nuclear localization of MR was unaffected. Adrenalectomy (ADX) resulted in removal of MR and GR from the cell nuclei of all epithelial cells. Aldosterone replacement rapidly relocated the receptors in the cell nuclei. In ASDN cells, low-dose corticosterone replacement caused nuclear localization of MR, but not of GR. The GR was redistributed to the nucleus only in PT, TAL, early DCT, and IC that express no or very little 11β-HSD2. In ASDN cells, nuclear GR localization was only achieved when corticosterone was replaced at high doses. Thus ligand-induced nuclear translocation of MR and GR are part of MR and GR regulation in the kidney and show remarkable segment- and cell type-specific characteristics. Differential regulation of MR and GR may alter the level of heterodimerization of the receptors and hence may contribute to the complexity of corticosteroid effects on ASDN function.

Localization of kallikrein in the rat kidney and its anatomical relationship to renin.

1982 ◽  
Vol 30 (4) ◽  
pp. 385-390 ◽  
Author(s):  
T B Orstavvik ◽  
T Inagami
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Distal Tubule ◽  
Juxtaglomerular Apparatus ◽  
Thick Ascending Limb ◽  
Loop Of Henle ◽  
Afferent Arteriole ◽  
Epitheloid Cells ◽  
Anatomical Relationship

The anatomical relationship between kallikrein and renin in the rat kidney was investigated immunohistochemically by the peroxidase-antiperoxidase method. Kallikrein was localized to the convoluted distal tubule, starting at a point, distal to the juxtaglomerular apparatus, where the thick ascending limb of loop of Henle transformed into the convoluted distal tubule. The thick ascending limb was identified by its content of uromucoid (Tamm-Horsfall glycoprotein). Kallikrein was never observed within the juxtaglomerular apparatus itself. The kallikrein-containing tubule ended where the distal tubule submerged into the collecting duct. Renin was found in epitheloid cells of the afferent arteriole. When neighboring sections were stained for kallikrein and renin, respectively, no close anatomical relationship was observed between the kallikrein-containing and the renin-containing structures.

scholarly journals Glucagon actions on the kidney revisited: possible role in potassium homeostasis

2016 ◽  
Vol 311 (2) ◽  
pp. F469-F486 ◽  
Author(s):  
Lise Bankir ◽  
Nadine Bouby ◽  
Bertrand Blondeau ◽  
Gilles Crambert
Keyword(s):  
Collecting Duct ◽  
Distal Tubule ◽  
Glucagon Secretion ◽  
Transepithelial Transport ◽  
Thick Ascending Limb ◽  
Receptor Antagonists ◽  
Renal Handling ◽  
Glucagon Receptor ◽  
Potassium Homeostasis ◽  
Treatment Of Diabetes

It is now recognized that the metabolic disorders observed in diabetes are not, or not only due to the lack of insulin or insulin resistance, but also to elevated glucagon secretion. Accordingly, selective glucagon receptor antagonists are now proposed as a novel strategy for the treatment of diabetes. However, besides its metabolic actions, glucagon also influences kidney function. The glucagon receptor is expressed in the thick ascending limb, distal tubule, and collecting duct, and glucagon regulates the transepithelial transport of several solutes in these nephron segments. Moreover, it also influences solute transport in the proximal tubule, possibly by an indirect mechanism. This review summarizes the knowledge accumulated over the last 30 years about the influence of glucagon on the renal handling of electrolytes and urea. It also describes a possible novel role of glucagon in the short-term regulation of potassium homeostasis. Several original findings suggest that pancreatic α-cells may express a “potassium sensor” sensitive to changes in plasma K concentration and could respond by adapting glucagon secretion that, in turn, would regulate urinary K excretion. By their combined actions, glucagon and insulin, working in a combinatory mode, could ensure an independent regulation of both plasma glucose and plasma K concentrations. The results and hypotheses reviewed here suggest that the use of glucagon receptor antagonists for the treatment of diabetes should take into account their potential consequences on electrolyte handling by the kidney.

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