bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of 20-HETE in mediating the effect of dietary K intake on the apical K channels in the mTAL
Authors: Gu Ruimin , Wei Yuan , Jiang Houli , Balazy Michael , Wang Wenhui
Published: December 2017

We have used the patch-clamp technique to study the effect of dietary K intake on the apical K channels in the medullary thick ascending limb (mTAL) of rat kidneys. The channel activity, defined by the number of channels in a patch and the open probability ( NPo), of the 30- and 70-pS K channels, was 0.18 and 0.11, respectively, in the mTAL from rats on a K-deficient diet. In contrast, NPoof the 30- and 70-pS K channels increased to 0.60 and 0.80, respectively, in the tubules from animals on a high-K diet. The concentration of 20-hydroxyeicosatetraenoic acid (20-HETE) measured with gas chromatography-mass spectrometry was 0.8 pg/μg protein in the mTAL from rats on a high-K diet and increased significantly to 4.6 pg/μg protein in the tubules from rats on a K-deficient diet. Addition of N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or 17-octadecynoic acid (17-ODYA), agents that inhibit the formation of 20-HETE, had no significant effect on the activity of the 30-pS K channels. However, DDMS/17-ODYA significantly increased the activity of the apical 70-pS K channel from 0.11 to 0.91 in the mTAL from rats on a K-deficient diet. In contrast, inhibition of the cytochrome P-450 metabolism of arachidonic acid increased NPofrom 0.64 to 0.81 in the tubules from animals on a high-K diet. Furthermore, the sensitivity of the 70-pS K channel to 20-HETE was the same between rats on a high-K diet and on a K-deficient diet. Finally, the pretreatment of the tubules with DDMS increased NPoof the 70-pS K channels in the mTAL from rats on a K-deficient diet to 0.76. We conclude that an increase in 20-HETE production is involved in reducing the activity of the apical 70-pS K channels in the mTAL from rats on a K-deficient diet.
Peritubular fluid viscosity modulates H+ flux in proximal tubules through NO release
Authors: Díaz-Sylvester Paula , Mac Laughlin Myriam , Amorena Carlos
Published: December 2017

We evaluated the effects of increasing the viscosity (η) in peritubular capillary perfusates (PCP; 20 mM HNaPO4 −-Ringer, pH 7.4) on proximal convoluted tubule (PCT) acidification. Micropuncture experiments were performed with simultaneous luminal and peritubular perfusion. Changes in pH of a 20 mM HNaPO4 −-Ringer (pH 7.4 at t = 0) droplet placed in PCT lumen were measured with H+-sensitive microelectrodes. By adding neutral dextran (molecular wt 300,000–400,000) to the PCP, η was increased. The effect of 10−5 M ATP added to normal-η PCP was evaluated. High η increased H+ flux (85 and 97% when η was increased 20 and 30%, respectively, above the control value). This increase was abolished by adding the nitric oxide antagonist N ω-nitro-l-arginine (l-NNA; 10−4 M) or the purinoreceptor antagonists suramin (10−4 M) and reactive blue 2 (3 × 10−5 M). Addition of 5 × 10−3 Ml-arginine to the peritubular perfusate overcame the inhibitory effect of l-NNA on high-η-induced increase in H+ flux. ATP increased H+ flux (80%), and this effect was blocked by l-NNA. These results suggest that changes in η can modulate proximal H+ flux, at least in part, through ATP-dependent nitric oxide release from the endothelial cells of the peritubular capillaries.
An endocytosis defect as a possible cause of proteinuria in polycystic kidney disease
Authors: Obermüller Nicholas , Kränzlin Bettina , Blum Werner F. , Gretz Norbert , Witzgall Ralph
Published: December 2017

Because proteinuria has been demonstrated in patients with autosomal-dominant polycystic kidney disease (ADPKD), we have investigated whether proteinuria also occurs in the ( cy/ +) rat, a widely used model for ADPKD. Increased urinary excretion of proteins, in particular of albumin, can be found in 16-wk-old ( cy/ +) rats, with a gel electrophoresis pattern compatible with a tubular origin of proteinuria. Using FITC-labeled dextran as an in vivo tracer for renal tubular endosomal function, we could show that portions of cyst-lining epithelia from proximal tubules have lost the ability to endocytose, which is necessary for the reabsorption of low-molecular-weight proteins. By immunohistochemistry, the expression of other proteins implicated in endocytosis, such as the chloride channel ClC-5 and the albumin receptor megalin, correlated well with the presence and absence of FITC-dextran in cysts. As an example of growth factor systems possibly being affected by this endocytosis defect, we could detect increased urinary levels of insulin-like growth factor-I protein in ( cy/ +) animals. These data indicate that proteinuria and albuminuria in the aforementioned rat model for ADPKD are due to a loss of the endocytic machinery in epithelia of proximal tubular cysts. This may also affect the concentration of different growth factors and hormones in cyst fluids and thus modulate cyst development.
Early streptozotocin-diabetes mellitus downregulates rat kidney AT2receptors
Authors: Wehbi George J. , Zimpelmann Joseph , Carey Robert M. , Levine David Z. , Burns Kevin D.
Published: December 2017

