bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of insulin-like growth factor I on the renal juxtamedullary microvasculature
Authors: Tönshoff Burkhard , Kaskel Frederick J. , Moore Leon C.
Published: December 2017

To characterize the effects on the rat renal preglomerular microvasculature of insulin-like growth factor I (IGF-I), experiments were performed using the in vitro blood-perfused juxtamedullary nephron preparation. IGF-I induced a reversible vasodilation of pre- but not postglomerular microvessels in a dose-dependent manner (10−9–10−7M). The IGF-I-induced vasodilation was similar in all preglomerular vascular segments: interlobular artery, 11.5 ± 1.2% of control ( n = 16); mid-afferent arterioles, 11.6 ± 1.7% ( n = 24); and juxtaglomerular afferent segments, 16.1 ± 2.8% ( n = 19). Renal autoregulatory capacity was not reduced by IGF-I. Pretreatment with the nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine methyl ester (10−4M) completely inhibited the vasodilatory response to IGF-I. IGF-I induced a rapid increase of NO concentration in intact renal microvessels, monitored by a NO-selective voltametric microelectrode. Pretreatment with the cyclooxygenase inhibitor indomethacin (10−5M) not only abrogated the IGF-I-induced dilation, but, moreover, IGF-I elicited a small but significant (∼10%) vasoconstriction in all preglomerular vessels. These results indicate that the renal vascular effects of IGF-I involve activation of two endogenous vasodilators (NO and vasodilatory prostaglandins). In addition, IGF-I may also release an undefined vasoconstrictor.
HCO 3 − absorption in rabbit outer medullary collecting duct: role of luminal carbonic anhydrase
Authors: Tsuruoka Shuichi , Schwartz George J.
Published: December 2017

Membrane-bound luminal carbonic anhydrase (CA) IV, by catalyzing the dehydration of carbonic acid into CO2 plus water, facilitates H+ secretion in the renal outer medullary collecting duct from the inner stripe (OMCDi). To examine the role of CA IV on H+ secretion, we measured net [Formula: see text] transport in perfused OMCDi segments and examined the effect on transport of two extracellular CA inhibitors, benzolamide and F-3500, aminobenzolamide coupled to a nontoxic polymer, polyoxyethylene bis(acetic acid) [synthesized and kindly provided by C. Conroy and T. Maren (C. W. Conroy, G. C. Wynns, and T. H. Maren. Bioorg. Chem. 24: 262–272, 1996)]. These agents would inhibit only the luminal CA enzyme. Dose titration curves for net[Formula: see text] flux were performed for each drug. Basal [Formula: see text] absorptive flux was 12 pmol ⋅ min−1 ⋅ mm−1in control segments and significantly increased to 16 pmol ⋅ min−1 ⋅ mm−1in segments from 3-day acid-treated animals. The concentrations of benzolamide and F-3500 that inhibited[Formula: see text] absorption by 50% were ∼0.1 and ∼5 μM, similar to the K i for CA IV inhibition by these agents (0.2 and 4.0 μM, respectively; T. Maren, C. W. Conroy, G. C. Wynns, and D. R. Godman. J. Pharmacol. Exp. Ther. 280: 98–104, 1997). Adding exogenous CA to the inhibitor in the perfusate nearly restored basal [Formula: see text] transport, suggesting that cytosolic CA II was not inhibited by these impermeant inhibitors. In OMCDi segments from acidotic rabbits, the concentrations of benzolamide and F-3500 that inhibited[Formula: see text] absorption by 50% were 50 and 500 μM, respectively, >100 times the K i for CA IV inhibition and for inhibition of [Formula: see text]transport in control tubules. Thus, in the OMCDi, doses of extracellular CA inhibitors that inhibited ∼50% of CA IV activity also comparably inhibited [Formula: see text] transport, indicating that H+ secretion depends in part on the availability of luminal CA IV activity. Acidosis substantially decreased the sensitivity of [Formula: see text]transport to CA inhibition.
Heat-shock protein 25 induction and redistribution during actin reorganization after renal ischemia
Authors: Aufricht Christoph , Ardito Thomas , Thulin Gunilla , Kashgarian Michael , Siegel Norman J. , Van Why Scott K.
Published: December 2017

The small heat-shock proteins appear to have a regulatory role in actin dynamics. Since cytoskeletal disruption is integral to ischemic renal injury, we evaluated expression and intracellular distribution of heat-shock protein 25 (HSP-25) in rat renal cortex after 45 min of renal ischemia. HSP-25 was constitutively expressed and induced by ischemia with peak levels reached by 6 h reflow. Ischemia caused a shift of HSP-25 from the detergent-soluble into the insoluble cytoskeletal fraction. By 2 h reflow, the majority of HSP-25 had redistributed into the soluble fraction. HSP-25 was predominantly localized in a subapical distribution in control proximal tubules, a pattern intermediate between deoxyribonuclease (DNase)-reactive and filamentous actin. After ischemia, HSP-25 dispersed through the cytoplasm with small punctate accumulations similar to DNase-reactive actin. During later reflow, all three proteins were found in coarse intracytoplasmic accumulations; however, HSP-25 and DNase-reactive actin were in separate accumulations. HSP-25 and microfilamentous actin staining returned to the subapical domain. Thus the temporal and spatial patterns of HSP-25 induction and distribution suggest specific interactions between HSP-25 and actin during the early postischemic reorganization of the cytoskeleton. HSP-25 may have additional roles distinct from actin dynamics later in the course of postischemic recovery.
Renal growth and development in mice lacking AT1Areceptors for angiotensin II
Authors: Oliverio Michael I. , Madsen Kirsten , Best Christopher F. , Ito Masaki , Maeda Nobuyo , Smithies Oliver , Coffman Thomas M.
Published: December 2017

