bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of P2X1 purinoceptors by autoradiography and immunohistochemistry in rat kidneys
Authors: Chan C. M. , Unwin R. J. , Bardini M. , Oglesby I. B. , Ford A. P. D. W. , Townsend-Nicholson A. , Burnstock G.
Published: December 2017

P2 receptors have been identified in rat kidney by autoradiography, using the radioligand [3H]α,β-methylene ATP, and by immunohistochemistry, using a polyclonal antibody to the P2X1 purinoceptor. They have been localized to the vascular smooth muscle of intrarenal arteries, including arcuate and interlobular arteries, and afferent arterioles, but not glomeruli, postglomerular efferent arterioles, or renal tubules. We conclude that at least some of the P2 receptors present on vascular smooth muscle are of the P2X1 subtype. The functional significance of these findings in the vascular control of the kidney is discussed.
Dose-dependent effects of CRF-like diuretic peptide on transcellular and paracellular transport pathways
Authors: Clark Thomas M. , Hayes Timothy K. , Beyenbach Klaus W.
Published: December 2017

The mechanism of action of synthetic Culexcorticotropin-releasing factor (CRF)-like diuretic peptide (CCRF-DP) was investigated in isolated, perfused Malpighian tubules of the yellow fever mosquito, Aedes aegypti. Low concentrations of CCRF-DP (10−10and 10−9M) caused depolarizing oscillations of the lumen-positive transepithelial voltage ( Vt) in Malpighian tubules, whereas high concentrations (10−8and 10−7M) first depolarized and then transiently hyperpolarized Vt; CCRF-DP always lowered transepithelial resistance ( Rt), regardless of voltage depolarization or hyperpolarization. The short-circuit current ( Isc), an electrical estimate of active transepithelial transport of Na and K, remained unchanged at low concentrations of CCRF-DP, but Iscmore than doubled at high concentrations. These effects of CCRF-DP suggest dose-dependent sites of action: low concentrations of CCRF-DP affect the paracellular pathway, and high concentrations affect both paracellular and transcellular pathways.
A mathematical model of the inner medullary collecting duct of the rat: pathways for Na and K transport
Authors: Weinstein Alan M.
Published: December 2017

A mathematical model of the inner medullary collecting duct (IMCD) of the rat has been developed representing Na+, K+, Cl−,[Formula: see text] CO2, H2CO3, phosphate, ammonia, and urea. Novel model features include: finite rates of hydration of CO2, a kinetic representation of the H-K-ATPase within the luminal cell membrane, cellular osmolytes that are regulated in defense of cell volume, and the repeated coalescing of IMCD tubule segments to yield the ducts of Bellini. Model transport is such that when entering Na+is 4% of filtered Na+, approximately 75% of this load is reabsorbed. This requirement renders the area-specific transport rate for Na+comparable to that for proximal tubule. With respect to the luminal membrane, there is experimental evidence for both NaCl cotransport and an Na+channel in parallel. The experimental constraints that transepithelial potential difference is small and that the fractional apical resistance is greater than 85% mandate that more than 75% of luminal Na+entry be electrically silent. When Na+delivery is limited, an NaCl cotransporter can be effective at reducing luminal Na+concentration to the observed low urinary values. Given the rate of transcellular Na+reabsorption, there is necessarily a high rate of peritubular K+recycling; also, given the lower bound on luminal membrane Cl−reabsorption, substantial peritubular Cl−flux must be present. Thus, if realistic limits on cell membrane electrical resistance are observed, then this model predicts a requirement for peritubular electroneutral KCl exit.
Renoprotective effects of nitric oxide in angiotensin II-induced hypertension in the rat
Authors: Chin So Yeon , Wang Chi-Tarng , Majid Dewan S. A. , Navar L. Gabriel
Published: December 2017

Experiments were performed in anesthetized male Sprague-Dawley rats to determine whether increased nitric oxide (NO) activity during the development of hypertension exerts a protective effect on renal cortical blood flow (CBF) and medullary blood flow (MBF). The effects of acute NO synthase inhibition on renal function and on CBF and MBF, measured by laser-Doppler flow probes, were evaluated in control and ANG II-infused hypertensive rats, prepared by the infusion of ANG II at a rate of 65 ng/min via osmotic minipumps implanted subcutaneously for 13 days. In normotensive rats ( n = 8), intravenous infusion of Nω-nitro-l-arginine (NLA; 20 μg ⋅ 100 g−1⋅ min−1) decreased CBF by 21 ± 4% and MBF by 49 ± 8% and increased blood pressure from 118 ± 1 to 140 ± 2 mmHg. In ANG II-infused rats ( n = 7), CBF and MBF decreased by 46 ± 5% and 25 ± 6%, respectively, during infusion of NLA. Arterial pressure increased from 160 ± 5 to 197 ± 7 mmHg, which was a greater absolute increase than in normotensive controls. Basal renal blood flow (RBF), estimated from p-aminohippurate clearance and hematocrit, was similar in both the control (6.0 ± 0.5 ml ⋅ min−1⋅ g−1) and hypertensive (6.0 ± 0.6 ml ⋅ min−1⋅ g−1) rats. However, NLA-induced reductions in RBF averaged 60 ± 5% in the hypertensive rats, compared with 31 ± 9% observed in control rats. GFR in control (0.97 ± 0.03 ml ⋅ min−1⋅ g−1) and hypertensive rats (0.78 ± 0.12 ml ⋅ min−1⋅ g−1) decreased to a similar extent during the first 30-min period of NLA infusion. GFR returned toward control levels in control rats; in contrast, GFR remained significantly decreased in the ANG II-infused rats (0.58 ± 0.11 ml ⋅ min−1⋅ g−1). Basal urinary sodium excretion (0.2 ± 0.08 μeq ⋅ min−1⋅ g−1), fractional excretion of sodium (0.3 ± 0.13%), and urine flow (4.9 ± 0.39 μl ⋅ min−1⋅ g−1) in hypertensive rats did not increase significantly after NLA treatment as occurred in normotensive controls. These data suggest that a compensatory increase in nitric oxide activity partially counteracts the vasoconstrictor influence of elevated ANG II levels to regulate renal hemodynamics and maintain cortical perfusion in the renal circulation.
Maturation of rabbit proximal straight tubule chloride/base exchange
Authors: Shah Mehul , Quigley Raymond , Baum Michel
Published: December 2017