The interaction of ANG II with intrarenal AT1receptors has been implicated in the progression of diabetic nephropathy, but the role of intrarenal AT2receptors is unknown. The present studies determined the effect of early diabetes on components of the glomerular renin-angiotensin system and on expression of kidney AT2receptors. Three groups of rats were studied after 2 wk: 1) control (C), 2) streptozotocin (STZ)-induced diabetic (D), and 3) STZ-induced diabetic with insulin implant (D+I), to maintain normoglycemia. By competitive RT-PCR, early diabetes had no significant effect on glomerular mRNA expression for renin, angiotensinogen, or angiotensin-converting enzyme (ACE). In isolated glomeruli, nonglycosylated (41-kDa) AT1receptor protein expression (AT1Aand AT1B) was increased in D rats, with no change in glycosylated (53-kDa) AT1receptor protein or in AT1receptor mRNA. By contrast, STZ diabetes caused a significant decrease in glomerular AT2receptor protein expression (47.0 ± 6.5% of C; P < 0.001; n = 6), with partial reversal in D+I rats. In normal rat kidney, AT2receptor immunostaining was localized to glomerular endothelial cells and tubular epithelial cells in the cortex, interstitial, and tubular cells in the outer medulla, and inner medullary collecting duct cells. STZ diabetes caused a significant decrease in AT2receptor immunostaining in all kidney regions, an effect partially reversed in D+I rats. In summary, early diabetes has no effect on glomerular mRNA expression for renin, angiotensinogen, or ACE. AT2receptors are present in glomeruli and are downregulated in early diabetes, as are all kidney AT2receptors. Our data suggest that alterations in the balance of kidney AT1and AT2receptor expression may contribute to ANG II-mediated glomerular injury in progressive diabetic nephropathy.
Role of 14–3-3ε, c-Myc/Max, and Akt phosphorylation in HIV-1 gp 120-induced mesangial cell proliferation
Authors: Kapasi Aditi A. , Fan Saijun , Singhal Pravin C.
Published: December 2017

Focal glomerulosclerosis (FGS) is the predominant glomerular lesion in patients with human immunodeficiency virus (HIV)-associated nephropathy. Initial mesangial cell hyperplasia and subsequent hypoplasia are common features of FGS. In the present study we evaluated the effect of HIV-1 glycoprotein (gp) 120 on human mesangial cell (HMC) growth. HIV-1 gp 120 stimulated HMC proliferation at lower concentrations, whereas it suppressed cell proliferation at higher concentrations. In parallel to the modulation of cell growth, gp 120 at low concentrations resulted in an increase in the expression of c-Myc, Max, and 14–3-3ε proteins and phosphorylation of ATP-dependent tyrosine kinases (Akt) at Ser473. However, the expression of these proteins decreased with increasing concentrations of gp 120. Furthermore, gp 120 also exhibited a dose-dependent inhibition of Akt phosphorylation at Ser-473 without any significant alteration of Akt expression. Little or no effects of gp 120 were observed on the expression of extracellular signal-regulated kinase (ERK), phospho-ERK, Bcl-2, and Bax proteins. At a higher concentration, gp 120 not only promoted HMC apoptosis but also enhanced expression of Fas and FasL. These results suggest that HIV-1 gp 120 induces alterations in conflicting survival signaling pathways that contribute to the potential dual effects of gp 120 in promoting or inhibiting HMC proliferation.
Remodeling the cellular profile of collecting ducts by chronic carbonic anhydrase inhibition
Authors: Bagnis Corinne , Marshansky Vladimir , Breton Sylvie , Brown Dennis
Published: December 2017

Factors regulating the differentiated phenotype of principal cells (PC) and A- and B-intercalated cells (IC) in kidney collecting ducts are poorly understood. However, we have shown previously that carbonic anhydrase II (CAII)-deficient mice have no IC in their medullary collecting ducts, suggesting a potential role for this enzyme in determining the cellular composition of this tubule segment. We now report that the cellular profile of the collecting ducts of adult rats can be remodeled by inhibiting CA activity in rats by using osmotic pumps containing acetazolamide. The 31-kDa subunit of the vacuolar H+-ATPase, the sodium/hydrogen exchanger regulatory factor NHE-RF, and the anion exchanger AE1 were used to identify IC subtypes by immunofluorescence staining, while aquaporin 2 and aquaporin 4 were used to identify PC. In the cortical collecting ducts of animals treated with acetazolamide for 2 wk, the percentage of B-IC decreased significantly (18 ± 2 vs. 36 ± 4%, P < 0.01) whereas the percentage of A-IC increased (82 ± 2 vs. 64 ± 4%, P < 0.01) with no change in the percentage of total IC in the epithelium. In some treated rats, B-IC were virtually undetectable. In the inner stripe of the outer medulla, the percentage of IC increased in treated animals (48 ± 2 vs. 37 ± 3%, P < 0.05) and the percentage of PC decreased (52 ± 2 vs. 63 ± 3%, P < 0.05). Moreover, IC appeared bulkier, protruded into the lumen, and showed a significant increase in the length of their apical (20.8 ± 0.5 vs. 14.6 ± 0.4 μm, P < 0.05) and basolateral membranes (25.8 ± 0.4 vs. 23.8 ± 0.5 μm, P < 0.05) compared with control rats. In the inner medullary collecting ducts of treated animals, the number of IC in the proximal third of the papilla was reduced compared with controls (11 ± 4 vs. 40 ± 11 IC/mm2, P < 0.05). These data suggest that CA activity plays an important role in determining the differentiated phenotype of medullary collecting duct epithelial cells and that the cellular profile of collecting ducts can be remodeled even in adult rats. The relative depletion of cortical B-IC and the relative increase in number and hyperplasia of A-IC in the medulla may be adaptive processes that would tend to correct or stabilize the metabolic acidosis that would otherwise ensue following systemic carbonic anhydrase inhibition.
Glucose scavenging of nitric oxide
Authors: Brodsky Sergey V. , Morrishow Albert Marcus , Dharia Nimish , Gross Steven S. , Goligorsky Michael S.
Published: December 2017

