bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin II receptor subtypes determine induced NO production in rat glomerular mesangial cells
Authors: Schwöbel Jörg , Fischer Tina , Lanz Bettina , Mohaupt Markus
Published: December 2017

Angiotensin II (ANG II) and nitric oxide (NO) have contrasting vascular effects, yet both sustain inflammatory responses. We investigated the impact of ANG II on lipopolysaccharide (LPS)/interferon-γ (IFN)-induced NO production in cultured rat mesangial cells (MCs). LPS/IFN-induced nitrite production, the inducible form of nitric oxide synthase (NOS-2) mRNA, and protein expression were dose dependently inhibited by ANG II on coincubation, which was abolished on ANG II type 2 (AT2) receptor blockade by PD-123319. Homology-based RT-PCR verified the presence of AT1A, AT1B, and AT2receptors. To shift the AT receptor expression toward the type 1 receptor, two sets of experiments were performed: LPS/IFN preincubation for 24 h was followed by 8-h coincubation with ANG II; or during 24-h coincubation of LPS/IFN and ANG II, dexamethasone was added for the last 6-h period. Both led to an amplified overall expression of NOS-2 protein and NO production that was inhibitable by actinomycin D in the first setup. Induced NO production was enhanced via the AT1receptor; however, it was diminished via the AT2receptor. In conclusion, induced NO production is negatively controlled by the AT2, whereas AT1receptor stimulation enhanced NO synthesis in MCs. The overall NO availability depended on the onset of the inflammatory stimuli with respect to ANG II exposure and the available AT receptors.
β-Adrenergic agonists stimulate Mg2+ uptake in mouse distal convoluted tubule cells
Authors: Kang Hyung Sub , Kerstan Dirk , Dai Long-Jun , Ritchie Gordon , Quamme Gary A.
Published: December 2017

β-Adrenergic agonists influence electrolyte reabsorption in the proximal tubule, loop of Henle, and distal tubule. Although isoproterenol enhances magnesium absorption in the thick ascending limb, it is unclear what effect, if any, β-adrenergic agonists have on tubular magnesium handling. The effects of isoproterenol were studied in immortalized mouse distal convoluted tubule (MDCT) cells by measuring cellular cAMP formation with radioimmunoassays and Mg2+ uptake with fluorescence techniques. Intracellular free Mg2+ concentration ([Mg2+]i) was measured in single MDCT cells by using microfluorescence with mag-fura-2. To assess Mg2+uptake, MDCT cells were first Mg2+ depleted to 0.22 ± 0.01 mM by culturing in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2, and the changes in [Mg2+]i were determined. [Mg2+]i returned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate, d([Mg2+]i)/d t, of 168 ± 11 nM/s. Isoproterenol stimulated Mg2+ entry in a concentration-dependent manner, with a maximal response of 252 ± 11 nM/s, at a concentration of 10−7 M, that represented a 50 ± 7% increase in uptake rate above control values. This was associated with a sixfold increase in intracellular cAMP generation. Isoproterenol-stimulated Mg2+ uptake was completely inhibited with RpcAMPS, a protein kinase A inhibitor, and U-73122, a phospholipase C inhibitor, and partially blocked by RO 31–822, a protein kinase C inhibitor. Accordingly, isoproterenol-mediated Mg2+ entry rates involve multiple intracellular signaling pathways. Aldosterone potentiated isoproterenol-stimulated Mg2+ uptake (326 ± 31 nM/s), whereas elevation of extracellular Ca2+ inhibited isoproterenol-mediated cAMP accumulation and Mg2+ uptake, 117 ± 37 nM/s. These studies demonstrate that isoproterenol stimulates Mg2+ uptake in a cell line of mouse distal convoluted tubules that is modulated by hormonal and extracellular influences.
Translational regulation of Na-K-ATPase subunit mRNAs by glucocorticoids
Authors: Devarajan Prasad , Benz Edward J.
Published: December 2017

Glucocorticoids (GC) regulate Na-K-ATPase-subunit mRNA transcription. However, GC-induced increases in Na-K-ATPase activity are not always paralleled by changes in subunit mRNA abundance. We therefore examined posttranscriptional mechanisms of subunit gene regulation by GC. cDNA-derived mRNAs encoding α1-, α3-, and β1-subunits were tested for stability and translation efficiency in a cell-free lysate, in the presence of hydrocortisone (HC) or dexamethasone (Dex). No effect of HC on subunit mRNA stability was noted. Translation efficiency of α1- and α3-mRNAs, but not of β1-mRNA, was significantly increased by HC and Dex. Deletion of the 5′untranslated region (5′UT) of α1-mRNA abolished this effect. Translation of a chimeric β1-mRNA, constructed by transposing the 5′UT of α1 onto the coding region of β1, was enhanced by HC. Transposition of a putative steroid-modulatory element conserved in the 5′UT of all α isoforms (ACAGGACCC) onto the coding region of β1-mRNA rendered it responsive to HC. A synthetic primer containing the ACAGGACCC sequence abolished the effect of HC on α1- and chimeric β1-mRNAs. Our results indicate that GC can directly enhance Na-K-ATPase translation in vitro in a subunit-specific manner, via a putative GC-modulatory element situated in a predicted loop structure within the 5′UT of α-mRNAs.
Embryonic epithelial membrane transporters
Authors: Horster Michael
Published: December 2017