The present in vitro microperfusion study compared the mechanism and rates of NaCl transport in neonatal and adult rabbit proximal straight tubules. In proximal straight tubules perfused with a late proximal tubular fluid and bathed in a serumlike albumin solution, the rate of volume absorption ( J V) was 0.54 ± 0.10 and 0.12 ± 0.05 nl ⋅ mm−1 ⋅ min−1in adults and neonates, respectively ( P < 0.05). With the addition of 10−5 M bath ouabain, J Vdecreased to 0.27 ± 0.07 and −0.03 ± 0.04 nl ⋅ mm−1 ⋅ min−1in adult and neonatal tubules, respectively ( P < 0.05), consistent with lower rates of active and passive NaCl transport in the neonatal proximal straight tubule. The effect of luminal sodium and chloride removal on intracellular pH was used to assess the relative rates of Na+/H+and Cl−/base exchange. The rates of Na+/H+and Cl−/base exchange were approximately fivefold less in neonatal proximal straight tubules than adult tubules. In both neonatal and adult proximal straight tubules, the rate of Cl−/base exchange was not affected by formate, bicarbonate, or cyanide and acetazolamide, consistent with Cl−/OH−exchange. These data demonstrate an increase in proximal straight tubule NaCl transport during postnatal renal development.
Dietary sodium affects systemic and renal hemodynamic response to NO inhibition in healthy humans
Authors: Bech J. N. , Nielsen C. B. , Ivarsen P. , Jensen K. T. , Pedersen E. B.
Published: December 2017

Animal studies have indicated that increased nitric oxide (NO) synthesis plays a significant role in the renal adaptation to increased sodium intake. To investigate the role of NO during increased sodium intake in humans, we studied the effect of acute, systemic injection of NG-monomethyl-l-arginine (l-NMMA) on renal hemodynamics [glomerular filtration rate and renal plasma flow (GFR and RPF, respectively)], urinary sodium excretion (FENa), systemic hemodynamics [mean arterial blood pressure and heart rate (MAP and HR)], and plasma levels of several vasoactive hormones in 12 healthy subjects during high (250 mmol/day) and low (77 mmol/day) sodium intake in a crossover design. The sodium diets were administered for 5 days before the l-NMMA treatments, in randomized order, with a washout period of 9 days between each diet and l-NMMA treatment. GFR and RPF were measured using the renal clearance of51Cr-labeled EDTA and125I-labeled hippuran by the constant infusion technique in clearance periods of 30-min duration. Two baseline periods were obtained, after whichl-NMMA was given (3 mg/kg over 10 min), and the effect of treatment was followed over the next five clearance periods. During high sodium intake,l-NMMA induced a more pronounced relative decrease in RPF ( P = 0.0417, ANOVA), a more pronounced relative decrease in FENa( P = 0.0032, ANOVA), and a more pronounced relative increase in MAP ( P= 0.0231, ANOVA). During low sodium intake, the effect ofl-NMMA on FENawas abolished. During low sodium intake, l-NMMA induced a sustained drop in plasma renin (31 ± 5 vs. 25 ± 5 μU/ml, P < 0.001), which was not seen during high sodium intake. The data indicate that increased production of NO is an important part of the adaptation to increased dietary sodium intake in healthy humans, with respect to renal hemodynamics, sodium excretion, and the secretion of renin.
Increased calcium oxalate monohydrate crystal binding to injured renal tubular epithelial cells in culture
Authors: Verkoelen Carl F. , Van Der Boom Burt G. , Houtsmuller Adriaan B. , Schröder Fritz H. , Romijn Johannes C.
Published: December 2017

The retention of crystals in the kidney is considered to be a crucial step in the development of a renal stone. This study demonstrates the time-dependent alterations in the extent of calcium oxalate (CaOx) monohydrate (COM) crystal binding to Madin-Darby canine kidney (MDCK) cells during their growth to confluence and during the healing of wounds made in confluent monolayers. As determined by radiolabeled COM crystal binding studies and confirmed by confocal-scanning laser microscopy, relatively large amounts of crystals (10.4 ± 0.4 μg/cm2) bound to subconfluent cultures that still exhibited a low transepithelial electrical resistance (TER < 400 Ω ⋅ cm2). The development of junctional integrity, indicated by a high resistance (TER > 1,500 Ω ⋅ cm2), was followed by a decrease of the crystal binding capacity to almost undetectable low levels (0.13 ± 0.03 μg/cm2). Epithelial injury resulted in increased crystal adherence. The highest level of crystal binding was observed 2 days postinjury when the wounds were already morphologically closed but TER was still low. Confocal images showed that during the repair process, crystals selectively adhered to migrating cells at the wound border and to stacked cells at sites were the wounds were closed. After the barrier integrity was restored, crystal binding decreased again to the same low levels as in undamaged controls. These results indicate that, whereas functional MDCK monolayers are largely protected against COM crystal adherence, epithelial injury and the subsequent process of wound healing lead to increased crystal binding.
Regulation of the polymeric immunoglobulin receptor by water intake and vasopressin in the rat kidney
Authors: Rice James C. , Spence Jeff S. , Megyesi Judit , Safirstein Robert L. , Goldblum Randall M.
Published: December 2017