Endothelial dysfunction accompanies suboptimal glucose control in patients with diabetes mellitus. A hallmark of endothelial dysfunction is a deficiency in production or bioavailability of vascular nitric oxide (NO). Here we demonstrate that acute exposure of human endothelial cells to glucose, at levels found in plasma of diabetic patients, results in a significant blunting of NO responses to the endothelial nitric oxide synthase (eNOS) agonists bradykinin and A-23187. Monitoring of NO generation by purified recombinant bovine eNOS in vitro, using amperometric electrochemical detection and an NO-selective porphyrinic microelectrode, showed that glucose causes a progressive and concentration-dependent attenuation of detectable NO. Addition of glucose to pure NO solutions similarly elicited a sharp decrease in NO concentration, indicating that glucose promotes NO loss. Electrospray ionization-tandem mass spectrometry, using negative ion monitoring, directly demonstrated the occurrence of a covalent reaction involving unitary addition of NO (or a derived species) to glucose. Collectively, our findings reveal that hyperglycemia promotes the chemical inactivation of NO; this glucose-mediated NO loss may directly contribute to hypertension and endothelial dysfunction in diabetic patients.
Chronic NOS inhibition reverses systemic vasodilation and glomerular hyperfiltration in pregnancy
Authors: Cadnapaphornchai Melissa A. , Ohara Mamiko , Morris Kenneth G. , Knotek Mladen , Rogachev Boris , Ladtkow Teri , Carter Ethan P. , Schrier Robert W.
Published: December 2017

The chronic role of nitric oxide (NO), independent of prostaglandin synthesis, in the primary peripheral vasodilation, increased glomerular filtration rate (GFR), and renal plasma flow (RPF) in normal pregnancy remains to be defined. The purpose of the present study was to chronically inhibit NOS to return systemic vascular resistance (SVR), cardiac output (CO), GFR, and RPF to nonpregnant values. Pregnant rats received the nitric oxide synthase (NOS) inhibitor, nitro-l-arginine methyl ester (l-NAME), orally from gestational days 7 through 14. Results were compared with nonpregnant and untreated pregnant rats. At 14 days gestation, CO significantly increased in pregnant vs. nonpregnant rats (187 ± 17 vs. 125 ± 10 ml/min, P < 0.05) as SVR decreased (0.64 ± 0.08 vs. 1.08 ± 0.08 mmHg · ml−1 · min, P < 0.05) and mean arterial pressure was unchanged (117 ± 5 vs. 125 ± 2 mmHg, not significant). Pregnant rats also demonstrated increased GFR (3,015 ± 33 vs. 2,165 ± 136 μl/min, P < 0.01) and RPF (7,869 ± 967 vs. 5,507 ± 290 μl/min, P < 0.05) vs. nonpregnant rats. l-NAME-treated pregnant rats had values for CO (118 ± 7 ml/min), SVR (1.09 ± 0.07 mmHg · ml−1 · min), GFR (2,264 ± 150 μl/min), and RPF (5,777 ± 498 μl/min), which were no different than nonpregnant animals. In summary, similar to human pregnancy, primary peripheral vasodilation occurs early in rat pregnancy. Furthermore, the hyperdynamic circulation and glomerular hyperfiltration of normal rat midterm pregnancy can be chronically reversed by NOS inhibition. These findings suggest a role for endothelial damage and decreased NO in the pathogenesis of preeclampsia.
Renal concentrating defect in mice lacking group IV cytosolic phospholipase A2
Authors: Downey Patricio , Sapirstein Adam , O'Leary Eileen , Sun Tian-Xiao , Brown Dennis , Bonventre Joseph V.
Published: December 2017

Eicosanoids regulate various cellular functions that are important in physiological and pathophysiological processes. Arachidonic acid is released from membranes by phospholipase A2(PLA2) activity. Activated macrophages derived from mice lacking the 85-kDa group IV cytosolic PLA2(cPLA2) have a markedly reduced release of prostaglandin E2and leukotrienes B4and C4. Under basal conditions and after furosemide, urinary prostaglandin E2excretion is reduced in cPLA2-knockout (cPLA2−/−) mice. Serum creatinine, Na+, K+, and Ca2+concentrations, glomerular filtration rate, and fractional excretion of Na+and K+are not different in cPLA2−/−and cPLA2+/+mice. Maximal urinary concentration is lower in 48-h water-deprived cPLA2−/−mice compared with cPLA2+/+animals (1,934 ± 324 vs. 3,541 ± 251 mmol/kgH2O). Plasma osmolality is higher (337 ± 5 vs. 319 ± 3 mmol/kgH2O) in cPLA2−/−mice that lose a greater percentage of their body weight (20 ± 2 vs. 13 ± 1%) compared with cPLA2+/+mice after water deprivation. Vasopressin does not correct the concentrating defect. There is progressive reduction in urinary osmolality with age in cPLA2−/−mice. Membrane-associated aquaporin-1 (AQP1) expression, identified by immunocytochemical techniques, is reduced markedly in proximal tubules of older cPLA2−/−animals but is normal in thin descending limbs. However, Western blot analysis of kidney cortical samples revealed an equivalent AQP1 signal intensity in cPLA2+/+and cPLA2−/−animals. Young cPLA2−/−mice have normal proximal tubule AQP1 staining. Collecting duct AQP2, -3, and -4 were normally expressed in the cPLA2−/−mice. Thus mice lacking cPLA2develop an age-related defect in renal concentration that may be related to abnormal trafficking and/or folding of AQP1 in the proximal tubule, implicating cPLA2in these processes.
Compensatory increase in AQP2, p-AQP2, and AQP3 expression in rats with diabetes mellitus
Authors: Nejsum Lene N. , Kwon Tae-Hwan , Marples David , Flyvbjerg Allan , Knepper Mark A. , Frøkiær Jørgen , Nielsen Søren
Published: December 2017