Embryonic epithelial membrane transporters are organized into transporter families that are functional in several epithelial organs, namely, in kidney, lung, pancreas, intestine, and salivary gland. Family members (subtypes) are developmentally expressed in plasma membranes in temporospatial patterns that are 1) similar for one subtype within different organs, like aquaporin-1 (AQP1) in lung and kidney; 2) different between subtypes within the same organ, like the amiloride-sensitive epithelial sodium channel (ENaC) in lung; and 3) apparently matched among members of different transporter families, as α-ENaC with AQP1 and -4 in lung and with AQP2 in kidney. Finally, comparison of temporal expression patterns in early embryonic development of transporters from different families [e.g., cystic fibrosis transmembrane conductance regulator (CFTR), ENaC, and outer medullary potassium channel] suggests regulatory activating or inactivating interactions in defined morphogenic periods. This review focuses on embryonic patterns, at the mRNA and immunoprotein level, of the following transporter entities expressed in epithelial cell plasma membranes: ENaC; the chloride transporters CFTR, ClC-2, bumetanide-sensitive Na-K-Cl cotransporter, Cl/OH, and Cl/HCO3; the sodium glucose transporter-glucose transporter; the sodium/hydrogen exchanger; the sodium-phosphate cotransporter; the ATPases; and AQP. The purpose of this article is to relate temporal and spatial expression patterns in embryonic and in early postnatal epithelia to developmental changes in organ structure and function.
Ammonium carriers in medullary thick ascending limb
Authors: Attmane-Elakeb Amel , Amlal Hassane , Bichara Maurice
Published: December 2017

Absorption of NH4+by the medullary thick ascending limb (MTAL) is a key event in the renal handling of NH4+, leading to accumulation of NH4+/NH3in the renal medulla, which favors NH4+secretion in medullary collecting ducts and excretion in urine. The Na+-K+(NH4+)-2Cl−cotransporter (BSC1/NKCC2) ensures ∼50–65% of MTAL active luminal NH4+uptake under basal conditions. Apical barium- and verapamil-sensitive K+/NH4+antiport and amiloride-sensitive NH4+conductance account for the rest of active luminal NH4+transport. The presence of a K+/NH4+antiport besides BSC1 allows NH4+and NaCl absorption by MTAL to be independently regulated by vasopressin. At the basolateral step, the roles of NH3diffusion coupled to Na+/H+exchange or Na+/NH4+exchange, which favors NH4+absorption, and of Na+/K+(NH4+)-ATPase, NH4+-Cl−cotransport, and NH4+conductance, which oppose NH4+absorption, have not been quantitatively defined. The increased ability of the MTAL to absorb NH4+during chronic metabolic acidosis involves an increase in BSC1 expression, but fine regulation of MTAL NH4+transport probably requires coordinated effects on various apical and basolateral MTAL carriers.
Distal tubular electrolyte transport during inhibition of renal 11β-hydroxysteroid dehydrogenase
Authors: Biller Katharine J. , Unwin Robert J. , Shirley David G.
Published: December 2017

To test the proposal that the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) confers aldosterone specificity on mineralocorticoid receptors in the distal nephron by inactivating glucocorticoids, we performed a free-flow micropuncture study of distal tubular function in adrenalectomized rats infused with high-dose corticosterone. One-half of the rats were additionally given intravenous carbenoxolone (CBX; 6 mg/h) to inhibit renal 11β-HSD activity. Although this maneuver lowered fractional Na+excretion (1.1 ± 0.2 vs. 1.9 ± 0.2%, P < 0.01), Na+reabsorption within the accessible distal tubule was found to be similar in the two groups of animals. In contrast, distal tubular K+secretion was enhanced in CBX-treated rats: fractional K+deliveries to the early and late distal collection sites in the corticosterone-alone group were 13 ± 1 and 20 ± 3%, respectively (not significant), whereas corresponding data in the CBX-treated group were 9 ± 1 and 24 ± 2% ( P < 0.01). This stimulation of distal K+secretion provides the first direct in vivo evidence that 11β-HSD normally prevents corticosterone from exerting a mineralocorticoid-like effect in the distal tubule. The reduction in fractional Na+excretion during inhibition of 11β-HSD, in the absence of a change in end-distal Na+delivery, suggests enhanced Na+reabsorption in the collecting ducts.
ETB receptor activation causes exocytic insertion of NHE3 in OKP cells
Authors: Peng Yan , Amemiya Morimasa , Yang Xiaojing , Fan Lingzhi , Moe Orson W. , Yin Helen , Preisig Patricia A. , Yanagisawa Masashi , Alpern Robert J.
Published: December 2017

Endothelin-1 (ET-1) activates sodium/hydrogen exchanger 3 (NHE3) in opossum kidney clone P (OKP) cells expressing ETB receptors. ET-1 (10−8 M) caused a two- to threefold increase in apical membrane NHE3 (assessed by surface biotinylation), in the absence of a change in total cellular NHE3. A maximal effect was achieved within 15 min. The increase in apical NHE3 was not blocked by cytochalasin D but was blocked by latrunculin B, which also prevented the ET-1-induced increase in NHE3 activity. Endocytic internalization of NHE3, measured as protection of biotinylated NHE3 from the membrane-impermeant, sulfhydryl-reducing agent MesNa was minimal within 35 min and was not regulated by ET-1. Exocytic insertion of NHE3, measured as the appearance of biotinylated NHE3 after the blockade of reactive sites with sulfo-NHS-acetate, was increased in response to ET-1. These studies demonstrate that ET-1 induces net trafficking of NHE3 to the apical membrane that is mediated by enhanced exocytic insertion and is required for increased NHE3 activity.
Location and function of the epithelial Na channel in the cochlea
Authors: Couloigner Vincent , Fay Michel , Djelidi Sabri , Farman Nicolette , Escoubet Brigitte , Runembert Isabelle , Sterkers Olivier , Friedlander Gérard , Ferrary Evelyne
Published: December 2017