The polymeric immunoglobulin receptor (pIgR) transports polymeric immunoglobulins (IgA) from the basolateral to the apical surface of epithelial cells. At the apical surface, its amino-terminal domain, termed secretory component (SC), is proteolytically cleaved and released either unbound (free SC) or bound to IgA. We examined the effects of changes in water balance and vasopressin on the production and secretion of the pIgR in the rat kidney in vivo. Water deprivation induced a 2.7-fold increase in the pIgR mRNA and a 2.2-fold increase in intracellular pIgR protein compared with water-loaded animals. Physiological doses of desmopressin reproduced the effects of water deprivation on mRNA and intracellular protein levels, suggesting that pIgR expression may be regulated by a vasopressin-coupled mechanism. Secretion of free SC and secretory IgA in the urine, however, correlated directly with water intake and urine flow. These results suggest that hydration status and vasopressin may affect the mucosal immunity of the kidney by regulating at different steps the epithelial cell production and secretion of the polymeric immunoglobulin transporter/secretory component.
Effect of metabolic acidosis on NaCl transport in the proximal tubule
Authors: Wang Tong , Egbert Allan L. , Aronson Peter S. , Giebisch Gerhard
Published: December 2017

In metabolic acidosis, the capacity of the proximal tubule for bicarbonate absorption is enhanced, whereas NaCl reabsorption is inhibited. Recent evidence indicates that transcellular NaCl absorption in the proximal tubule is mediated by apical membrane Cl−/formate exchange and Cl−/oxalate exchange, in parallel with recycling of these organic anions. We evaluated whether the effect of metabolic acidosis to inhibit NaCl reabsorption in the proximal tubule is due at least in part to inhibition of organic anion-dependent NaCl transport in this nephron segment. Absorption rates of bicarbonate ([Formula: see text]), chloride ( JCl), and fluid ( Jv) were measured in rat proximal tubule segments microperfused in situ. We confirmed that metabolic acidosis stimulates[Formula: see text]in tubules microperfused with 25 mM[Formula: see text], pH 7.4. For measurements of JCl, tubules were microperfused with a low-bicarbonate (5 mM), high-chloride solution, simulating conditions in the late proximal tubule. Under these conditions, baseline JCland Jvmeasured in the absence of formate and oxalate were not significantly different between control and acidotic rats. However, whereas addition of 50 μM formate or 1 μM oxalate to luminal and capillary perfusates markedly stimulated JCland Jvin control rats, formate and oxalate failed to stimulate JCland Jvin acidotic rats. We conclude that metabolic acidosis markedly downregulates organic anion-stimulated NaCl absorption, thereby allowing differential regulation of proximal tubule NaHCO3and NaCl transport.
Role of heat stress response in the tolerance of immature renal tubules to anoxia
Authors: Gaudio Karen M. , Thulin Gunilla , Mann Andrea , Kashgarian Michael , Siegel Norman J.
Published: December 2017

The stress response was studied in suspensions of tubules from immature (IT) and mature (MT) rats after noninjury, heat, oxygen, and anoxia. Under all conditions, IT exhibited more exuberant activation of heat shock transcription factor (HSF) than MT. Characterization of activated HSF in immature cortex revealed HSF1. Also, 2 h after each condition, heat shock protein-72 (HSP-72) mRNA was twofold in IT. As the metabolic response to 45 min of anoxia, 20-min reoxygenation was assessed by measuring O2consumption (O2C). Basal O2C was manipulated with ouabain, nystatin, and carbonylcyanide p-chloromethyoxyphenylhydrazone (CCCP). Basal O2C in IT were one-half the value of MT. After anoxia, basal O2C was reduced by a greater degree in MT. Ouabain reduced O2C to half the basal value in both noninjured and anoxic groups. Basal O2C was significantly stimulated by nystatin but not to the same level following anoxia in MT and IT. Basal O2C was also stimulated by CCCP, but after anoxia, CCCP O2C was significantly less in MT with no decrease in IT, suggesting mitochondria are better preserved in IT. Also, O2C devoted to nontransport activity was better maintained in IT.
Functional and molecular evidence for P2X receptors in LLC-PK1cells
Authors: Filipovic Dragana M. , Adebanjo Olugbenga A. , Zaidi Mone , Reeves W. Brian
Published: December 2017

Extracellular ATP affects a wide variety of cells via purinergic membrane receptors. One class of purinergic receptors, P2X, consists of ATP-gated, calcium-permeable, cation-selective channels. We performed whole cell patch-clamp studies, intracellular calcium concentration ([Ca2+]i) measurements, and reverse transcription-polymerase chain reaction (RT-PCR) to determine whether P2X receptors are expressed in LLC-PK1cells. First, in patch-clamp studies, 100 μM ATP depolarized the cell membrane and increased the whole cell conductance of LLC-PK1cells. This response was dose dependent and inhibited by 100 μM suramin, a P2 receptor antagonist. The ATP-induced conductance was cation selective but did not discriminate between Na+and K+. ADP, α,β-methylene-ATP, and β,γ-methylene-ATP had no effect on the whole cell conductance. Next, 10 μM ATP caused a rapid rise in [Ca2+]iin LLC-PK1cells. This effect of ATP was inhibited by the absence of extracellular calcium and by suramin but not by pretreatment with pertussis toxin. ADP and β,γ-methylene- ATP had little or no effect on [Ca2+]i. Finally, RT-PCR produced a 330-bp fragment from LLC-PK1cell RNA, whose sequence was 80% identical to the rat P2X1receptor. We conclude that LLC-PK1cells express purinergic receptors of the P2X class, which mediate depolarization and calcium entry when activated.
Regulation of B-type intercalated cell apical anion exchange activity by CO2/ HCO 3 −
Authors: Milton Amy E. , Weiner I. David
Published: December 2017