Diabetes mellitus (DM) is associated with osmotic diuresis and natriuresis. At day 15, rats with DM induced by streptozotocin ( n = 13) had severe hyperglycemia (27.1 ± 0.4 vs. 4.7 ± 0.1 mM in controls) and had a fivefold increase in water intake (123 ± 5 vs. 25 ± 2 ml/day) and urine output. Semiquantitative immunoblotting revealed a significant increase in inner medullary AQP2 (201 ± 12% of control rats, P < 0.05) and phosphorylated (Ser256) AQP2 (p-AQP2) abundance (299 ± 32%) in DM rats. Also, the abundance of inner medullary AQP3 was markedly increased to 171 ± 19% of control levels (100 ± 4%, n = 7, P < 0.05). In contrast, the abundance of whole kidney AQP1 (90 ± 3%) and inner medullary AQP4 (121 ± 16%) was unchanged in rats with DM. Immunoelectron microscopy further revealed an increased labeling of AQP2 in the apical plasma membrane of collecting duct principal cells (with less labeling in the intracellular vesicles) of DM rats, indicating enhanced trafficking of AQP2 to the apical plasma membrane. There was a marked increase in urinary sodium excretion in DM. Only Na+/H+exchanger NHE3 was downregulated (67 ± 10 vs. 100 ± 11%) whereas there were no significant changes in abundance of type 2 Na-phosphate cotransporter (128 ± 6 vs. 100 ± 10%); the Na-K-2Cl cotransporter (125 ± 19 vs. 100 ± 10%); the thiazide-sensitive Na-Cl cotransporter (121 ± 9 vs. 100 ± 10%); the α1-subunit of the Na-K-ATPase (106 ± 7 vs. 100 ± 5%); and the proximal tubule Na-HCO3cotransporter (98 ± 16 vs. 100 ± 7%). In conclusion, DM rats had an increased AQP2, p-AQP2, and AQP3 abundance as well as high AQP2 labeling of the apical plasma membrane, which is likely to represent a vasopressin-mediated compensatory increase in response to the severe polyuria. In contrast, there were no major changes in the abundance of AQP1, AQP4, and several major proximal and distal tubule Na+transporters except NHE3 downregulation, which may participate in the increased sodium excretion.
Acquired lecithin-cholesterol acyltransferase deficiency in nephrotic syndrome
Authors: Vaziri N. D. , Liang K. , Parks J. S.
Published: December 2017

Lecithin-cholesterol acetyltransferase (LCAT) is involved in the synthesis of plasma cholesteryl esters and is pivotal in the maturation of plasma high-density lipoprotein (HDL) and conversion of HDL3 to HDL2. In nephrotic syndrome (NS), the ratio of HDL2 to HDL3 is low even though the total concentration of HDL is generally normal. We hypothesize that the reduced HDL2/HDL3 ratio in NS is due to urinary losses of LCAT, leading to plasma LCAT deficiency. To test this hypothesis, Sprague-Dawley rats were randomized to NS (given 130 mg puromycin aminonucleoside on day 1 and 60 mg ip on day 14) or control groups and were studied on day 30. To dissect the effect of proteinuria from hypoalbuminemia, a group of Nagase rats with inherited hypoalbuminemia was included. Hepatic LCAT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA abundance and plasma and urine LCAT activity were measured. The NS group showed a fourfold rise in serum cholesterol and triglycerides, a fivefold rise in free cholesterol, and a fourfold fall in the HDL-to-total cholesterol ratio. Despite severe hypoalbuminemia, the Nagase rats showed only a mild elevation of serum cholesterol and triglycerides with a normal serum free cholesterol and HDL-to-total cholesterol ratio. The NS group exhibited a normal hepatic LCAT-to-GAPDH mRNA ratio, a marked reduction in plasma LCAT activity, and a significant increase in urinary LCAT excretion. LCAT/GAPDH mRNA and plasma and urine LCAT were normal in Nagase rats. Thus NS led to heavy urinary losses and reduced plasma concentration of LCAT, despite normal hepatic LCAT mRNA abundance. However, hypoalbuminemia, per se, without proteinuria as seen in the Nagase rats had no effect on plasma LCAT or the HDL-to-total cholesterol ratio. Therefore, proteinuria, not hypoalbuminemia, causes LCAT deficiency and a depressed HDL-to-total cholesterol ratio in NS.
Endothelial nitric oxide synthase plays an essential role in regulation of renal oxygen consumption by NO
Authors: Adler Stephen , Huang Harer , Loke Kit E. , Xu Xiaobin , Tada Hideo , Laumas Arupam , Hintze Thomas H.
Published: December 2017

Nitric oxide (NO) regulates renal O2consumption, but the source of NO mediating this effect is unclear. We explored the effects of renal NO production on O2consumption using renal cortex from mice deficient (−/−) in endothelial (e) nitric oxide synthase (NOS). O2consumption was determined polarographically in slices of cortex from control and eNOS(−/−) mice. NO production was stimulated by bradykinin (BK) or ramiprilat (Ram) in the presence or absence of an NOS inhibitor. Basal O2consumption was higher in eNOS(−/−) mice than in heterozygous controls (919 ± 46 vs. 1,211 ± 133 nmol O2· min−1· g−1; P < 0.05). BK and Ram decreased O2consumption significantly less in eNOS(−/−) mice [eNOS(−/−): BK −19.0 ± 2.8%, Ram −20.5 ± 3.3% at 10−4M; control: BK −29.5 ± 2.5%, Ram −34 ± 1.6% at 10−4M]. The NO synthesis inhibitor nitro-l-arginine methyl ester (l-NAME) attenuated this decrease in control but not eNOS(−/−) mice. An NO donor inhibited O2consumption similarly in both groups independent of the presence of l-NAME. These results demonstrate that NO production by eNOS is responsible for regulation of renal O2consumption in mouse kidney.
Localization of prostaglandin E2EP2 and EP4 receptors in the rat kidney
Authors: Jensen Boye L. , Stubbe Jane , Hansen Pernille B. , Andreasen Ditte , Skøtt Ole
Published: December 2017