In the cochlea, endolymph is a K-rich and Na-poor fluid. The purpose of the present study was to check the presence and to assess the role of epithelial Na channel (ENaC) in this organ. α-, β-, and γ-ENaC subunit mRNA, and proteins were detected in rat cochlea by RT-PCR and Western blot. α-ENaC subunit mRNA was localized by in situ hybridization in both epithelial (stria vascularis, spiral prominence, spiral limbus) and nonepithelial structures (spiral ligament, spiral ganglion). The α-ENaC-positive tissues were also positive for β-subunit mRNA (except spiral ganglion) or for γ-subunit mRNA (spiral limbus, spiral ligament, and spiral ganglion), but the signals of β- and γ-subunits were weaker than those observed for α-subunit. In vivo, the endocochlear potential was recorded in guinea pigs under normoxic and hypoxic conditions after endolymphatic perfusion of ENaC inhibitors (amiloride, benzamil) dissolved either in K-rich or Na-rich solutions. ENaC inhibitors altered the endocochlear potential when Na-rich but not when K-rich solutions were perfused. In conclusion, ENaC subunits are expressed in epithelial and nonepithelial cochlear structures. One of its functions is probably to maintain the low concentration of Na in endolymph.
Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells
Authors: Kifor Olga , MacLeod R. John , Diaz Ruben , Bai Mei , Yamaguchi Toru , Yao Tham , Kifor Imre , Brown Edward M.
Published: December 2017

Regulation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway by the extracellular calcium (Cao2+)-sensing receptor (CaR) was investigated in bovine parathyroid and CaR-transfected human embryonic kidney (HEKCaR) cells. Elevating Cao2+or adding the selective CaR activator NPS R-467 elicited rapid, dose-dependent phosphorylation of ERK1/2. These phosphorylations were attenuated by pretreatment with pertussis toxin (PTX) or by treatment with the phosphotyrosine kinase (PTK) inhibitors genistein and herbimycin, the phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor U-73122, or the protein kinase C (PKC) inhibitor GF109203X and were enhanced by the PKC activator phorbol 12-myristate 13-acetate. Combined treatment with PTX and inhibitors of both PKC and PTK nearly abolished high Cao2+-evoked ERK1/2 activation in HEKCaR cells, demonstrating CaR-mediated coupling via both Gqand Gi. High Cao2+increased serine phosphorylation of the 85-kDa cytosolic phospholipase A2(cPLA2) in both parathyroid and HEKCaR cells. The selective mitogen-activated protein kinase (MAPK) inhibitor PD98059 abolished high-Cao2+-induced ERK1/2 activation and reduced cPLA2phosphorylation in both cell types, documenting MAPK's role in cPLA2activation. Thus our data suggest that the CaR activates MAPK through PKC, presumably through Gq/11-mediated activation of PI-PLC, as well as through Gi- and PTK-dependent pathway(s) in bovine parathyroid and HEKCaR cells and indicate the importance of MAPK in cPLA2activation.
Pendrin: an apical Cl−/OH−/HCO3−exchanger in the kidney cortex
Authors: Soleimani Manoocher , Greeley Tracey , Petrovic Snezana , Wang Zhaohui , Amlal Hassane , Kopp Peter , Burnham Charles E.
Published: December 2017

The identities of the apical Cl−/base exchangers in kidney proximal tubule and cortical collecting duct (CCD) cells remain unknown. Pendrin (PDS), which is expressed at high levels in the thyroid and its mutation causes Pendred's syndrome, is shown to be an anion exchanger. We investigated the renal distribution of PDS and its function. Our results demonstrate that pendrin mRNA expression in the rat kidney is abundant and limited to the cortex. Proximal tubule suspensions isolated from kidney cortex were highly enriched in pendrin mRNA. Immunoblot analysis studies localized pendrin to cortical brush-border membranes. Nephron segment RT-PCR localized pendrin mRNA to proximal tubule and CCD. Expression studies in HEK-293 cells demonstrated that pendrin functions in the Cl−/OH−, Cl−/HCO3−, and Cl−/formate exchange modes. The conclusion is that pendrin is an apical Cl−/base exchanger in the kidney proximal tubule and CCD and mediates Cl−/OH−, Cl−/HCO3−, and Cl−/formate exchange.
Estrogen-induced cardiorenal protection: potential cellular, biochemical, and molecular mechanisms
Authors: Dubey Raghvendra K. , Jackson Edwin K.
Published: December 2017

A number of cellular and biochemical processes are involved in the pathophysiology of glomerular and vascular remodeling, leading to renal and vascular disorders, respectively. Although estradiol protects the renal and cardiovascular systems, the mechanisms involved remain unclear. In this review we provide a discussion of the cellular, biochemical, and molecular mechanisms by which estradiol may exert protective effects on the kidneys and vascular wall. In this regard, we consider the possible role of genomic vs. nongenomic mechanisms and estrogen receptor-dependent vs. estrogen receptor-independent mechanisms in mediating the protective effects of estradiol on the renal and cardiovascular systems.
Molecular characterization of a novel urea transporter from kidney inner medullary collecting ducts
Authors: Shayakul Chairat , Tsukaguchi Hiroyasu , Berger Urs V. , Hediger Matthias A.
Published: December 2017

In the terminal part of the kidney collecting duct, rapid urea reabsorption is essential to maintaining medullary hypertonicity, allowing maximal urinary concentration to occur. This process is mediated by facilitated urea transporters on both apical and basolateral membranes. Our previous studies have identified three rat urea transporters involved in the urinary concentrating mechanism, UT1, UT2 and UT3 , herein renamed UrT1-A, UrT1-B, and UrT2, which exhibit distinct spatial distribution in the kidney. Here we report the molecular characterization of an additional urea transporter isoform, UrT1-C, from rat kidney that encodes a 460-amino acid residue protein. UrT1-C has 70 and 62% amino acid identity to rat UrT1-B and UrT2 (UT3), respectively, and 99% identity to a recently reported rat isoform (UT-A3; Karakashian A, Timmer RT, Klein JD, Gunn RB, Sands JM, and Bagnasco SM. J Am Soc Nephrol 10: 230–237, 1999). We report the anatomic distribution of UrT1-C in the rat kidney tubule system as well as a detailed functional characterization. UrT1-C m RNA is primarily expressed in the deep part of the inner medulla. When expressed in Xenopus laevis oocytes, UrT1-C induced a 15-fold stimulation of urea uptake, which was inhibited almost completely by phloretin (0.7 mM) and 60–95% by thiourea analogs (150 mM). The characteristics are consistent with those described in perfusion studies with inner medullary collecting duct (IMCD) segments, but, contrary to UrT1-A, UrT1-C-mediated urea uptake was not stimulated by activation of protein kinase A. Our data show that UrT1-C is a phloretin-inhibitable urea transporter expressed in the terminal collecting duct that likely serves as an exit mechanism for urea at the basolateral membrane of IMCD cells.
Stimulation of pro-α1(I) collagen by TGF-β1in mesangial cells: role of the p38 MAPK pathway
Authors: Chin Beek Yoke , Mohsenin Amir , Li Su Xia , Choi Augustine M. K. , Choi Mary E.
Published: December 2017