To examine the role of the type 1A (AT1A) angiotensin receptor in renal growth and development, we analyzed F2 progeny from a series of crosses between F1 mice that were heterozygous for a targeted disruption of the AT1Areceptor gene [ Agtr1A-(+/−)]. Among 21-day-old weanling F2 mice, we found that 194 (32%) were homozygous for the wild-type allele Agtr1A-(+/+), 299 (49%) were Agtr1A-(+/−), and 119 (19%) were Agtr1A-(−/−). This differed significantly from the proportions predicted by Mendelian genetics ( P = 0.01), suggesting that the complete absence of AT1Areceptors is associated with a mild survival disadvantage. Agtr1A-(−/−) mice grew normally, and we found no significant differences in body weight or heart and kidney weights in Agtr1A-(+/+) and Agtr1A-(−/−) mice examined at 21, 60, and 100 days. Protein and DNA content of kidneys and hearts were also similar in weanling or adult Agtr1A-(+/+) and Agtr1A-(−/−) mice. By light microscopy with immunohistochemistry, kidneys from Agtr1A-(−/−) were essentially normal, with two exceptions: 1) there was marked hypertrophy of the juxtaglomerular apparatus (JGA) and proximal expansion of renin-producing cells along the afferent arterioles, and 2) some glomeruli showed evidence of mesangial expansion. We did not find the severe renal vascular lesions or papillary atrophy that have been observed in angiotensinogen- or angiotensin converting enzyme-deficient animals. We conclude that the AT1Areceptor is not essential for the normal organogenesis of the kidney; however, its absence is associated with mild mesangial expansion and JGA hypertrophy.
Characterization of binding sites for amylin, calcitonin, and CGRP in primate kidney
Authors: Chai Siew Yeen , Christopoulos George , Cooper Mark E. , Sexton Patrick M.
Published: December 2017

Analysis of receptor distributions for125I-labeled amylin,125I-labeled calcitonin, and125I-labeled calcitonin gene-related peptide (CGRP) in Macaca fascicularis kidney by in vitro autoradiography revealed distinct patterns of binding for each peptide.125I-rat amylin bound primarily to the cortex, being associated with the distal tubule, including apparent binding to the juxtaglomerular apparatus.125I-salmon calcitonin displayed high-density binding in the cortex with low-density binding to the medulla. Emulsion autoradiography indicated that binding was associated with both distal tubule and thick ascending limb of the loop of Henle. Intense binding was also found often over juxtaglomerular apparatus.125I-rat CGRP-α exhibited low- to moderate-density binding to the inner medulla/papilla with high-density binding over small-, medium-, and large-caliber arteries. Weak binding to the glomerulus was also seen, but no binding was associated with cortical tubules. Competition binding studies, performed with each of the radioligands, revealed peptide specificity profiles for CGRP and calcitonin receptors that were similar to those described in rat. However, the monkey amylin receptors differed from those in rat, exhibiting relatively higher affinity for calcitonin peptides but reduced affinity for CGRP peptides. These studies suggest potential roles for amylin, calcitonin, and CGRP in primate renal function.
Urea activates ribosomal S6 kinase (RSK) in a MEK-dependent fashion in renal mIMCD3 cells
Authors: Zhang Zheng , Cohen David M.
Published: December 2017

Urea activates a characteristic subset of signaling pathways in a tissue-specific fashion, including transcription of immediate early genes through activation of the mitogen-activated protein kinase (MAPK), ERK (extracellular signal-regulated kinase), and activation of its transcription factor substrate, Elk-1. The ability of urea to activate the ERK effector and pivotal regulatory kinase, ribosomal S6 kinase (RSK), was investigated in mIMCD3 renal inner medullary collecting duct cells. Urea upregulated RSK activity in a time-dependent fashion in serum-deprived mIMCD3 cells; the effect was maximal at 5 min. Activation by hypertonic NaCl, in contrast, was negligible at 5 min and peaked at 15 min. Both stimuli induced the nuclear translocation of cytosolic RSK, as determined via immunofluorescence. Importantly, activation of RSK by both solutes was MAPK/ERK kinase (MEK) dependent, as determined by the ability of the specific MEK inhibitor, PD-98059, to abrogate the response. Taken together, these data indicate that urea activates the ERK effector, RSK, in cells of the renal medulla in an ERK-dependent fashion, further emphasizing the functional significance of urea signaling through ERK activation in renal medullary cells.
Expression and localization of epithelial sodium channel in mammalian urinary bladder
Authors: Smith Peter R. , Mackler Scott A. , Weiser Philip C. , Brooker David R. , Ahn Yoon J. , Harte Brian J. , McNulty Kathleen A. , Kleyman Thomas R.
Published: December 2017

The mammalian urinary bladder exhibits transepithelial Na+absorption that contributes to Na+gradients established by the kidney. Electrophysiological studies have demonstrated that electrogenic Na+absorption across the urinary bladder is mediated in part by amiloride-sensitive Na+channels situated within the apical membrane of the bladder epithelium. We have used a combination of in situ hybridization, Northern blot analysis, and immunocytochemistry to examine whether the recently cloned epithelial Na+channel (ENaC) is expressed in the rat urinary bladder. In situ hybridization and Northern blot analyses indicate that α-, β-, and γ-rat ENaC (rENaC) are expressed in rat urinary bladder epithelial cells. Quantitation of the levels of α-, β-, and γ-rENaC mRNA expression in rat urinary bladder, relative to β-actin mRNA expression, indicates that, although comparable levels of α- and β-rENaC subunits are expressed in the urinary bladder of rats maintained on standard chow, the level of γ-rENaC mRNA expression is 5- to 10-fold lower than α- or β-rENaC mRNA. Immunocytochemistry, using an antibody directed against α-rENaC, revealed that ENaCs are predominantly localized to the luminal membrane of the bladder epithelium. Together, these data demonstrate that ENaC is expressed in the mammalian urinary bladder and suggest that amiloride-sensitive Na+transport across the apical membrane of the mammalian urinary bladder epithelium is mediated primarily by ENaC.
Apical and basolateral uptake and intracellular fate of dopamine precursorl-dopa in LLC-PK1cells
Authors: Soares-Da-Silva P. , Serrão M. P. , Vieira-Coelho M. A.
Published: December 2017