We investigated the localization of cAMP-coupled prostaglandin E2EP2 and EP4 receptor expression in the rat kidney. EP2 mRNA was restricted to the outer and inner medulla in rat kidney, as determined by RNase protection assay. RT-PCR analysis of microdissected resistance vessels and nephron segments showed EP2 expression in descending thin limb of Henle's loop (DTL) and in vasa recta of the outer medulla. The EP4 receptor was expressed in distal convoluted tubule (DCT) and cortical collecting duct (CCD) in preglomerular vessels, and in outer medullary vasa recta. Butaprost, an EP2 receptor-selective agonist, dose dependently raised cAMP levels in microdissected DTL and outer medullary vasa recta specimens but had no effect in EP2-negative outer medullary collecting duct segments. Dietary salt intake did not alter EP2 expression in the kidney medulla. These results suggest that PGE2may act in the resistance vessels and in the DTL and DCT-CCD segments as a paracrine, cAMP-dependent regulator of vascular resistance and tubular transport, respectively.
Epithelial Na+ channels are regulated by flow
Authors: Satlin Lisa M. , Sheng Shaohu , Woda Craig B. , Kleyman Thomas R.
Published: December 2017

Na+ absorption in the renal cortical collecting duct (CCD) is mediated by apical epithelial Na+ channels (ENaCs). The CCD is subject to continuous variations in intraluminal flow rate that we speculate alters hydrostatic pressure, membrane stretch, and shear stress. Although ENaCs share limited sequence homology with putative mechanosensitive ion channels in Caenorhabditis elegans,controversy exists as to whether ENaCs are regulated by biomechanical forces. We examined the effect of varying the rate of fluid flow on whole cell Na+ currents ( I Na) in oocytes expressing mouse α,β,γ-ENaC (mENaC) and on net Na+ absorption in microperfused rabbit CCDs. Oocytes injected with mENaC but not water responded to the initiation of superfusate flow (to 4–6 ml/min) with a reversible threefold stimulation of I Nawithout a change in reversal potential. The increase in I Na was variable among oocytes. CCDs responded to a threefold increase in rate of luminal flow with a twofold increase in the rate of net Na+ absorption. An increase in luminal viscosity achieved by addition of 5% dextran to the luminal perfusate did not alter the rate of net Na+ absorption, suggesting that shear stress does not influence Na+ transport in the CCD. In sum, our data suggest that flow stimulation of ENaC activity and Na+ absorption is mediated by an increase in hydrostatic pressure and/or membrane stretch. We propose that intraluminal flow rate may be an important regulator of channel activity in the CCD.
cAMP-dependent fluid secretion in rat inner medullary collecting ducts
Authors: Wallace Darren P. , Rome Lorraine A. , Sullivan Lawrence P. , Grantham Jared J.
Published: December 2017

We used an unambiguous in vitro method to determine if inner medullary collecting ducts (IMCD) have intrinsic capacities to absorb and secrete solutes and fluid in an isotonic medium. IMCD1, IMCD2, and IMCD3were dissected from kidneys of young Sprague-Dawley rats. 8-Bromo-3′,5′-cyclic monophosphate (8-BrcAMP) stimulated lumen formation and progressive dilation in all IMCD subsegments; lumen formation was greatest in IMCD1.Benzamil potentiated the rate of lumen expansion in response to 8-BrcAMP. Fluid entered tubule lumens by transcellular secretion rather than simple translocation of intracellular fluid. Secreted lumen solutes were osmometrically active. Inhibition of protein kinase A with H-89 and Rp diastereomer of adenosine 3′,5′-cyclic monophosphorothioate blocked fluid secretion. The rate of lumen expansion was reduced by the selective addition of ouabain, barium, diphenyl-2-carboxylate, bumetanide, glybenclamide, or DIDS, or reduction of extracellular Cl−. We conclude that IMCD absorb and secrete electrolytes and fluid in vitro and that secretion is accelerated by cAMP. We suggest that salt and fluid secretion by the terminal portions of the renal collecting system may have a role in modulating the composition and volume of the final urine.
In vitro phosphorylation of COOH termini of the epithelial Na+channel and its effects on channel activity inXenopusoocytes
Authors: Chigaev Alexander , Lu Gang , Shi Haikun , Asher Carol , Xu Rong , Latter Hedva , Seger Rony , Garty Haim , Reuveny Eitan
Published: December 2017

Recent findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na+channel (ENaC). This study reports the in vitro phosphorylation of the COOH termini of ENaC subunits expressed as glutathione S-transferase fusion proteins. Channel subunits were specifically phosphorylated by kinase-enriched cytosolic fractions derived from rat colon. The phosphorylation observed was not mediated by the serum- and glucocorticoid-regulated kinase sgk. For the γ-subunit, phosphorylation occurred on a single, well-conserved threonine residue located in the immediate vicinity of the PY motif (T630). The analogous residue on β(S620) was phosphorylated as well. The possible role of γT630 and βS620 in channel function was studied in Xenopus laevis oocytes. Mutating these residues to alanine had no effect on the basal channel-mediated current. They do, however, inhibit the sgk-induced increase in channel activity but only in oocytes that were preincubated in low Na+and had a high basal Na+current. Thus mutating γT630 or βS620 may limit the maximal channel activity achieved by a combination of sgk and low Na+.
Immunocytochemical and immunoelectron microscopic localization of α-, β-, and γ-ENaC in rat kidney
Authors: Hager Henrik , Kwon Tae-Hwan , Vinnikova Anna K. , Masilamani Shyama , Brooks Heddwen L. , Frøkiaer Jørgen , Knepper Mark A. , Nielsen Søren
Published: December 2017