The cortical collecting duct (CCD) B cell possesses an apical anion exchanger dissimilar to AE1, AE2, and AE3. The purpose of these studies was to characterize this transporter more fully by examining its regulation by CO2and[Formula: see text]. We measured intracellular pH (pHi) in single intercalated cells of in vitro microperfused CCD using the fluorescent, pH-sensitive dye, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In the absence of extracellular CO2/[Formula: see text], luminal Cl−removal caused reversible intracellular alkalinization, identifying this transporter as a Cl−/base exchanger able to transport bases other than [Formula: see text]. Adding extracellular CO2/[Formula: see text]decreased B cell pHiwhile simultaneously increasing Cl−/base exchange activity. Since intracellular acidification inhibits AE1, AE2, and AE3, we examined mechanisms other than pHiby which the stimulation occurred. These studies showed that B cell apical anion exchange activity was CO2stimulated and carbonic anhydrase dependent. Moreover, the stimulation was independent of luminal bicarbonate, luminal pH or pHi, and changes in buffer capacity. We conclude that the B cell possesses an apical Cl−/base exchanger whose activity is regulated by CO2-stimulated, carbonic anhydrase-dependent cytoplasmic [Formula: see text]formation.
Human proximal tubular epithelial cells express somatostatin: regulation by growth factors and cAMP
Authors: Turman Martin A. , Apple Courtney A.
Published: December 2017

Somatostatin modulates several renal tubular cell functions, including gluconeogenesis and proliferation. In this study, we demonstrate that cultured human proximal tubular epithelial cells (PTEC) express somatostatin. We also demonstrate positive and negative regulation of PTEC somatostatin production. We found that PTEC derived from 14 different human donors consistently expressed somatostatin mRNA and/or peptide as detected by RT-PCR and enzyme-linked immunoassay. Furthermore, Northern blot analysis revealed that PTEC express the same size mRNA transcript (750 nucleotides) as human thyroid carcinoma (TT) cells. The PTEC mitogens, epidermal growth factor (EGF) and hydrocortisone, inhibit PTEC somatostatin secretion, whereas forskolin (a direct stimulator of adenylate cyclase) and fetal bovine serum stimulate secretion. These findings raise the possibility that renal-derived somatostatin modulates tubular cell function in an autocrine/paracrine manner. Manipulation of this pathway may lead to novel methods with which to alter tubular cell proliferation and function in vivo.
NCC27, a homolog of intracellular Cl−channel p64, is expressed in brush border of renal proximal tubule
Authors: Tulk Barry M. , Edwards John C.
Published: December 2017

NCC27, a 27-kDa homolog of the intracellular chloride channel p64, was recently described as a chloride channel in nuclear membrane. We probed human Northern blots for NCC27 and found an ∼1.7-kb message in all tissues examined, including kidney, the transcript being most abundant in heart and skeletal muscle. NCC27-specific antisera was raised to a COOH-terminal peptide derived from the NCC27 coding region. Using this antisera, we find NCC27 is expressed in an intracellular vesicular compartment in HeLa cells, PancI cells, and macrophages. In human and mouse kidney, NCC27 is expressed at low levels in most cells of the kidney. NCC27 is highly expressed in glomeruli, in periarterial smooth muscle, and in the apical membrane of a subset of cortical tubule cells. Double staining with nephron segment-specific lectins indicates that the NCC27-expressing cells are proximal tubule cells.
A serum-free in vitro model of renal microvessel development
Authors: Antes Lisa M. , Villar Monica M. , Decker Sylvia , Nicosia Roberto F. , Kujubu Dean A.
Published: December 2017

The differentiation and organization of the embryonic renal vasculature is a crucial event in renal development. To study this process, we developed a serum-free in vitro model of renal microvessel development. Mouse embryonic kidney explants, when embedded specifically in type I collagen, demonstrate outgrowth of microvascular structures when stimulated by the phorbol ester 12- O-tetradecanoylphorbol 13-acetate (TPA, 10–50 ng/ml). Other polypeptide growth factors stimulated little, if any, microvessel outgrowth from the explants. Similar outgrowths were not observed when other embryonic tissue explants were used. The number of microvessels observed depended on the gestational age of the explants. We hypothesize that TPA induces the in situ differentiation of metanephric mesenchymal cells into endothelial cell precursors and that specific matrix proteins and cell-matrix interactions are necessary for the organization of these precursors into microvessels. Our model will allow us to examine in detail the responsiveness of metanephric kidney cells to both growth factors and extracellular matrix molecules and to understand how they influence renal endothelial cell differentiation.
Escape from vasopressin-induced antidiuresis: role of vasopressin resistance of the collecting duct
Authors: Ecelbarger Carolyn A. , Chou Chung-Lin , Lee Alanna J. , DiGiovanni Susan R. , Verbalis Joseph G. , Knepper Mark A.
Published: December 2017