The present study was aimed at the uptake ofl-3,4-dihydroxyphenylalanine (l-dopa) and its intracellular decarboxylation to dopamine. The accumulation ofl-dopa from the apical side in cells cultured in collagen-treated plastic was found to be a saturable process with a Michaelis constant ( Km) of 123 ± 17 μM and a maximal velocity ( Vmax) of 6.0 ± 0.2 nmol ⋅ mg protein−1⋅ 6 min−1. The uptake ofl-dopa applied from either the apical or basal cell borders in cells cultured in polycarbonate filters was also found to be saturable; nonlinear analysis of saturation curves for apical and basal application revealed Kmvalues of 63.8 ± 17.0 and 42.5 ± 9.6 μM and Vmaxvalues of 32.0 ± 5.8 and 26.2 ± 3.4 nmol ⋅ mg protein−1⋅ 6 min−1, respectively. Cell monolayers incubated withl-dopa, applied from either the apical or the basal side, in the absence of benserazide, led to the accumulation of newly formed dopamine. The intracellular accumulation of newly formed dopamine was a saturable process with apparent Kmvalues of 20.5 ± 8.2 and 247.3 ± 76.8 μM when the substrate was applied from the apical and basal side, respectively. Some of the newly formed dopamine escaped to the extracellular milieu. The basal outward transfer of dopamine was five- to sevenfold of that occurring at the apical side and was uniform over a wide range of concentrations of intracellular dopamine; the apical outward transfer of the amine depended on the intracellular concentration of dopamine and was a nonsaturable process. The apical and basal outward transfers of dopamine were insensitive to cocaine (10 and 30 μM) and GBR-12909 (1 and 3 μM). The accumulation of exogenous dopamine in LLC-PK1cells was found to be saturable; nonlinear analysis of the saturation curves revealed for the apical and basal application of dopamine a Kmof 17.7 ± 4.3 and 96.0 ± 28.1 μM and a Vmaxof 2.0 ± 0.1 and 2.2 ± 0.3 nmol ⋅ mg protein−1⋅ 6 min−1, respectively. However, both cocaine (10, 30, or 100 μM) and GBR-12909 (1 or 3 μM) were found not to affect the uptake of 100 μM dopamine applied from either the apical or the basal cell border. In conclusion, the data presented here show that LLC-PK1cells are endowed with considerable aromaticl-amino acid decarboxylase (AADC) activity and transportl-dopa quite efficiently through both the apical and basal cell borders. On the other hand, our observations support the possibility of a basal-to-apical gradient of AADC activity and the possibility that LLC-PK1cells might constitute an interesting in vitro model for the study of the renal dopaminergic physiology.
Carbonic anhydrase II and IV mRNA in rabbit nephron segments: stimulation during metabolic acidosis
Authors: Tsuruoka Shuichi , Kittelberger Ann M. , Schwartz George J.
Published: December 2017

Carbonic anhydrase (CA) facilitates renal bicarbonate reabsorption and acid excretion. Cytosolic CA II catalyzes the buffering of intracellular hydroxyl ions by CO2, whereas membrane-bound CA IV catalyzes the dehydration of carbonic acid generated from the secretion of protons. Although CA II and IV are expressed in rabbit kidney, it is not entirely clear which segments express which isoforms. It was the purpose of this study to characterize the expression of CA II and CA IV mRNAs by specific segments of the nephron using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and to determine the effect of chronic metabolic acidosis on CA expression by those segments. Individual nephron segments (usually 1–2 mm) were isolated by microdissection and subjected to RT-PCR. Amplification was performed simultaneously for CA IV, CA II, and malate dehydrogenase (MDH), a housekeeping gene. The intensities of the PCR products were quantitated by densitometry. CA IV mRNA was expressed by S1 and S2 proximal tubules and by outer medullary collecting duct from inner stripe (OMCDi) and outer stripe and initial inner medullary collecting duct (IMCDi). CA II mRNA was expressed by S1, S2, and S3 proximal tubules, thin descending limb, connecting segment (CNT), and all collecting duct segments. Acid loading induced CA IV mRNA expression in S1 and S2 proximal tubules and in OMCDi and IMCDi. CA II mRNA was induced by acidosis in all three proximal segments and nearly all distal segments beginning with CNT. No upregulation of MDH mRNA expression occurred. These adaptive increases in CA II and IV mRNAs are potentially important in the kidney’s adaptation to chronic metabolic acidosis.
Local upregulation of colonic angiotensin II receptors enhances potassium excretion in chronic renal failure
Authors: Hatch Marguerite , Freel Robert W. , Vaziri N. D.
Published: December 2017