Epithelial sodium channel (ENaC) subunit (α, β, and γ) mRNA and protein have been localized to the principal cells of the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) in rat kidney. However, the subcellular localization of ENaC subunits in the principal cells of these cells is undefined. The cellular and subcellular localization of ENaC subunits in rat kidney was therefore examined. Immunocytochemistry demonstrated the presence of all three subunits in principal cells of the CNT, CCD, OMCD, and IMCD. In cortex and outer medulla, confocal microscopy demonstrated a difference in the subcellular localization of subunits. α-ENaC was localized mainly in a zone in the apical domains, whereas β- and γ-ENaC were found throughout the cytoplasm. Immunoelectron microscopy confirmed the presence of ENaC subunits in both the apical plasma membrane and intracellular vesicles. In contrast to the labeling pattern seen in cortex, α-ENaC labeling in IMCD cells was distributed throughout the cytoplasm. In the urothelium covering pelvis, ureters, and bladder, immunoperoxidase and confocal microscopy revealed differences the presence of all ENaC subunits. As seen in CCD, α-ENaC was present in a narrow zone near the apical plasma membrane, whereas β- and γ-ENaC were dispersed throughout the cytoplasm. In conclusion, all three subunits of ENaC are expressed throughout the collecting duct (CD), including the IMCD as well as in the urothelium. The intracellular vesicular pool in CD principal cells suggests ENaC trafficking as a potential mechanism for the regulation of Na+reabsorption.
Angiotensin IV induces tyrosine phosphorylation of focal adhesion kinase and paxillin in proximal tubule cells
Authors: Chen Jian-Kang , Zimpelmann Joe , Harris Raymond C. , Burns Kevin D.
Published: December 2017

Angiotensin IV (ANG IV), the COOH-terminal hexapeptide fragment of angiotensin II (ANG II), binds to specific sites in the kidney, distinct from type 1 (AT1) and type 2 (AT2) receptors and designated type 4 (AT4) receptors. We determined signaling pathways for ANG IV in a proximal tubular cell line, LLC-PK1/Cl4. In these cells, we found no specific binding of [125I]-ANG II. In contrast, ANG IV dose dependently competed for [125I]-labeled ANG IV binding, with no displacement by either ANG II, the AT1receptor antagonist losartan, or the AT2 antagonist PD-123319. Saturation binding indicated the presence of AT4receptors of high affinity [dissociation constant ( K d) = 1.4 nM]. ANG IV did not affect cAMP or cGMP production and did not increase cytosolic calcium concentration in these cells. In contrast, immunoprecipitation and immunoblotting studies revealed that ANG IV caused dose-dependent tyrosine phosphorylation of p125-focal adhesion kinase (p125-FAK) and p68-paxillin within 2 min, with maximal stimulation at 30 min. ANG IV-stimulated tyrosine phosphorylation of p125-FAK and paxillin was not affected by pretreatment with either losartan or PD-123319, and ANG II (10−7 M) did not induce protein tyrosine phosphorylation. Our results indicate that LLC-PK1/Cl4 cells express ANG IV receptors, which we demonstrate for the first time are linked to tyrosine phosphorylation of focal adhesion-associated proteins. This suggests that ANG IV, a product of ANG II metabolism, may regulate function of the focal adhesion complex in proximal tubule cells.
Uremic levels of BUN do not cause nitric oxide deficiency in rats with normal renal function
Authors: Xiao Shen , Erdely Aaron , Wagner Laszlo , Baylis Chris
Published: December 2017

In vitro, 7 days of high blood urea nitrogen (BUN) inhibits endothelial l-arginine transport and nitric oxide synthase (NOS) activity. The present study investigates whether 7 days of high BUN in vivo influences renal hemodynamics, blood pressure (BP), and/or the nitric oxide (NO) system. Normal rats were fed low-nitrate food containing 30% urea for 7 days, which increased BUN (15 ± 1 to 69 ± 4 mg/100 ml, P < 0.001). High BUN did not reduce 24-hour urinary nitrite/nitrate excretion (a measure of total NO production). Baseline BP and renal hemodynamics were unaffected by high BUN as were the pressor and renal vasoconstrictor responses to acute NOS inhibition with NG-nitro-l-arginine-methyl ester. In addition, high BUN had no impact on renal corticall-arginine concentration, density of either endothelial NOS or neuronal NOS protein, or renal cortical NOS activity. NOS activity in the brain cerebellum was also unaffected. In conclusion, high BUN did not lead to vasoconstriction or NO deficiency in rats with normal renal function. Further studies are needed to evaluate the effect of high BUN on the NO system in rats with progressive renal functional insufficiency.
Physiological regulation of cyclooxygenase-2 in the kidney
Authors: Harris Raymond C. , Breyer Matthew D.
Published: December 2017

In adult mammalian kidney, cyclooxygenase-2 (COX-2) expression is found in a restricted subpopulation of cells. The two sites of renal COX-2 localization detected in all species to date are the macula densa (MD) and associated cortical thick ascending limb (cTALH) and medullary interstitial cells (MICs). Physiological regulation of COX-2 in these cellular compartments suggests functional roles for eicosanoid products of the enzyme. COX-2 expression increases in high-renin states (salt restriction, angiotensin-converting enzyme inhibition, renovascular hypertension), and selective COX-2 inhibitors significantly decrease plasma renin levels, renal renin activity, and mRNA expression. There is evidence for negative regulation of MD/cTALH COX-2 by angiotensin II and by glucocorticoids and mineralocorticoids. Conversely, nitric oxide generated by neuronal nitric oxide synthase is a positive modulator of COX-2 expression. Decreased extracellular chloride increases COX-2 expression in cultured cTALH, an effect mediated by increased p38 mitogen-activated protein kinase activity, and, in vivo, a sodium-deficient diet increases expression of activated p38 in MD/cTALH. In contrast to COX-2 in MD/cTALH, COX-2 expression increases in MICs in response to a high-salt diet as well as water deprivation. Studies in cultured MICs have confirmed that expression is increased in response to hypertonicity and is mediated, at least in part, by nuclear factor-κB activation. COX-2 inhibition leads to apoptosis of MICs in response to hypertonicity in vitro and after water deprivation in vivo. In addition, COX-2 metabolites appear to be important mediators of medullary blood flow and renal salt handling. Therefore, there is increasing evidence that COX-2 is an important physiological mediator of kidney function.
Optimized suspension culture: the rotating-wall vessel
Authors: Hammond T. G. , Hammond J. M.
Published: December 2017