Previously, we demonstrated that escape from vasopressin-induced antidiuresis (“vasopressin escape”) in rats is associated with a large, selective decrease in whole kidney expression of aquaporin-2, the vasopressin-regulated water channel. Here, we show that isolated perfused inner medullary collecting ducts (IMCDs) from vasopressin-escape rats {desamino-[d-arginine]vasopressin (DDAVP)/water-loaded} have dramatically reduced vasopressin-dependent osmotic water permeabilities [46% of control rats (DDAVP alone)], which coincides with a fall in inner medullary aquaporin-2 protein abundance as measured by immunoblotting in the opposite kidney. Furthermore, we demonstrate in IMCD suspensions that cAMP accumulation in response to DDAVP is substantially reduced in the vasopressin-escape rats both in the presence and absence of the phosphodiesterase inhibitor IBMX. By immunoblotting, we show that the abundance of two proteins important in cAMP generation: the stimulatory heterotrimeric G protein subunit Gsα and adenylyl cyclase type VI, do not change. We conclude that vasopressin escape is associated with relative vasopressin resistance of the collecting duct cells manifested by decreased intracellular cAMP levels. The decreased cAMP levels can contribute to the demonstrated decrease in collecting duct water permeability in two ways: 1) by causing a decrease in aquaporin-2 expression and 2) by limiting the acute action of vasopressin to increase collecting duct water permeability.
Activation of purinergic P2Y2 receptors inhibits inducible NO synthase in cultured rat mesangial cells
Authors: Mohaupt Markus G. , Fischer Tina , Schwöbel Jörg , Sterzel R. Bernd , Schulze-Lohoff Eckhard
Published: December 2017

Cytokine-induced nitric oxide (NO) is produced on glomerular inflammation. Glomerular injury and thrombocyte aggregation result in the release of nucleotides, which may regulate induced NO synthesis in cultured rat mesangial cells (MCs). ATP (10−3M) inhibited 24-h nitrite production induced by lipopolysaccharide (LPS, 10 μg/ml)/interferon-γ (IFN-γ, 100 U/ml) by 48.2 ± 6.3%, as well as induction of inducible NOS (iNOS) protein and mRNA. Also, coincubation with either 10−4M of UTP, ATP, or ATPγS inhibited LPS/IFN-γ-induced nitrite production by 29.9 ± 5.8, 36.4 ± 4.3, and 50.3 ± 6.5%, respectively, indicating involvement of purinergic P2Y2 receptors. Correspondingly, cultured MCs expressed P2Y2 receptor mRNA. Agonists for other purinergic receptors [α,β-methylene-ATP, 3′- O-(4-benzoyl)-benzoyl-ATP, 2-methylthio-ATP, ADP, UDP, adenosine] were ineffective. Treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 10−8M) reproduced the inhibitory effect of ATP on iNOS protein expression and nitrite inhibition (by 46.6 ± 10.4%). The effect of ATP or PMA was reversed by the PKC inhibitors Ro-31-8220 (10−8M) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (10−5M), indicating that suppression of iNOS is mediated via activation of PKC through stimulated P2Y2 receptors. In conclusion, the release of purine mediators may play a critical role for iNOS expression and synthesis of NO during glomerular inflammatory disorders.
Ligand-dependent regulation of NPR-A gene expression in inner medullary collecting duct cells
Authors: Cao Li , Chen Song Cang , Cheng Tong , Humphreys Michael H. , Gardner David G.
Published: December 2017

Atrial natriuretic peptide (ANP) interacts with high-affinity, guanylyl cyclase-linked receptors in the inner medullary collecting duct (IMCD), where it exerts important regulatory control over sodium handling. We sought to determine whether receptor activity in these cells would be modulated (downregulated) by prolonged exposure to ligand. A number of natriuretic peptides (ANP, brain natriuretic peptide, and urodilatin) were found to decrease ligand-dependent natriuretic peptide receptor A (NPR-A) activity in IMCD cells. This inhibition was in direct proportion to their capacity to increase basal cGMP levels in this cell population. The reduction in receptor activity was accompanied by a dose- and time-dependent reduction in NPR-A mRNA levels in these cells. The decrease in transcript levels arose, in part, from a reduction in NPR-A gene transcription. ANP reduced NPR-A gene promoter activity in a transiently transfected IMCD cell population. 8-Bromo-cGMP was also effective in inhibiting NPR-A mRNA levels and NPR-A promoter activity, suggesting that the second messenger (i.e., cGMP) rather than ANP, itself, is responsible for downregulation of NPR-A gene expression.
Expression of synaptotagmin VIII in rat kidney
Authors: Kishore Bellamkonda K. , Wade James B. , Schorr Kristel , Inoue Takeaki , Mandon Béatrice , Knepper Mark A.
Published: December 2017

The synaptotagmins are a family of integral membrane proteins proposed to function as regulators of both exocytosis and endocytosis. Here, we have used immunochemical techniques and RT-PCR to assess sites of renal expression of synaptotagmin VIII. A polyclonal antibody was raised to a synthetic peptide corresponding to the carboxy-terminal 21 amino acids of mouse synaptotagmin VIII. On immunoblots of membrane fractions from renal cortex and medulla (and in several other tissues), the antibody labeled a 52-kDa band (absent with preimmune IgG). Immunofluorescence localization was carried out in tissue sections from rat kidney. The synaptotagmin VIII antibody labeled early proximal tubules, thin ascending limbs, thick ascending limbs, connecting tubules, and collecting ducts. In collecting ducts, both type A and B intercalated cells exhibited basolateral labeling, whereas principal cells were labeled chiefly in the apical and subapical portion of the cells. Thick ascending limbs were labeled in both the basolateral and apical regions. RT-PCR experiments using total RNA extracted from cortex and medulla or microdissected inner medullary collecting ducts gave a single band of appropriate size. Sequencing of the PCR product confirmed that the amplified target is synaptotagmin VIII. We conclude that synaptotagmin VIII is broadly expressed among renal tubule epithelia, raising the possibility that it is involved in regulation of transport and/or cell remodeling at several sites in the nephron and collecting duct.
Cadmium is more toxic to LLC-PK1cells than to MDCK cells acting on the cadherin-catenin complex
Authors: Zimmerhackl L. B. , Momm F. , Wiegele G. , Brandis M.
Published: December 2017