The role of angiotensin II (ANG II) in colonic secretion of K+was examined in rats with chronic renal failure (CRF). The basal net secretory flux of86Rb+(as a tracer for K+) across the CRF distal colon (−0.20 ± 0.04 μeq ⋅ cm−2⋅ h−1) was reversed to an absorptive flux (0.35 ± 0.05 μeq ⋅ cm−2⋅ h−1) by injecting the rats with the AT1receptor antagonist, losartan. A similar result was observed when losartan was added to the CRF colonic tissue in vitro. In contrast, an AT2receptor antagonist, PD-123319, did not reverse the CRF-induced alterations in Rb+transport across the short-circuited colonic tissue. Plasma concentrations of ANG II, aldosterone, and K+, as well as the ANG II content of colonic tissues from CRF and normal rats, were similar. However, specific125I-labeled ANG II binding sites in rat distal colon increased twofold in CRF [maximal specific binding (Bmax) = 28.6 ± 1.6 fmol/mg protein] compared with normal (Bmax= 15.2 ± 0.4 fmol/mg protein). These studies suggest that CRF-induced secretion of K+by the colon is mediated by an upregulation of AT1receptors present in CRF.
Angiotensin IV AT4-receptor system in the rat kidney
Authors: Handa Rajash K. , Krebs Luke T. , Harding Joseph W. , Handa Shelly E.
Published: December 2017

Angiotensin IV, {[des-Asp1,Arg2]ANG II or ANG-(3—8)}, has been shown to preferentially bind to a novel angiotensin binding site (AT4receptor). The cellular location and function of this receptor in the rat kidney is unknown. Autoradiography localized AT4receptors to the cell body and apical membrane of convoluted and straight proximal tubules in the cortex and outer stripe of the outer medulla. ANG IV (0.1 pM-1 μM) elicited a concentration-dependent decrease in transcellular Na+transport (as measured by proximal tubule O2consumption rates) in fresh suspensions of control or nystatin-stimulated (bypasses rate-limiting step of apical Na+entry) rat proximal tubules. The inhibitory effect of 1 pM ANG IV was unaltered by either 1 μM losartan (AT1-receptor antagonist) or 1 μM PD-123319 (AT2-receptor antagonist) and yet was abolished by 1 μM divalinal-ANG IV (AT4-receptor antagonist) or ouabain pretreatment. These results demonstrate that the kidney AT4-receptor system is localized to the proximal tubule and suggests that one potential biological role of this system is in the regulation of Na+transport by inhibiting a ouabain-sensitive component of Na+-K+-adenosinetriphosphatase activity in the rat.
Aldosterone potentiates hormone-stimulated Mg2+uptake in distal convoluted tubule cells
Authors: Dai Long-Jun , Ritchie Gordon , Bapty Brian , Quamme Gary A.
Published: December 2017

The distal convoluted tubule reabsorbs significant amounts of filtered magnesium that is under hormonal control. In this study, we describe the effects of aldosterone on Mg2+uptake in an immortalized mouse distal convoluted tubule (MDCT) cell line. Intracellular free Mg2+concentration ([Mg2+]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To determine Mg2+entry rate into MDCT cells, they were first Mg2+depleted ([Mg2+]i, 0.22 ± 0.01 mM) by culturing in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2. The rate of change in [Mg2+]ias measured as a function of time, d([Mg2+]i)/d t, was 164 ± 5 nM/s in control cells. We have shown that glucagon or arginine vasopressin (AVP) stimulates Mg2+entry by 63% and 15%, respectively. Incubation of MDCT cells with aldosterone for 16 h did not change the rate of Mg2+uptake (172 ± 8 nM/s). However, aldosterone potentiated glucagon- and AVP-stimulated Mg2+uptake rate up to 330 ± 39 and 224 ± 6 nM/s, respectively. Aldosterone also potentiated glucagon- and AVP-induced intracellular cAMP accumulation in a concentration-independent manner. As cAMP stimulates Mg2+entry in MDCT cells, it is inferred that aldosterone may stimulate Mg2+uptake through intracellular signaling pathways involving cAMP. The actions of aldosterone were dependent on de novo protein synthesis, as pretreatment of the cells with cycloheximide inhibited aldosterone potentiation of hormone stimulation of Mg2+uptake and cAMP accumulation. These studies with MDCT cells suggest that aldosterone may modulate the effects of hormones acting within the distal convoluted tubule to control magnesium absorption.
Expression of Cl−/ HCO 3 − exchanger in the basolateral membrane of mouse medullary thick ascending limb
Authors: Sun Adam M.
Published: December 2017

Although a basolateral Cl−/[Formula: see text]exchanger (AE) has been implicated in the arginine vasopressin (AVP)-dependent hypertonic regulatory increase in the medullary thick ascending limb (MTAL), there are conflicting data regarding whether this exchanger is indeed present in this tubule segment. In this study, mouse MTAL was examined whether Cl−/[Formula: see text]exchange activity was present in the basolateral membrane and whether mRNAs from the known AE genes are expressed. Cl−/[Formula: see text]exchange activity was examined in isolated perfused MTAL tubules under isotonic conditions and in the absence of arginine vasopressin. 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein was used to monitor intracellular pH. Removal of basolateral Cl−induced reversible cell alkalization that was independent of external Na+and completely inhibited by peritubular 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (200 μM). The rate and extent of cell alkalinization were significantly greater in the presence than absence of external CO2/[Formula: see text]. A voltage clamp did not inhibit cell alkalinization induced by basolateral Cl−removal. Consistently, addition of basolateral Cl−induced reversible cell acidification in MTAL depleted of intracellular Cl−. Furthermore, mRNA encoding two members (AE2 and AE3) of the AE gene family were demonstrated in microdissected mouse MTAL tubules by reverse transcription-polymerase chain reaction. It is concluded that AE is present in the basolateral membrane of mouse MTAL.
Increased glomerular capillary pressure and size mediate glomerulosclerosis in SHR juxtamedullary cortex
Authors: Iversen Bjarne M. , Amann Kerstin , Kvam Fred I. , Wang Xuemei , Ofstad Jarle
Published: December 2017