Suspension culture remains a popular modality, which manipulates mechanical culture conditions to maintain the specialized features of cultured cells. The rotating-wall vessel is a suspension culture vessel optimized to produce laminar flow and minimize the mechanical stresses on cell aggregates in culture. This review summarizes the engineering principles, which allow optimal suspension culture conditions to be established, and the boundary conditions, which limit this process. We suggest that to minimize mechanical damage and optimize differentiation of cultured cells, suspension culture should be performed in a solid-body rotation Couette-flow, zero-headspace culture vessel such as the rotating-wall vessel. This provides fluid dynamic operating principles characterized by 1) solid body rotation about a horizontal axis, characterized by colocalization of cells and aggregates of different sedimentation rates, optimally reduced fluid shear and turbulence, and three-dimensional spatial freedom; and 2) oxygenation by diffusion. Optimization of suspension culture is achieved by applying three tradeoffs. First, terminal velocity should be minimized by choosing microcarrier beads and culture media as close in density as possible. Next, rotation in the rotating-wall vessel induces both Coriolis and centrifugal forces, directly dependent on terminal velocity and minimized as terminal velocity is minimized. Last, mass transport of nutrients to a cell in suspension culture depends on both terminal velocity and diffusion of nutrients. In the transduction of mechanical culture conditions into cellular effects, several lines of evidence support a role for multiple molecular mechanisms. These include effects of shear stress, changes in cell cycle and cell death pathways, and upstream regulation of secondary messengers such as protein kinase C. The discipline of suspension culture needs a systematic analysis of the relationship between mechanical culture conditions and biological effects, emphasizing cellular processes important for the industrial production of biological pharmaceuticals and devices.
Downregulation of AQP1, -2, and -3 after ureteral obstruction is associated with a long-term urine-concentrating defect
Authors: Li Chunling , Wang Weidong , Kwon Tae-Hwan , Isikay Levent , Wen Jian Guo , Marples David , Djurhuus Jens Christian , Stockwell Anette , Knepper Mark A. , Nielsen Søren , Frøkiær Jørgen
Published: December 2017

Previously, we demonstrated that 24 h of bilateral ureteral obstruction (BUO) and short-term release of BUO was associated with a decrease in the expression of aquaporin-2 (AQP2), polyuria, and a reduced urinary concentrating capacity (10). The purposes of the present study were to examine whether BUO and the long-term release of BUO (BUO-R) for 3, 14, and 30 days were associated with changes in the expression of renal AQP1, AQP2, and AQP3 and whether such changes were associated with parallel changes in urinary output and urinary concentrating capacity. Rats ( n = 4–7 in each group) were kept in metabolic cages for measurements of urinary output. Kidneys were removed to determine the expression levels of AQP1, AQP2, and AQP3 by semiquantitative immunoblotting. AQP2 was downregulated after 24 h of BUO (42 ± 3%). Downregulation of AQP2 persisted 3 (43 ± 14%; P < 0.01) and 15 days after BUO-R (48 ± 11%; P < 0.01) but was normalized 30 days after BUO-R. AQP3 showed a similar pattern. Moreover, AQP1 was downregulated in response to BUO (65 ± 7%) and remained downregulated 3 days after BUO-R (41 ± 5%), 14 days after BUO-R (57 ± 8%), and 30 days after BUO-R (59 ± 5%). BUO-R resulted in a significant polyuria that gradually decreased, although it remained significant at day 30. Urinary concentrating capacity remained significantly impaired when determined 3, 14, and 30 days after BUO-R in response to a 24-h period of thirst (1,712 ± 270 vs. 2,880 ± 91 mosmol/kgH2O at day 30, P < 0.05). In conclusion, the expression of AQP1, AQP2, and AQP3 were long-term downregulated after BUO-R, suggesting that dysregulation of aquaporins located at the proximal tubule, thin descending limb of the loop of Henle, and the collecting duct may contribute to the long-term polyuria and impairment of urinary concentrating capacity associated with obstructive nephropathy.
HGF promotes adhesion of ATP-depleted renal tubular epithelial cells in a MAPK-dependent manner
Authors: Liu Zhen-Xiang , Nickel Christian H. , Cantley Lloyd G.
Published: December 2017

Hepatocyte growth factor (HGF) has been shown to enhance recovery from renal tubular ischemia. We investigated the possibility that HGF improves recovery by preventing ischemia-induced loss of cell adhesion. Murine inner medullary collecting duct-3 (mIMCD-3) cells subjected to 90% ATP depletion demonstrated a 55% decrease in adhesion, an effect that was completely reversed by the addition of HGF. Assays examining release of adherent cells revealed similar results with 30 min of ATP depletion causing loss of adhesion of 25% of mIMCD-3 cells and HGF completely reversing this effect. In contrast, HGF was unable to reverse the loss of adhesion of cells exposed to 99% ATP depletion. Examination of the mitogen-activated protein kinase (MAPK) signaling pathway revealed that HGF could induce extracellular signal-regulated kinase (ERK) phosphorylation in control and 90% ATP-depleted cells but not in 99% ATP-depleted cells. Inhibition of ERK activation with U0126 completely blocked the HGF-dependent reversal of ATP-depleted cell adhesion. Thus ATP-depleted cells demonstrate a marked decrease in cell adhesion that is reversible by the addition of HGF. This effect of HGF requires activation of the MAPK pathway.
Cell surface expression and turnover of the α-subunit of the epithelial sodium channel
Authors: Kleyman Thomas R. , Zuckerman Jonathan B. , Middleton Pamela , McNulty Kathleen A. , Hu Baofeng , Su Xuefeng , An Bing , Eaton Douglas C. , Smith Peter R.
Published: December 2017