Transforming growth factor-β1(TGF-β1) is a potent inducer of extracellular matrix protein synthesis and a key mediator of renal fibrosis. However, the intracellular signaling mechanisms by which TGF-β1stimulates this process remain incompletely understood. In this report, we examined the role of a major stress-activated intracellular signaling cascade, belonging to the mitogen-activated protein kinase (MAPK) superfamily, in mediating TGF-β1responses in rat glomerular mesangial cells, using dominant-negative inhibition of TGF-β1signaling receptors. We first stably transfected rat glomerular mesangial cells with a kinase-deleted mutant TGF-β type II receptor (TβR-IIM) designed to inhibit TGF-β1signaling in a dominant-negative fashion. Next, expression of TβR-IIMmRNA was confirmed by Northern analysis. Cell surface expression and ligand binding of TβR-IIMprotein were demonstrated by affinity cross-linking with125I-labeled-TGF-β1. TGF-β1rapidly induced p38 MAPK phosphorylation in wild-type and empty vector (pcDNA3)-transfected control mesangial cells. Interestingly, transfection with dominant-negative TβR-IIMfailed to block TGF-β1-induced p38 MAPK phosphorylation. Moreover, dominant-negative TβR-IIMfailed to block TGF-β1-stimulated pro-α1(I) collagen mRNA expression and cellular protein synthesis, whereas TGF-β1-induced extracellular signal-regulated kinase (ERK) 1/ERK2 activation and antiproliferative responses were blocked by TβR-IIM. In the presence of a specific inhibitor of p38 MAPK, SB-203580, TGF-β1was unable to stimulate pro-α1(I) collagen mRNA expression in the control and TβR-IIM-transfected mesangial cells. Finally, we confirmed that both p38 MAPK activation and pro-α1(I) collagen stimulation were TGF-β1effects that were abrogated by dominant-negative inhibition of TGF-β type I receptor. Thus we show first demonstration of p38 MAPK activation by TGF-β1in mesangial cells, and, given the rapid kinetics, this TGF-β1effect is likely a direct one. Furthermore, our findings suggest that the p38 MAPK pathway functions as a component in the signaling of pro-α1(I) collagen induction by TGF-β1in mesangial cells.
The renal nerve is required for regulation of proximal tubule transport by intraluminally produced ANG II
Authors: Quan Albert , Baum Michel
Published: December 2017

The proximal tubule synthesizes and luminally secretes high levels of angiotensin II, which modulate proximal tubule transport independently of systemic angiotensin II. The purpose of this in vivo microperfusion study is to examine whether the renal nerves modulate the effect of intraluminal angiotensin II on proximal tubule transport. The decrement in volume reabsorption after addition of 10−4M luminal enalaprilat is a measure of the role of luminal angiotensin II on transport. Acute denervation decreased volume reabsorption (2.97 ± 0.14 vs. 1.30 ± 0.21 nl · mm−1· min−1, P < 0.001). Although luminal 10−4M enalaprilat decreased volume reabsorption in controls (2.97 ± 0.14 vs. 1.61 ± 0.26 nl · mm−1· min−1, P < 0.001), it did not after acute denervation (1.30 ± 0.21 vs. 1.55 ± 0.19 nl · mm−1· min−1). After chronic denervation, volume reabsorption was unchanged from sham controls (2.26 ± 0.28 vs. 2.70 ± 0.19 nl · mm−1· min−1). Addition of luminal 10−4M enalaprilat decreased volume reabsorption in sham control (2.70 ± 0.19 vs. 1.60 ± 0.10 nl · mm−1· min−1, P < 0.05) but not with chronic denervation (2.26 ± 0.28 vs. 2.07 ± 0.20 nl · mm−1· min−1). Addition of 10−8M angiotensin II to the lumen does not affect transport due to the presence of luminal angiotensin II. However, addition of 10−8M angiotensin II to the tubular lumen increased the volume reabsorption after both acute (1.30 ± 0.21 vs. 2.67 ± 0.18 nl · mm−1· min−1, P < 0.05) and chronic denervation (2.26 ± 0.28 vs. 3.57 ± 0.44 nl · mm−1· min−1, P < 0.01). These data indicate that renal denervation abolished the luminal enalaprilat-sensitive component of proximal tubule transport, which is consistent with the renal nerves playing a role in the modulation of the intraluminal angiotensin II mediated component of proximal tubule transport.
The distal convoluted tubule of rabbit kidney does not express a functional sodium channel
Authors: Velázquez Heino , Silva Teresa , Andújar Eleanor , Desir Gary V. , Ellison David H. , Greger Rainer
Published: December 2017