To gain insight into the mechanisms in the development of glomerulosclerosis in juxtamedullary cortex, the degree of glomerulosclerosis, glomerular tuft diameter, glomerular capillary pressure (Pgc), and local renal blood flow (RBF) autoregulation were measured in superficial and juxtamedullary cortex of 10- and 70-wk-old spontaneously hypertensive rat (SHR), using aged matched Wistar-Kyoto (WKY) rats as controls. Pgcwas measured after corticotomy by direct micropuncture of glomeruli in superficial and juxtamedullary cortex. Total RBF was measured by a transit-time flowmeter (Transonic) and local blood flow by use of laser-Doppler flowmetry. The degree of glomerulosclerosis measured by a semiquantitative histological technique was significantly increased in juxtamedullary compared with superficial cortex in all groups. The difference was most pronounced in the juxtamedullary cortex of 70-wk-old SHR. Pgcwas significantly increased in juxtamedullary cortex compared with superficial cortex in 70-wk SHR (57.1 ± 2.7 vs. 46.5 ± 0.5 mmHg, P < 0.01). The corresponding data set from 70-wk WKY was 45.5 ± 0.43 vs. 41.6 ± 1.5 ( P < 0.05). The Pgcin juxtamedullary cortex of 10-wk SHR was slightly higher than in superficial cortex (45.1 ± 2.3 vs. 50.1 ± 1.2 mmHg, P = 0.05), whereas there was no difference in 10-wk WKY. Glomerular diameter was larger in juxtamedullary cortex in old animals but not significantly different in 10-wk WKY rats and 10-wk SHR. Total RBF was reset to higher perfusion pressures in hypertensive rats. Juxtamedullary and superficial blood flow autoregulation were not significantly different from total RBF autoregulation in all groups. These results suggest that hypertrophy as well as increased Pgcmight contribute to the development of manifest glomerulosclerosis. Changes in local blood flow autoregulation do not seem to play a major role in the development of glomerulosclerosis.
Dynein and dynactin colocalize with AQP2 water channels in intracellular vesicles from kidney collecting duct
Authors: Marples David , Schroer Trina A. , Ahrens Nikolai , Taylor Ann , Knepper Mark A. , Nielsen Søren
Published: December 2017

We investigated whether the motor protein cytoplasmic dynein and dynactin, a protein complex thought to link dynein with vesicles, are present in rat renal collecting ducts and associated with aquaporin-2 (AQP2)-bearing vesicles. Immunoblotting demonstrated cytoplasmic dynein heavy and intermediate chains in kidney, with relative expression levels of inner medulla > outer medulla > cortex. In addition to being present in cytoplasmic fractions, dynein was abundant in membrane fractions enriched for intracellular vesicles. Dynactin was also abundant in membrane fractions enriched for intracellular vesicles. Furthermore, both dynactin and dynein were present in vesicles specifically immunoisolated using anti-AQP2 antibodies. Immunocytochemistry revealed labeling for dynein in the collecting duct principal cells with a pattern consistent with labeling of intracellular vesicles. Moreover, quantitative double immunogold labeling confirmed colocalization of AQP2 and dynein in the same vesicles at the electron microscopic level. Thus the microtubule-associated motor protein dynein and the associated dynactin complex are present in rat renal collecting duct principal cells and are associated with intracellular vesicles, including those bearing AQP2, consistent with the view that dynein and dynactin are involved in vasopressin-regulated trafficking of AQP2-bearing vesicles.
Localization of cytochromeP-450 4A isoforms along the rat nephron
Authors: Ito Osamu , Alonso-Galicia Magdalena , Hopp Kathleen A. , Roman Richard J.
Published: December 2017

The expression of P-450 4A isoforms responsible for the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) was examined using the reverse transcription and polymerase chain reaction in various nephron segments and preglomerular arterioles microdissected from the kidneys of Sprague-Dawley rats. Expression of cytochrome P-450 4A1, 4A2, 4A3, and 4A8 mRNA could be detected in RNA extracted from the whole kidney. The expression of P-450 4A1, 4A3, and 4A8 mRNA was similar in the kidney of male and female rats, whereas the expression of 4A2 mRNA was fourfold greater in the kidney of male vs. female rats. At the single-nephron level, P-450 4A1 mRNA could not be detected in either preglomerular arterioles or any nephron segments. P-450 4A2 mRNA was readily detected in preglomerular arterioles, glomeruli, proximal convoluted tubule (PCT), proximal straight tubule (PST), medullary thick ascending limb (MTAL), cortical thick ascending limb (CTAL), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD). P-450 4A3 mRNA was also detected in every nephron segment, but the expression of this isoform was barely detectable in preglomerular arterioles. The expression of P-450 4A8 mRNA was detected in the glomerulus, PCT, PST, CTAL, and CCD. It was not detectable in preglomerular arterioles, MTAL, OMCD, or IMCD. Immunoblot analysis using a P-450 4A antibody exhibited a strong signal for P-450 4A protein in the proximal tubule. Smaller signals were also observed in glomerulus, MTAL, and preglomerular arterioles, but no signal could be detected in the IMCD. A similar pattern of P-450 4A protein expression was seen in kidney sections immunostained with this antibody. These results indicate that the expression of P-450 4A isoforms in the kidney of rats is sex dependent and that different P-450 4A isoforms are expressed throughout various nephron segments and the renal vasculature of rats.
The human plasminogen activator inhibitor type I gene promoter targets to kidney
Authors: Emert Martin P. , Sorenson Christine M. , Basile David P. , Rogers Joseph G. , Hammerman Marc R. , Billadello Joseph J.
Published: December 2017