Cadmium toxicity to renal cells was investigated in Madin-Darby canine kidney (MDCK) and LLC-PK1cells as models of the distal tubule/collecting duct and proximal tubule, respectively. Cells were grown on two-compartment filters and exposed to 0.1–50 μM Cd2+. In MDCK cells, Cd2+was more toxic from the basolateral than from the apical side and dependent on the extracellular Ca2+concentration. Toxicity was evident within 24 h, as shown by a decrease in transepithelial resistance (TER), reduced proliferation (bromodeoxyuridine incorporation), reduction in ATP concentration, and morphological changes. On confocal microscopy, E-cadherin and α-catenin staining patterns indicated interference with the cadherin-catenin complex. LLC-PK1cells showed a similar toxicity pattern, which was evident at lower Cd2+concentrations. An increase of E-cadherin and α-catenin molecules in the Triton X-100-insoluble fraction was detectable at high Cd2+concentrations in LLC-PK1cells but not in MDCK cells. Lactate dehydrogenase release indicated membrane leakage in LLC-PK1cells. Rhodamine-phalloidin staining, a probe for F-actin filaments, demonstrated alterations of the actin cytoskeleton in both cell lines. In conclusion, cadmium caused ATP depletion and interfered with the cadherin-catenin complex and probably the tight junctions changing renal cell morphology and function.
Effects of dietary salt on adrenomedullin and its receptor mRNAs in rat kidney
Authors: Jensen Boye L. , Gambaryan Stepan , Schmaus Elke , Kurtz Armin
Published: December 2017

There is accumulating evidence that adrenomedullin (ADM) is involved in the control of salt and water homeostasis. ADM is considered to act primarily in a paracrine fashion, and since the kidneys are target organs for ADM, we investigated the localization and regulation of ADM and ADM receptor (ADM-R) mRNAs in the kidney. mRNAs for ADM and ADM-R were colocalized in renal vessels, glomeruli, and inner medullary collecting ducts. ADM mRNA was also detected in proximal tubules, whereas ADM-R mRNA was found in distal convoluted tubules. By ribonuclease protection assay, the abundance of ADM mRNA was fourfold higher in cortex than in outer medulla and papilla. In isolated glomeruli, ADM mRNA was threefold higher compared with cortex. Conversely, ADM-R mRNA was fourfold higher in papilla than in renal cortex. This distribution of mRNAs for ADM and ADM-R suggests a cortical source of ADM and a preferential action of ADM in the papilla. Ten days of feeding a low-salt (0.02%) or a high-salt diet (4%) did not change ADM mRNA or ADM-R mRNA in any kidney zone.
Dopamine inhibits vasopressin-dependent cAMP production in the rat cortical collecting duct
Authors: Li Li , Schafer James A. ,   (With the Technical Assistance of Mary L. Watkins)
Published: December 2017

Dopamine inhibits Na+ and water reabsorption in the rat cortical collecting duct (CCD) in the presence of arginine vasopressin (AVP). This inhibition appears to involve the D4 dopamine receptor isoform, which inhibits cAMP production; however, the D1A receptor, which stimulates cAMP production, is also expressed in the CCD. To discriminate between these opposing effects, we measured cAMP production in intact CCD segments. The basal rate of cAMP production ranged from 6.5 to 10 fmol/mm of tubule length over a 7-min incubation period, and it was unaffected by either dopamine or the D1A-specific agonist fenoldopam. AVP increased cAMP production to the range of 85–153 fmol ⋅ mm−1 ⋅ 7 min−1. Whereas neither 0.1 nor 1.0 μM fenoldopam affected AVP-dependent cAMP production, dopamine reduced it in a dose-dependent manner, achieving a maximum inhibition of 50% at 10 μM. This effect was reversed by the D4 receptor antagonist clozapine but not by pimozide or spiperone (antagonists of D2 and D3 receptors) or by calphostin C or chelerythrine (inhibitors of protein kinase C). We conclude that dopamine inhibits transepithelial Na+ transport and osmotic water permeability in the presence of AVP by inhibition of cAMP production, which is mediated by the D4receptor isoform linked via the inhibitory G protein Gi.
Immunolocalization of sat-1 sulfate/oxalate/bicarbonate anion exchanger in the rat kidney
Authors: Karniski Lawrence P. , Lötscher Marius , Fucentese Monica , Hilfiker Helen , Biber Jürg , Murer Heini
Published: December 2017

The rat liver sulfate/bicarbonate/oxalate exchanger (sat-1) transports sulfate across the canalicular membrane in exchange for either bicarbonate or oxalate. Sulfate/oxalate exchange has been detected in the proximal tubule of the kidney, where it is probably involved in the reabsorption of filtered sulfate and the secretion of oxalate and may contribute to oxalate-dependent chloride reabsorption. Screening of a renal cortex cDNA library determined that sat-1 is expressed in the rat kidney. To evaluate this anion exchanger, the sat-1 protein was expressed in Sf9 cells. Sodium-independent sulfate and oxalate uptake was enhanced 7.3-fold and 13.1-fold, respectively, in Sf9 cells expressing the sat-1 protein compared with cells infected with wild-type virus. We determined that sat-1 is glycosylated in the kidney; however, anion exchange via sat-1 is observed despite incomplete glycosylation of sat-1 in Sf9 cells. The sat-1 protein, with an added COOH-terminal 6-histidine tag, was purified on a metal affinity column and used to generate anti-sat-1 monoclonal antibodies. The sat-1 protein was localized to the basolateral membrane, but not the apical membrane, of the proximal tubule by both Western blot analysis and immunohistochemistry. These studies demonstrate that sulfate/oxalate exchange on the apical and basolateral membranes of the proximal tubule represents transport on two different anion exchangers.
Modulation of polymyxin B effects on mammalian urinary bladder
Authors: Berg Jamie R. , Spilker Christian M. , Lewis Simon A.
Published: December 2017