The renal epithelial cell line A6, derived from Xenopus laevis, expresses epithelial Na+channels (ENaCs) and serves as a model system to study hormonal regulation and turnover of ENaCs. Our previous studies suggest that the α-subunit of Xenopus ENaC (α- xENaC) is detectable as 150- and 180-kDa polypeptides, putative immature and mature α-subunit heterodimers. The 150- and 180-kDa α- xENaC were present in distinct fractions after sedimentation of A6 cell lysate through a sucrose density gradient. Two anti-α- xENaC antibodies directed against distinct domains demonstrated that only 180-kDa α- xENaC was expressed at the apical cell surface. The half-life of cell surface-expressed α- xENaC was 24–30 h, suggesting that once ENaC matures and is expressed at the plasma membrane, its turnover is similar to that reported for mature cystic fibrosis transmembrane conductance regulator. No significant changes in apical surface expression of α- xENaC were observed after treatment of A6 cells with aldosterone for 24 h, despite a 5.3-fold increase in short-circuit current. This lack of change in surface expression is consistent with previous observations in A6 cells and suggests that aldosterone regulates ENaC gating and increases channel open probability.
Noninvasive measurement of concurrent single-kidney perfusion, glomerular filtration, and tubular function
Authors: Krier James D. , Ritman Erik L. , Bajzer Zeljko , Romero J. Carlos , Lerman Amir , Lerman Lilach O.
Published: December 2017

To assess the reliability of electron beam computed tomography (EBCT), measurements of single-kidney renal blood flow (RBF), glomerular filtration rate (GFR), and intratubular contrast medium concentration (ITC) of radiographic contrast media were quantified in anesthetized pigs before and after acetylcholine-induced vasodilation and diuresis. EBCT measurements were compared with those obtained with intravascular Doppler and inulin clearance. The capability of EBCT to detect chronic changes in single-kidney function was evaluated in pigs with unilateral renal artery stenosis, and their long-term reproducibility in normal pigs was studied repeatedly at 1-mo intervals. EBCT-RBF (ml/min) correlated with Doppler-RBF as RBFEBCT= 45 + 1.07 ∗ RBFDoppler, r = 0.81. EBCT-GFR (ml/min) correlated with inulin clearance as GFREBCT= 11.7 + 1.02 ∗ GFRinulin, r = 0.80. During vasodilation, RBF and GFR increased, whereas ITC decreased along the nephron. In renal artery stenosis, single-kidney GFR decreased linearly with the degree of stenosis, and ITC increased along the nephron, indicating increased fluid reabsorption. EBCT-RBF, GFR, and ITC were similar among repeated measurements. This approach might be invaluable for simultaneous quantification of regional hemodynamics and function in the intact kidneys, in a manner potentially applicable to humans.
Autoradiographic analysis and regulation of angiotensin receptor subtypes AT4, AT1, and AT(1—7)in the kidney
Authors: Handa Rajash K. , Handa Shelly E. , Elgemark Monica K. S.
Published: December 2017

Receptor autoradiography revealed that angiotensin AT4receptors were abundantly expressed in normal mammalian (mouse, rat, gerbil, guinea pig, rabbit) and avian (sparrow, chicken, turkey) kidneys and were more extensively distributed than previously reported (including proximal and distal segments of the nephron, interstitium, renal artery, vein, and ureter). Angiotensin AT4receptors were generally found to be more abundant than angiotensin AT1receptors in mammalian kidneys, whereas angiotensin AT(1—7)receptors were not detected in either mammalian or avian kidneys. Rats subjected to various chronic treatments were found to preferentially decrease kidney AT4receptor density (furosemide, puromycin aminonucleoside, nitro-l-arginine methyl ester), decrease kidney AT1receptor density (bilateral ureteral obstruction), or increase kidney AT1receptor distribution in the inner medulla (water diuresis). These results indicate that the AT4receptor can be expressed in numerous cell types within the normal kidney of several species. Furthermore, several models of renal dysfunction and injury have been identified that selectively alter kidney AT4density and may potentially aid in elucidating the role of this novel angiotensin receptor system in renal function.
Role of COX-2-derived metabolites in regulation of the renal hemodynamic response to norepinephrine
Authors: Llinás Maria T. , López Ruth , Rodríguez Francisca , Roig Francisco , Salazar F. Javier
Published: December 2017

The objective of this study was to examine the role of cylcooxygenase (COX)-2-derived prostaglandins (PG) in modulating the renal hemodynamic effects of norepinephrine (NE) during low or normal sodium intake. The relative contribution of each COX isoform in producing the PG that attenuate the renal NE effects during normal sodium intake was also evaluated. The renal response to three doses of NE (50, 100, and 250 ng · kg−1· min−1) was evaluated in anesthetized dogs pretreated with vehicle, a selective COX-2 inhibitor (nimesulide), or a nonselective COX inhibitor (meclofenamate). Intrarenal infusion of the two lower doses of NE in vehicle-pretreated dogs with normal sodium intake ( n = 8) elicited an increase in renal vascular resistance (RVR; 21 and 34%) without inducing changes in glomerular filtration rate (GFR). The highest dose of NE in this group induced a further increment in RVR (113%) and a decrease in GFR (33%). Pretreatment with nimesulide in dogs with normal sodium intake ( n = 7) did not modify the NE-induced increments in RVR but enhanced the decreases in GFR induced by the three NE doses (12, 26, and 64%). The renal hemodynamic response to NE in meclofenamate-pretreated dogs with normal sodium intake ( n = 7) was similar to that found in dogs pretreated with nimesulide. Infusion of the lowest dose of NE to vehicle-pretreated dogs with low sodium intake ( n = 6) did not modify GFR and elicited an increase in RVR (42%). Infusion of the second and third doses of NE led to a decrease in GFR (35 and 91%) and a rise in RVR (82 and 587%). Infusion of the first two doses of NE in nimesulide-pretreated dogs with low sodium intake ( n= 5) induced a fall in GFR (64 and 92%) and an increase in RVR (174 and 2,293%) that were greater ( P < 0.05) than those induced by NE in vehicle-pretreated dogs. The elevation in the urinary excretion rates of PGE2and 6-keto-PGF1αelicited by NE was prevented in the nimesulide-pretreated dogs. Our results show that COX-2 inhibition potentiates the renal hemodynamic effects of NE and propose that the PG involved in modulating them are mainly derived from COX-2 activity.