The plasminogen activator inhibitor type 1 (PAI-1) gene encodes the physiological inhibitor of tissue-type and urokinase-type plasminogen activators and is induced by cytokines such as transforming growth factor-β (TGF-β). Studies have identified DNA sequence elements within the first 1.3 kb of the 5′-upstream DNA that mediate cytokine responsiveness in transfected cells in vitro. However, the DNA sequences that mediate PAI-1 expression in vivo have not yet been delineated. To define these regulatory sequences, we generated transgenic mice that expressed a hybrid gene comprising sequences between −1,272 and +75 of the human PAI-1 gene ligated to a LacZ reporter gene. Transgene expression detected in two independent lines was observed only in kidney from embryonic day 13 to adult and was seen primarily in proximal tubule cells of the outer medulla. Transgene expression and activity were unchanged in response to TGF-β and remained restricted to kidney. Thus we have identified a promoter region within the PAI-1 gene that targets transgene expression to kidney but, unlike the native promoter, is unresponsive to TGF-β in the experimental protocol used.
Outer medullary anatomy and the urine concentrating mechanism
Authors: Wang X. , Thomas S. R. , Wexler A. S.
Published: December 2017

In earlier work, mathematical models of the urine concentration mechanism were developed incorporating the features of renal anatomy. However, several anatomic observations showed inconsistencies in the modeling representation of the outer stripe (OS) anatomy. In this study, based on observations from comparative anatomy and morphometric studies, we propose a new structural model of outer medullary anatomy, different from that previously presented [A. S. Wexler, R. E. Kalaba, and D. J. Marsh. Am. J. Physiol. 260 ( Renal Fluid Electrolyte Physiol. 29): F368–F383, 1991]. The modifications include the following features of rat outer medullary anatomy, for example, 1) in the OS, the limbs of long loops of Henle surround the descending and ascending vasa recta that develop into the vascular bundles in the inner stripe (IS), whereas the limbs of short loops are close to the collecting ducts; and 2) the descending limbs of short loops shift from the tubular region in the OS to near the vascular bundle in the IS, whereas the limbs of long loops are situated away from the vascular bundles in the tubular region. The sensitivity of the concentrating process to the relative position of loops and vessels was investigated in the different medullary regions. With these modifications, the model predicts a more physiological, axial osmolarity gradient in both outer and inner medulla with membrane parameters that are all in the range of measured physiological values, including the urea permeabilities of descending vasa recta reported by Pallone and co-workers (T. L. Pallone, J. Work, R. L. Myers, and R. L. Jamison. J. Clin.Invest. 93: 212–222, 1994).
Arginase activity is modulated by IL-4 and HOArg in nephritic glomeruli and mesangial cells
Authors: Waddington Simon N. , Tam Frederick W. K. , Cook H. Terence , Cattell Victoria
Published: December 2017

Arginase shares a common substrate, l-arginine, with nitric oxide synthase (NOS). Both enzymes are active at inflammatory sites. To understand regulation of arginase and its relationship to nitric oxide (NO) production, we studied effects of NG-hydroxy-l-arginine (HOArg) and interleukin-4 (IL-4) on urea and[Formula: see text] synthesis by glomeruli during rat immune glomerulonephritis and compared these with macrophages and glomerular mesangial cells (MC). In nephritic glomeruli, elicited macrophages, and MC stimulated with IL-1 and adenosine 3′,5′-cyclic monophosphate agonists, increased arginase and induced NOS activity was found. Urea production was inhibited by HOArg and increased by IL-4. NO inhibition [ NG-monomethyl-l-arginine (l-NMMA)] increased arginase activity in nephritic glomeruli and macrophages but not MC.[Formula: see text] synthesis was inhibited byl-NMMA and IL-4. It was increased with HOArg under conditions of NO inhibition. In contrast, in normal glomeruli and basal MC, where there was no induced NO synthesis, IL-4 had no effect on arginase activity, whereas HOArg consistently reduced it in glomeruli only. Type II arginase (Arg II) mRNA was detected in normal glomeruli; nephritic glomeruli expressed both Arg I and Arg II mRNAs. This is the first demonstration of arginase modulation in glomeruli and MC and of the expression of arginase isoforms in glomeruli. The differential responses to two endogenous compounds generated by inflammation suggest this may be part of coordinated regulation of arginase and inducible NOS in immune injury, whereby arginase is inhibited during high-output NO production and stimulated with NO suppression. This, together with control of arginase and NOS isoforms, may be important in controlling the balance of inflammatory and repair mechanisms.
Nitroflurbiprofen, a new nonsteroidal anti-inflammatory, ameliorates structural injury in the remnant kidney
Authors: Fujihara Clarice Kazue , Malheiros Denise Maria Avancini Costa , Donato JoséLuís , Poli Anicleto , De Nucci Gilberto , Zatz Roberto
Published: December 2017

Cyclooxygenase derivatives and nitric oxide (NO) may influence the pathogenesis of progressive nephropathies. We investigated the effect of nitroflurbiprofen (NOF), a NO-releasing nonsteroidal anti-inflammatory drug (NSAID) without gastrointestinal toxicity, in rats with 5/6 ablation (NX). The following four groups were studied: Sham, sham-operated rats; Sham + NOF, Sham receiving oral NOF two times daily; NX, rats subjected to NX; and NX + NOF, NX receiving NOF. NOF was barely detected in plasma but released the parent compound flurbiprofen. At 30 days, glomerular hydraulic pressure (PGC) was 76 ± 3 mmHg in NX (52 ± 1 in Sham, P < 0.05). NOF slightly reduced PGCto 69 ± 2 mmHg in NX + NOF ( P > 0.05 vs. NX). Glomerular volumes behaved similarly. At 60 days, tail cuff pressure was 152 ± 6 mmHg, glomerulosclerosis index was 22.1 ± 9.5, and interstitial fractional area was 9.9 ± 1.2% in NX. NOF reduced these parameters to 137 ± 4 mmHg, 3.5 ± 0.7, and 6.4 ± 0.8%, respectively ( P < 0.05), without causing growth stunting or anemia. These beneficial effects could not be ascribed to NO donation and may reflect cyclooxygenase inhibition. This is the first evidence that chronic NSAID treatment may ameliorate progressive nephropathies.
Isolation and characterization of kidney-specific ClC-K1 chloride channel gene promoter
Authors: Uchida Shinichi , Rai Tatemitsu , Yatsushige Hiroshi , Matsumura Yoshihiro , Kawasaki Masanobu , Sasaki Sei , Marumo Fumiaki
Published: December 2017