We sought to assess whether the distal convoluted tubule (DCT) segment of the rabbit nephron expresses a functional epithelial sodium channel. First, the transepithelial voltage ( V te, lumen vs. bath) was measured in isolated perfused DCT segments (assessed separately in the upstream half and the downstream half of the DCT). V tewas zero and not affected by amiloride or barium in the upstream DCT. V te was sometimes negative in the downstream DCT and depolarized by amiloride and hyperpolarized by barium, suggesting inclusion of connecting tubule (CNT) cells. To determine expression of epithelial sodium channel (ENaC) mRNA subunits by the upstream DCT, rabbit α-, β-, and γ-ENaC cDNA fragments were cloned and primers were selected for single-nephron RT-PCR analysis. Although α-ENaC was expressed by the DCT, β- and γ-ENaC were not detected in the DCT. In contrast, the CNT, CCD, and outer medullary collecting duct (OMCD) expressed all three subunits. Nedd4 was also not detected in the DCT but was expressed by the CNT, CCD, and OMCD. When upstream DCT fragments were grown to confluent monolayers in primary culture, the epithelia exhibited negative voltages and high transepithelial resistances and expressed mRNA for all three ENaC subunits as well as for Nedd4. The absence of a negative voltage and failure to detect transcript for β- and γ-ENaC and Nedd4 in the native rabbit DCT suggest that the sodium channel is not a significant pathway for sodium absorption by this segment. The phenotype conversion observed when DCT cells are grown in culture does not rule out the possibility that there may be conditions in which the DCT in the intact kidney expresses sodium channel activity. The results are consistent with the notion that DCT sodium transport is predominantly, if not exclusively, electroneutral.
Megalin and cubilin: synergistic endocytic receptors in renal proximal tubule
Authors: Christensen Erik Ilsø , Birn Henrik
Published: December 2017

The multiligand, endocytic receptors megalin and cubilin are colocalized in the renal proximal tubule. They are heavily expressed in the apical endocytic apparatus. Megalin is a 600-kDa transmembrane protein belonging to the low-density lipoprotein-receptor family. The cytoplasmic tail contains three NPXY motifs that mediate the clustering in coated pits and are possibly involved in signaling functions. Cubilin, also known as the intestinal intrinsic factor-cobalamin receptor, is a 460-kDa receptor with no transmembrane domain and no known signal for endocytosis. Because the two receptors bind each other with high affinity and colocalize in several tissues, it is highly conceivable that megalin mediates internalization of cubilin and its ligands. Both receptors are important for normal tubular reabsorption of proteins, including albumin. Among the proteins normally filtered in the glomeruli, cubilin has been shown to bind albumin, immunoglobulin light chains, and apolipoprotein A-I. The variety of filtered ligands identified for megalin include vitamin-binding proteins, hormones, enzymes, apolipoprotein H, albumin, and β2- and α1-microglobulin. Loss of these proteins and vitamins in the urine of megalin-deficient mice illustrates the physiological importance of this receptor.
Influence of Ca2+-activated K+ channels on rat renal arteriolar responses to depolarizing agonists
Authors: Fallet Rachel W. , Bast Joseph P. , Fujiwara Keiji , Ishii Naohito , Sansom Steven C. , Carmines Pamela K.
Published: December 2017

Experiments were performed to evaluate the hypothesis that opening of Ca2+-activated K+ channels (BKCachannels) promotes juxtamedullary arteriolar dilation and curtails constrictor responses to depolarizing agonists. Under baseline conditions, afferent and efferent arteriolar lumen diameters averaged 23.4 ± 0.9 ( n = 36) and 22.8 ± 1.1 ( n= 13) μm, respectively. The synthetic BKCa channel opener NS-1619 evoked concentration-dependent afferent arteriolar dilation. BKCa channel blockade (1 mM tetraethylammonium; TEA) decreased afferent diameter by 15 ± 3% and prevented the dilator response to 30 μM NS-1619. ANG II (10 nM) decreased afferent arteriolar diameter by 44 ± 4%, a response that was reduced by 30% during NS-1619 treatment; however, TEA failed to alter afferent constrictor responses to either ANG II or arginine vasopressin. Neither NS-1619 nor TEA altered agonist-induced constriction of the efferent arteriole. Thus, although the BKCa channel agonist was able to curtail afferent (but not efferent) arteriolar constrictor responses to ANG II, BKCa channel blockade did not allow exaggerated agonist-induced arteriolar constriction. These observations suggest that the BKCa channels evident in afferent arteriolar smooth muscle do not provide a prominent physiological brake on agonist-induced constriction under our experimental conditions.
Endothelin is a potent inhibitor of matrix metalloproteinase-2 secretion and activation in rat mesangial cells
Authors: Yao Jian , Morioka Tetsuo , Li Bing , Oite Takashi
Published: December 2017

We examined the effects of endothelin (ET) on the activity of matrix metalloproteinase-2 (MMP-2) in cultured MCs. Addition of the ETAreceptor antagonists or neutralizing anti-endothelin antibody into MC cultures markedly augmented the secretion and activation of MMP-2. On the contrary, addition of the exogenous ET-1 into MC culture significantly inhibited the synthesis of MMP-2 in both basal and cytokines (tumor necrosis factor-α and interferon-γ) plus lipopolysaccharide-stimulated conditions. Furthermore, pretreatment of cells with exogenous ET-1 obviously prevented cytochalasinD-elicited activation of MMP-2, an effect that was completely abolished by ETAreceptor antagonist, FR139317. In addition, ET-1 was found to be able to suppress the expression of membrane type-1 MMP (MT1-MMP) and promote the conversion of tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) from cell associated form to secreted form. The addition of recombinant TIMP-2 into the culture abrogated dose-dependently the cytochalasinD-elicited activation of MMP-2. These results suggest that ET is a potent inhibitor of MMP-2 secretion and activation in MCs. These novel findings may help us understand the subtle regulation of the synthesis and activation of MMP-2 in MCs. It also provides us with further insight into the pathophysiological mechanisms involving ET in the regulation of matrix turnover in glomerulus.
Antisense GLUT-1 protects mesangial cells from glucose induction of GLUT-1 and fibronectin expression
Authors: Heilig Charles W. , Kreisberg Jeffrey I. , Freytag Svend , Murakami Takashi , Ebina Yousuke , Guo Lirong , Heilig Kathleen , Loberg Robert , Qu Xuan , Jin Ying , Henry Douglas , Brosius Frank C.
Published: December 2017