This report demonstrates that Ca2+, Mg2+, and protons alter the ability of polymyxin B (PX, a cationic antibiotic used clinically as a bactericidal agent) to increase the apical membrane conductance of the rabbit urinary bladder. Using electrophysiological methods, we determine that these alterations occur by two mechanisms. First, they blocked the PX-induced conductance in a rapid and reversible manner; second, they competed with PX for a membrane binding site. In addition, Ca2+(but not Mg2+or protons) altered the rate at which the induced conductance could be reversed. When solution pH was greater than 8.8, PX was not able to induce a conductance. This ability of high pH to inhibit the action of PX was due to a decrease in the number of positive charges on PX. Further studies demonstrated that for maximal activity, PX required its fatty acid tail. These data were used to develop a model describing the mechanism by which PX can induce a conductance in the apical membrane of the rabbit urinary bladder.
Adrenergic- and capsaicin-evoked nitric oxide release from urothelium and afferent nerves in urinary bladder
Authors: Birder Lori A. , Apodaca Gerard , De Groat William C. , Kanai Anthony J.
Published: December 2017

Nitric oxide (NO) has been implicated in the regulation of the lower urinary tract. However, the source(s) of NO production in the urinary bladder (UB) has not been determined. Accordingly, we used a porphyrinic microsensor placed on the surface of UB strips in vitro to directly measure endogenous NO production. The afferent neurotoxin, capsaicin, and the mixed α/β-adrenergic agonist, norepinephrine (NE), both evoked transient (1–3 s) NO release (range 50 nM to 1.4 μM). Adrenergic-mediated release was not decreased following denervation of the UB but was abolished following selective removal of the mucosa. On the other hand, release evoked by capsaicin (range 50–900 nM) was significantly decreased after UB denervation. These data indicate that NE releases NO from UB epithelium, and capsaicin releases NO from epithelium as well as nervous tissue in the UB. In light of reports that NO may regulate epithelial integrity and function in other tissues, agonist regulation of a constitutive nitric oxide synthase activity in the UB may provide a novel mechanism for modulation of bladder and urothelial function.
Regulation of expression of type II sodium-phosphate cotransporters by protein kinases A and C
Authors: Lederer Eleanor D. , Sohi Sameet S. , Mathiesen Jeanine M. , Klein Jon B.
Published: December 2017

The purpose of the present study was to determine the effect of protein kinase A and protein kinase C activation on the membrane expression of NaPi-4, the type II sodium-phosphate cotransporter in OK cells. NaPi-4 expression was measured using polyclonal antisera produced in rabbits against a peptide identical to the carboxy-terminal 12-amino acid sequence of NaPi-4. The antisera identified an apically localized protein by confocal imaging of intact OK cells and a broad band of 110–140 kDa by immunoblot analysis of OK cell membranes. Treatment of OK cells with parathyroid hormone (PTH) decreased the intensity of the 110- to 140-kDa band, which was detectable by 2 h, maximal by 4 h at 62%, and sustained for 24 h. 8-Bromo-cAMP (8-BrcAMP) inhibited NaPi-4 expression for up to 24 h by over 90%. However, phorbol 12-myristate 13-acetate inhibited NaPi-4 expression by less than 10%. PTH-(3–34), a fragment which stimulates only protein kinase C, inhibited phosphate transport but also had no effect on NaPi-4 expression. We conclude that protein kinase A but not protein kinase C inhibits sodium-phosphate uptake in OK cells by downregulation of NaPi-4 expression.
Cloning, functional characterization, and localization of a rat renal Na+-dicarboxylate transporter
Authors: Sekine Takashi , Cha Seok Ho , Hosoyamada Makoto , Kanai Yoshikatsu , Watanabe Nobuaki , Furuta Yoshitake , Fukuda Kuniaki , Igarashi Takashi , Endou Hitoshi
Published: December 2017

We report here the isolation, functional characterization, tissue distribution, and membrane localization of rat renal Na+-dicarboxylate transporter (rNaDC-1). rNaDC-1 consists of 2,245 nucleotides, and the deduced amino acid sequence showed 73% and 75% identity to rabbit and human NaDC-1, respectively. When expressed in Xenopus laevis oocytes, rNaDC-1 mediated sodium-dependent uptake of di- and tricarboxylates. Substrates of rNaDC-1 evoked inward currents in oocytes expressed with rNaDC-1; succinate, α-ketoglutarate, and glutarate were relatively high-affinity substrates, and citrate was a low-affinity substrate of rNaDC-1. The coupling ratio of citrate to charge was determined to be 1:1 at pH 7.4; influx of one positive charge per citrate molecule suggests a symport of three Na+with a divalent citrate. Expression of rNaDC-1 mRNA was detected in the kidney and the small and large intestines. Immunohistochemistry using polyclonal antibodies raised against the 14 amino acids at the COOH terminus of rNaDC-1 revealed that rNaDC-1 is localized exclusively in the luminal membrane of S2 and S3.
Urea transporters in kidney: molecular analysis and contribution to the urinary concentrating process
Authors: Tsukaguchi Hiroyasu , Shayakul Chairat , Berger Urs V. , Hediger Matthias A.
Published: December 2017