The rat ClC-K1 chloride channel is a kidney-specific member of the ClC chloride channel family found exclusively in the thin ascending limb of Henle’s loop in the kidney. To gain insight into the mechanism(s) of kidney-specific expression of ClC-K1, a genomic clone that contains the 5′-flanking region of the rat ClC-K1 gene was isolated. A single transcription start site was located 84 bp upstream of the start codon. The sequence of the proximal 5′-flanking region contained an activator protein (AP)-3 site, a glucocorticoid-responsive element, several AP-2 sites, and several E-boxes, but it lacked a TATA box. To functionally express the promoter, the ∼2.5-kb pair 5′-flanking region was ligated to a luciferase reporter gene and transfected into inner medullary (IM) cells, a stable ClC-K1-expressing cell line derived from the inner medulla of simian virus 40 transgenic mouse, and ClC-K1-nonexpressing cell lines. Luciferase activity was 7- to 24-fold greater in IM cells than those in nonexpressing cell lines, suggesting that the ∼2.5-kb fragment contained cis-acting regulatory elements for cell-specific expression of the ClC-K1 gene. Deletion analysis revealed that this cell-specific promoter activity in IM cells was still present in the construct containing 51 bp of the 5′-flanking region but was lost in the −29 construct, clearly demonstrating that the 22 bp from −51 to −30 have a major role in the cell-specific activity of the ClC-K1 promoter. These 22 bp consist of purine-rich sequence (GGGGAGGGGGAGGGGAG), and gel-retardation analysis demonstrated the existence of a specific protein(s) binding to this element in IM cells. These results suggest that the novel purine-rich element may play a key role in the activity of the ClC-K1 gene promoter.
Documentation of angiotensin II receptors in glomerular epithelial cells
Authors: Sharma Mukut , Sharma Ram , Greene Andrew. S. , McCarthy Ellen T. , Savin Virginia J.
Published: December 2017

Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1and AT2receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1and AT2, respectively. The presence of both AT1and AT2receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial cells participate in angiotensin II-mediated control of the glomerular filtration barrier.
Axial heterogeneity of sodium-bicarbonate cotransporter expression in the rabbit proximal tubule
Authors: Abuladze N. , Lee I. , Newman D. , Hwang J. , Pushkin A. , Kurtz I.
Published: December 2017

It is generally accepted that Na(HCO3) n cotransport is the most important mechanism mediating basolateral bicarbonate efflux in the early proximal tubule. The presence of basolateral Na(HCO3) n cotransport in the late proximal tubule (S3 segment) and in the juxtamedullary S1 and S2 segments has been controversial. The renal sodium-bicarbonate cotransporter (NBC) has been recently cloned from rat (M. F. Romero, M. A. Hediger, E. L. Boulpaep, and W. F. Boron. J. Am. Soc. Nephrol. 7: 1259, 1996), salamander (M. F. Romero, M. A. Hediger, E. L. Boulpaep, and W. F. Boron. Nature 387: 409–413, 1997), and human (C. E. Burnham, H. Amlal, Z. Wang, G. E. Shull, and M. Soleimani. J. Biol. Chem. 272: 19111–19114, 1997). The localization of NBC in the kidney is unknown. The present study was designed to localize NBC mRNA expression in the rabbit proximal tubule. In situ hybridization studies were combined with functional studies of basolateral Na(HCO3) n cotransport in superficial and juxtamedullary S1, S2, and S3 segments of the rabbit proximal tubule. The results demonstrate that NBC mRNA is localized predominantly to the cortex, with less expression in the outer medulla. NBC expression was not detected in the inner medulla. The highest level of NBC mRNA is in the S1 proximal tubule. NBC is expressed at a low levels in the S3 segment, with intermediate expression in the S2 segment. In bicarbonate-buffered solutions, the rate of base efflux mediated by Na(HCO3) n cotransport followed a similar pattern in superficial and juxtamedullary proximal tubule segments, i.e., S1 > S2 > S3. The juxtamedullary S1 segment had the greatest rate of basolateral Na(HCO3) n cotransport and the highest level of NBC expression in the proximal tubule.
Arachidonic acid potentiates the feedback response of mesangial BKCachannels to angiotensin II
Authors: Stockand James D. , Silverman Meredith , Hall David , Derr Thomas , Kubacak Brian , Sansom Steven C.
Published: December 2017