A stable clone of rat mesangial cells expressing antisense GLUT-1 (i.e., MCGT1AS cells) was developed to protect them from high glucose exposure. GLUT-1 protein was reduced 50%, and the 2-deoxy-[3H]glucose uptake rate was reduced 33% in MCGT1AS. MCLacZ control cells and MCGT1 GLUT-1-overexpressing cells were used for comparisons. In MCLacZ, 20 mMd-glucose increased GLUT-1 transcription 90% vs. no increase in MCGT1AS. Glucose (8 mM) and 12 mM xylitol [a hexose monophosphate (HMP) shunt substrate] did not stimulate GLUT-1 transcription. An 87% replacement of the standard 8 mMd-glucose with 3- O-methylglucose reduced GLUT-1 transcription 80%. d-Glucose (20 mM) increased fibronectin mRNA and protein by 47 and 100%, respectively, in MCLacZ vs. no increases in MCGT1AS. Fibronectin synthesis was elevated 48% in MCGT1 and reduced 44% in MCGT1AS. We conclude that 1) transcription of GLUT-1 in response to d-glucose depends on glucose metabolism, although not through the HMP shunt, and 2) antisense GLUT-1 treatment of mesangial cells blocksd-glucose-induced GLUT-1 and fibronectin expression, thereby demonstrating a protective effect that could be beneficial in the setting of diabetes.
Inflammation is probably not a prerequisite for renal interstitial fibrosis in normoglycemic obese rats
Authors: Lavaud Stéphanie , Poirier Bruno , Mandet Chantal , Bélair Marie-France , Irinopoulou Théano , Heudes Didier , Bazin Raymond , Bariéty Jean , Myara Isaac , Chevalier Jacques
Published: December 2017

We examined the role of inflammation in the development of renal interstitial fibrosis in Zucker obese rats, which rapidly present kidney lesions in the absence of hypertension and hyperglycemia. Type I and III collagens were quantified using a polarized light and computer-assisted image analyzer. The expression of mRNA encoding matrix components, adhesion molecules, chemokines, and growth factors was followed by RT-PCR. The presence of synthesized proteins as well as lymphocytes and macrophages was determined by immunohistochemistry. Interstitial fibrosis developed in two phases. The first phase occurred as early as 3 mo and resulted from a neosynthesis of type III collagen and fibronectin and a reduction of extracellular matrix catabolism, in parallel with an overexpression of transforming growth factor-β1and in the absence of any lymphocyte or macrophage infiltration. After 6 mo, interstitial fibrosis worsened with a large accumulation of type I collagen, concomitantly with a large macrophage infiltration. Thus inflammation cannot explain the onset of interstitial fibrosis that developed in young, insulinoresistant, normoglycemic, obese Zucker rats but aggravated this process afterward.
Upregulation of juxtaglomerular NOS1 and COX-2 precedes glomerulosclerosis in fawn-hooded hypertensive rats
Authors: Weichert Wilko , Paliege Alexander , Provoost Abraham P. , Bachmann Sebastian
Published: December 2017

This study describes elevated histochemical signals for nitric oxide synthase-1 (NOS1) and cyclooxygenase-2 (COX-2) in juxtaglomerular apparatus (JGA) and adjacent thick ascending limb of the kidney of fawn-hooded hypertensive rats (FHH). Two different age groups of FHH (8 and 16 wk; FHH8 and FHH16, respectively) were compared with genetically related fawn-hooded rats with normal blood pressure (FHL) that served as controls. Histopathological changes in FHH comprised focal segmental glomerulosclerosis (FSGS), focal matrix overexpression, and a moderate arteriolopathy with hypertrophy of the media, enhanced immunoreactivity for α-smooth muscle actin, and altered distribution of myofibrils. Macula densa NOS activity, as expressed by NADPH-diaphorase staining, and NOS1 mRNA abundance were significantly elevated in FHH8 (+153 and +88%; P < 0.05) and FHH16 (+93 and +98%; P < 0.05), respectively. Even higher elevations were registered for COX-2 immunoreactivity in FHH8 (+166%; P < 0.05) and FHH16 (+157%; P < 0.05). The intensity of renin immunoreactivity and renin mRNA expression in afferent arterioles was also elevated in FHH8 (+51 and +166%; P < 0.05) and FHH16 (+105 and +136%; P < 0.05), respectively. Thus we show that coordinate upregulation of tubular NOS1, COX-2, and renin expression precedes, and continues after, the manifestation of glomerulosclerotic damage in FHH. These observations may have implications in understanding the role of local paracrine mediators in glomerular disease.
Cellular localization of divalent metal transporter DMT-1 in rat kidney
Authors: Ferguson C. J. , Wareing M. , Ward D. T. , Green R. , Smith C. P. , Riccardi D.
Published: December 2017

We have demonstrated that the kidney plays an important role in iron balance and that metabolically significant reabsorption of this ion occurs in the loop of Henle and the collecting ducts [Wareing M, Ferguson CJ, Green R, Riccardi D, and Smith CP. J Physiol (Lond) 524: 581–586, 2000]. To test the possibility that the divalent metal transporter DMT1 (Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, and Hediger MA. Nature 388: 482–488, 1997) could represent the apical route for iron entry in the kidney, we raised and affinity-purified an anti-DMT-1 polyclonal antibody and determined DMT-1 distribution in rat kidney by Western analysis, immunofluorescence, and confocal microscopy. The strongest DMT1-specific (i.e., peptide-protectable) immunoreactivity was found in the collecting ducts, in both principal and intercalated cells. Thick ascending limbs of Henle's loop and, more intensely, distal convoluted tubules exhibited apical immunostaining. Considerable intracellular DMT-1 immunoreactivity was seen throughout the nephron, particularly in S3 segments. The described distribution of DMT-1 protein is in agreement with our previous identification of nephron sites of iron reabsorption, suggesting that DMT-1 provides the molecular mechanism for apical iron entry in the distal nephron but not in the proximal tubule. Basolateral iron exit may be facilitated by a different system.
Overexpression of protein kinase G using adenovirus inhibits cyclin E transcription and mesangial cell cycle
Authors: Hanada Satoko , Terada Yoshio , Inoshita Seiji , Sasaki Sei , Lohmann Suzanne M. , Smolenski Albert , Marumo Fumiaki
Published: December 2017