Facilitated urea transporters (UTs) are responsible for urea accumulation in the renal inner medulla of the mammalian kidney and therefore play a central role in the urinary concentrating process. Recently, the cDNAs encoding three members of the UT family, UT1, UT2, and UT3 have been cloned. These transporters are expressed in different structures of the mammalian kidney. In rat, UT1 resides in the apical membrane of terminal inner medullary collecting ducts, where it mediates vasopressin-regulated urea reabsorption. UT2 and UT3 are located in descending thin limbs of Henle’s loop and descending vasa recta, respectively, and participate in urinary recycling processes, which minimize urea escape from the inner medulla. UT1 and UT2 are regulated independently and respond differently to changes in dietary protein content and hydration state. Identification and characterization of these urea transporters advances our understanding of the molecular basis and regulation of the urinary concentrating mechanism.
Long-term regulation of urinary concentrating capacity
Authors: Knepper Mark A.
Published: December 2017

Urinary concentrating capacity is regulated in part by a long-term adaptational process involving changes in the absolute abundance of the aquaporin-2 water channel in collecting duct cells. Alterations in aquaporin-2 abundance play key roles in the pathophysiology of several water balance disorders. Escape from the antidiuretic action of vasopressin, e.g. in the syndrome of inappropriate antidiuretic hormone secretion, involves a selective downregulation of aquaporin-2 expression. Excessive water retention causing hyponatremia in volume-expanded states such as congestive heart failure appears to be due in part to a failure of this escape mechanism.
Mg2+/Ca2+sensing inhibits hormone-stimulated Mg2+ uptake in mouse distal convoluted tubule cells
Authors: Bapty Brian W. , Dai Long-Jun , Ritchie Gordon , Canaff Lucie , Hendy Geoffrey N. , Quamme Gary A.
Published: December 2017

The distal convoluted tubule plays a significant role in renal magnesium conservation. An immortalized mouse distal convoluted tubule (MDCT) cell line has been extensively used to study the cellular mechanisms of magnesium transport in this nephron segment. MDCT cells possess an extracellular polyvalent cation-sensing mechanism responsive to Mg2+, Ca2+, and neomycin. The present studies determined the effect of Mg2+/Ca2+sensing on hormone-mediated cAMP formation and Mg2+ uptake in MDCT cells. MDCT cells were Mg2+ depleted by culturing in Mg2+-free media for 16 h, and Mg2+ uptake was measured by microfluorescence after placing the depleted cells in 1.5 mM MgCl2. The mean rate of Mg2+ uptake was 164 ± 5 nM/s in control MDCT cells. Activation of Mg2+/Ca2+sensing with neomycin did not affect basal Mg2+ uptake (155 ± 5 nM/s). We have previously reported that treatment of MDCT cells with either glucagon or arginine vasopressin (AVP) stimulated Mg2+ entry. In the present studies, the addition of extracellular Mg2+ or Ca2+ inhibited glucagon- and AVP-stimulated cAMP formation and Mg2+ uptake in concentration-dependent manner with half-maximal concentrations of ∼1.5 and 3.0 mM, respectively. Exogenous cAMP or forskolin stimulated Mg2+ uptake in the presence of Mg2+/Ca2+sensing activation. We infer from these studies that Mg2+/Ca2+-sensing mechanisms located in the distal convoluted tubule may play a role in control of distal magnesium absorption.
Immunolocalization of the Na+/H+exchanger isoform NHE2 in rat kidney
Authors: Chambrey Régine , Warnock David G. , Podevin René-Alexandre , Bruneval Patrick , Mandet Chantal , Bélair Marie-France , Bariéty Jean , Paillard Michel
Published: December 2017

Four Na+/H+exchangers (NHE1 to NHE4) have been detected in the kidney. Renal NHE2 expression sites have not been fully established. We have raised rabbit antisera against an oligopeptide related to the amino acids 652 to 661 of rat NHE2. Western blot analysis of plasma membrane fractions isolated from rat renal cortex showed that affinity-purified anti-NHE2 antibody detected an 85-kDa protein in apical but not in basolateral membranes. The labeling of this 85-kDa protein was specifically blocked by preincubation of the antibody with its monomeric peptide, indicating specific recognition. Indirect immunolabeling was performed on sections of paraformaldehyde-fixed rat kidney embedded in paraffin. Strong staining was seen in the apical membrane of cortical thick ascending limbs, distal convoluted tubules, and connecting tubules. Much weaker apical staining was found in medullary thick ascending limbs of Henle. In the inner medulla, some thin limbs were intensively labeled by the anti-NHE2 antibody. No staining could be detected in any segments of the proximal tubule and collecting duct.
Neuronal nitric oxide synthase is expressed in principal cell of collecting duct
Authors: Wang Xianhong , Lu Ming , Gao Yongzong , Papapetropoulos Andreas , Sessa William C. , Wang Wenhui
Published: December 2017

We used the RT-PCR technique and immunocytochemical methods to determine the expression of endothelial nitric oxide synthase (eNOS) or neuronal nitric oxide synthase (nNOS) in the cortical collecting duct (CCD) in rats on high-K+diet. The microdissected CCDs of the rat kidney were lysed, and RT-PCR was carried out using rat nNOS and eNOS gene-specific primers. Southern analysis showed the presence of mRNA of nNOS but not eNOS in the CCD. The presence of nNOS in the CCD was further confirmed by light microscopy. We used the polyclonal nNOS antibody in immunocytochemical studies of the isolated CCD. We found that immunoreactivity to nNOS was present in the CCD and heterogeneous with positive and negative immunostaining. We performed the immunocytochemical studies in the split-open CCD and found that the immunoreactivity to nNOS was detected only in principal cells but not in intercalated cells. We conclude that nNOS is expressed in the rat CCD in rats on high-K+diet. The presence of nNOS in the CCD is heterogeneous and mainly located in principal cells.