The cGMP-cGMP-dependent protein kinase (protein kinase G) system plays an important role in the pathogenesis of mesangial proliferative glomerulonephritis. However, the molecular mechanisms of the inhibitory effects of the cGMP-protein kinase G system in the cell cycle progression of mesangial cells are not well known. To determine the inhibitory pathway of cGMP-protein kinase G in cultured mesangial cells, we investigated the effects of cGMP- and adenovirus-mediated overexpression of protein kinase G on the promoter activities of cyclin E, cyclin D1, and cyclin A. 8-Bromo-cGMP (8-BrcGMP) and overexpression of protein kinase G reduced [3H]thymidine uptake, reduced the numbers of cells in S and G2/M phases, and decreased the phosphorylation of retinoblastoma (Rb) protein. 8-BrcGMP (10−3M), protein kinase G adenovirus (Ad-cGKIβ; 1010plaque-forming units/ml), atrial natriuretic peptide (ANP), and C-type natriuretic peptide (CNP) inhibited the promoter activity of cyclin E to 49, 57, 77, and 78%, respectively. On the other hand, the promoter activities of cyclin D1 and cyclin A were not changed significantly. In Western blot analysis, 8-BrcGMP, Ad-cGKIβ, ANP, and CNP also inhibited cyclin E protein expression dose and time dependently. The p44/p42 mitogen-activated protein kinase (MAPK) kinase 1-p44/p42 MAPK had no effect on cyclin E promoter activities, and the cGMP-protein kinase G pathway did not change MAPK activity. In conclusion, our findings suggest that the reduction of the cyclin E promoter activity that downregulates G1/S transition plays a dominant role in the cGMP- and protein kinase G-induced inhibition of mesangial cell proliferation.
Effect of water deprivation and hypertonic saline infusion on urinary AQP2 excretion in healthy humans
Authors: Pedersen R. S. , Bentzen H. , Bech J. N. , Pedersen E. B.
Published: December 2017

Arginine vasopressin (AVP) mediates water transport in the renal collecting ducts by forming water channels of aquaporin-2 (AQP2) in the apical plasma membrane. AQP2 is excreted in human urine. We wanted to test the hypothesis that urinary excretion of AQP2 (u-AQP2) reflects the effect of AVP on the renal collecting ducts during water deprivation and hypertonic saline infusion in healthy subjects. Fifteen healthy subjects underwent a 24-h period of fluid restriction. Urine and blood samples were collected at timed intervals. Fifteen healthy subjects were given 7 ml/kg 3% hypertonic saline infusion for 30 min. Urine and blood samples were collected at timed intervals. During fluid restriction, the u-AQP2 rate increased from 3.9 (25th percentile: 3.1; 75th percentile: 5.2) to 7.6 (5.9–9.1; P < 0.001) ng/min, and the plasma AVP (p-AVP) level increased from 0.5 (0.4–0.6) to 3 (1.7–3.3) pmol/l. There was a positive correlation between the maximum change in u-AQP2 rate and the maximum change in p-AVP ( r = 0.57, P < 0.03). During the infusion study, u-AQP2 rate was at maximum 90 min after the infusion [baseline: 4.5 ng/min (3.5–4.8); 90 min: 5 ng/min (4.5–6.0) P < 0.02]. p-AVP increased from 1.0 (0.9–1.1) to 1.5 (1.2–1.8; P < 0.002) pmol/l. There was a positive correlation between the maximum change in u-AQP2 rate and the maximum change in p-AVP ( r = 0.83; P < 0.0001). It can be concluded that p-AVP and u-AQP2 are increased during thirst and hypertonic saline infusion and that u-AQP2 reflects the action of AVP on the collecting ducts.
Ischemic injury induces ADF relocalization to the apical domain of rat proximal tubule cells
Authors: Ashworth Sharon L. , Sandoval Ruben M. , Hosford Melanie , Bamburg James R. , Molitoris Bruce A.
Published: December 2017

Breakdown of proximal tubule cell apical membrane microvilli is an early-occurring hallmark of ischemic acute renal failure. Intracellular mechanisms responsible for these apical membrane changes remain unknown, but it is known that actin cytoskeleton alterations play a critical role in this cellular process. Our laboratory previously demonstrated that ischemia-induced cell injury resulted in dephosphorylation and activation of the actin-binding protein, actin depolymerizing factor [(ADF); Schwartz, N, Hosford M, Sandoval RM, Wagner MC, Atkinson SJ, Bamburg J, and Molitoris BA. Am J Physiol Renal Fluid Electrolyte Physiol 276: F544–F551, 1999]. Therefore, we postulated that ischemia-induced ADF relocalization from the cytoplasm to the apical microvillar microfilament core was an early event occurring before F-actin alterations. To directly investigate this hypothesis, we examined the intracellular localization of ADF in ischemic rat cortical tissues by immunofluorescence and quantified the concentration of ADF in brush-border membrane vesicles prepared from ischemic rat kidneys by using Western blot techniques. Within 5 min of the induction of ischemia, ADF relocalized to the apical membrane region. The length of ischemia correlated with the time-related increase in ADF in isolated brush-border membrane vesicles. Finally, depolymerization of microvillar F-actin to G-actin was documented by using colocalization studies for G- and F-actin. Collectively, these data indicate that ischemia induces ADF activation and relocalization to the apical domain before microvillar destruction. These data further suggest that ADF plays a critical role in microvillar microfilament destruction and apical membrane damage during ischemia.
Contribution of the Na+-K+-2Cl−cotransporter NKCC1 to Cl−secretion in rat OMCD
Authors: Wall Susan M. , Fischer Michael P. , Mehta Pramod , Hassell Kathryn A. , Park Stanley J.
Published: December 2017

In rat kidney the “secretory” isoform of the Na+-K+-2Cl−cotransporter (NKCC1) localizes to the basolateral membrane of the α-intercalated cell. The purpose of this study was to determine whether rat outer medullary collecting duct (OMCD) secretes Cl−and whether transepithelial Cl−transport occurs, in part, through Cl−uptake across the basolateral membrane mediated by NKCC1 in series with Cl−efflux across the apical membrane. OMCD tubules from rats treated with deoxycorticosterone pivalate were perfused in vitro in symmetrical HCO[Formula: see text]/CO2-buffered solutions. Cl−secretion was observed in this segment, accompanied by a lumen positive transepithelial potential. Bumetanide (100 μM), when added to the bath, reduced Cl−secretion by 78%, although the lumen positive transepithelial potential and fluid flux were unchanged. Bumetanide-sensitive Cl−secretion was dependent on extracellular Na+and either K+or NH[Formula: see text], consistent with the ion dependency of NKCC1-mediated Cl−transport. In conclusion, OMCD tubules from deoxycorticosterone pivalate-treated rats secrete Cl−into the luminal fluid through NKCC1-mediated Cl−uptake across the basolateral membrane in series with Cl−efflux across the apical membrane. The physiological role of NKCC1-mediated Cl−uptake remains to be determined. However, the role of NKCC1 in the process of fluid secretion could not be demonstrated.
A mathematical model of rat cortical collecting duct: determinants of the transtubular potassium gradient
Authors: Weinstein Alan M.
Published: December 2017

In assessing disorders of potassium excretion, urine composition is used to calculate the transtubular gradient (TTKG), as an estimate of tubule fluid concentration, at a point when the fluid was last isotonic to plasma, namely, within the cortical collecting duct (CCD). A mathematical model of the CCD has been developed, consisting of principal cells and α- and β-intercalated cells, and which includes Na+, K+, Cl−, HCO[Formula: see text], CO2, H2CO3, phosphate, ammonia, and urea. Parameters have been selected to achieve fluxes and permeabilities compatible with data obtained from perfusion studies of rat CCD under the influence of both antidiuretic hormone and mineralocorticoid. Both epithelial (flat sheet) and tubule models have been configured, and model calculations have focused on the determinants of the TTKG. Using the epithelial model, luminal K+concentrations can be computed at which K+secretion ceases (0-flux equilibrium), and this luminal concentration derives from the magnitude of principal cell peritubular uptake of K+via the Na-K-ATPase, relative to principal cell peritubular membrane K+permeability. When the model is configured as a tubule and examined in the context of conditions in vivo, osmotic equilibration of luminal fluid produces a doubling of the initial K+concentration, which, depending on delivered load, may be substantially greater than the zero-flux equilibrium value. Under such circumstances, the CCD will be a site for K+reabsorption, although the relatively low permeability ensures that this reabsorptive flux is likely to be small. Osmotic equilibration may also raise luminal NH3concentrations well above those in cortical blood. In this situation, diffusive reabsorption of NH3provides a mechanism for base reclamation without the metabolic cost of active proton secretion.
Extracellular nucleotide signaling along the renal epithelium
Authors: Schwiebert Erik M. , Kishore Bellamkonda K.
Published: December 2017

During the past two decades, several cell membrane receptors, which preferentially bind extracellular nucleotides, and their analogs have been identified. These receptors, collectively known as nucleotide receptors or “purinergic” receptors, have been characterized and classified on the basis of their biological actions, their pharmacology, their molecular biology, and their tissue and cell distribution. For these receptors to have biological and physiological relevance, nucleotides must be released from cells. The field of extracellular ATP release and signaling is exploding, as assays to detect this biological process increase in number and ingenuity. Studies of ATP release have revealed a myriad of roles in local regulatory (autocrine or paracrine) processes in almost every tissue in the body. The regulatory mechanisms that these receptors control or modulate have physiological and pathophysiological roles and potential therapeutic applications. Only recently, however, have ATP release and nucleotide receptors been identified along the renal epithelium of the nephron. This work has set the stage for the study of their physiological and pathophysiological roles in the kidney. This review provides a comprehensive presentation of these issues, with a focus on the renal epithelium.
Functional characterization of basolateral and luminal dopamine receptors in rabbit CCD
Authors: Saito Osamu , Ando Yasuhiro , Kusano Eiji , Asano Yasushi
Published: December 2017

Previous studies reported the existence of both D1- and D2-like receptors in the cortical collecting duct (CCD). However, especially with regard to natriuresis, it remains controversial. In the present study, rabbit CCD was perfused to characterize the receptor subtypes responsible for the tubular actions. Basolateral dopamine (DA) induced a dose-dependent depolarization of transepithelial voltage. Basolateral domperidone, a D2-like receptor antagonist, abolished depolarization, whereas SKF-81297, a D1-like receptor agonist, showed no significant change. In addition, bromocriptine, a D2-like receptor agonist, also caused depolarization, whereas SKF-81297, a D1-like receptor agonist, did not depolarize significantly. Moreover, RBI-257, a D4-specific antagonist, reversed the basolateral DA-induced depolarization. In contrast to the basolateral side, luminal DA caused depolarization via a D1-like receptor; however the change was less than that for basolateral DA. For further evaluation,22Na+flux ( JNa) was measured to confirm the effect of DA on Na+transport. Basolateral DA also caused a suppression of JNa, and this reaction was abolished by domperidone. These results suggested that the basolateral D2-like receptor is mainly responsible for the natriuretic action of DA in rabbit CCD.