The influence of arachidonic acid (AA) on the feedback regulation of mesangial contraction by large Ca2+-activated K+channels (BKCa) was determined through single-channel analysis using the patch clamp method. The mesangial BKCais a low-gain negative feedback inhibitor of contraction that is activated in response to agonist-induced Ca2+transients and membrane depolarization. AA activated BKCain cell-attached patches in a dose-dependent manner with a maximal effect at 400 nM and a half-maximal response at 49 nM. In inside-out patches, AA directly activated BKCawith a maximal effect at 400 nM. BKCawas activated significantly in response to addition of 100 nM ANG II in the presence but not the absence of AA. Since it was shown previously that fatty acids stimulated both soluble and membrane-bound guanylyl cyclase, we determined whether AA activated BKCaby interfering with cGMP-mediated signal transduction pathways. It was previously shown that 10 μM cGMP, via cGMP-dependent protein kinase, activated BKCain a biphasic manner with an early increase in probability of a channel existing in an open state ( Po) and a subsequent inactivation mediated by protein phosphatase 2A (PP2A). We found that 10 μM dibutyryl-cGMP enhanced BKCaactivity in an additive manner with saturating concentrations (400 nM) of AA. Moreover, the inactivation phase mediated by PP2A was not abolished. Thus AA does not affect the phosphorylation/dephosphorylation regulatory cycle for BKCa. It is concluded that AA potentiates the ANG II feedback response of BKCaby a mechanism that is independent of the phosphorylation cycle.
K+depletion increases HCO 3 − reabsorption in OMCD by activation of colonic H+-K+-ATPase
Authors: Nakamura Suguru , Wang Zhaohui , Galla John H. , Soleimani Manoocher
Published: December 2017

To probe the role of the isoforms of H+-K+-ATPase (HKA) in potassium depletion (KD), rats were placed on a KD diet for 2 wk. Colonic HKA (cHKA) mRNA levels increased ∼30-fold in outer medulla, and net [Formula: see text] flux ([Formula: see text]) in outer medullary collecting duct (OMCD) increased (13.1 pmol ⋅ min−1⋅ mm tubule length−1in control to 17.7 pmol ⋅ min−1⋅ mm tubule length−1in KD; P < 0.01). In normal rats, 1 mM ouabain in perfusate had no effect on[Formula: see text], whereas 10 μM Sch-28080 decreased[Formula: see text]to 5.1 pmol ⋅ min−1⋅ mm tubule length−1( P < 0.001). In KD rats, ouabain 1 mM decreased[Formula: see text]to 6.3 pmol ⋅ min−1⋅ mm tubule length−1( P < 0.001). Although 10 μM Sch-28080 also decreased[Formula: see text]to 4.6 pmol ⋅ min−1⋅ mm tubule length−1( P < 0.001), the inhibitory effects of Sch-28080 and ouabain were not additive. Removal of K+from perfusate blocked Sch-28080-sensitive[Formula: see text]in both normal and KD tubules. The data suggest that, in KD, cHKA is induced and mediates increased [Formula: see text]reabsorption in OMCD, cHKA in vivo is sensitive to both Sch-28080 and ouabain, and cHKA activity is dominant.
Regulation of intrarenal blood flow in experimental heart failure: role of endothelin and nitric oxide
Authors: Abassi Zaid , Gurbanov Konstantin , Rubinstein Irith , Better Ori S. , Hoffman Aaron , Winaver Joseph
Published: December 2017

Congestive heart failure (CHF) is associated with a marked decrease in cortical blood flow and preservation of medullary blood flow. In the present study we tested the hypothesis that changes in the endothelin (ET) and nitric oxide (NO) systems in the kidney may contribute to the altered intrarenal hemodynamics in rats with aortocaval fistula, an experimental model of CHF. Cortical and medullary blood flow were measured simultaneously by laser-Doppler flowmetry in controls and rats with compensated and decompensated CHF. As previously reported [K. Gurbanov, I. Rubinstein, A. Hoffman, Z. Abassi, O. S. Better, and J. Winaver. Am. J. Physiol. 271 ( Renal Fluid Electrolyte Physiol. 40): F1166–F1172, 1996], administration of ET-1 in control rats produced a sustained cortical vasoconstriction and a transient medullary vasodilatory response. In rats with decompensated CHF, cortical vasoconstriction was severely blunted, whereas ET-1-induced medullary vasodilation was significantly prolonged. This prolonged response was mimicked by IRL-1620, a specific ETB agonist, and partially abolished by NO synthase (NOS) blockade. In line with these findings, expression of ET-1, ETA and ETB receptors, and endothelial NOS (eNOS), assessed by RT-PCR, and eNOS immunoreactivity, assessed by Western blotting, was significantly higher in the medulla than in the cortex. Moreover, expression of ET-1 mRNA in the cortex and eNOS mRNA in the cortex and the medulla increased in proportion to the severity of heart failure. These findings indicate that CHF is associated with altered regulation of intrarenal blood flow, which reflects alterations in expression and activity of the ET and NO systems. It is further suggested that exaggerated NO activity in the medulla contributes to preservation of medullary blood flow in the face of cortical vasoconstriction in CHF.
Role of NO in cyclosporin nephrotoxicity: effects of chronic NO inhibition and NO synthases gene expression
Authors: Bobadilla Norma A. , Gamba Gerardo , Tapia Edilia , García-Torres Romeo , Bolio Alexis , López-Zetina Pedro , Herrera-Acosta Jaime
Published: December 2017

The role of nitric oxide (NO) during cyclosporin renal vasoconstriction was evaluated by glomerular hemodynamic and histological changes produced by chronic NO synthesis inhibition and neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) NO synthases mRNA expression in renal cortex and medulla. Uninephrectomized rats treated during 7 days with vehicle (Veh), cyclosporin A (CsA) 30 mg/kg, CsA + nitro-l-arginine methyl ester (l-NAME), and Veh +l-NAME (10 mg/dl) in the drinking water were studied. Increase in arterial pressure and afferent and efferent resistances, as well as decrease in glomerular plasma flow, ultrafiltration coefficient, and single-nephron glomerular filtration rate were significantly greater with CsA +l-NAME than with CsA alone. The increase in afferent resistance was higher with CsA +l-NAME than with Veh +l-NAME. In addition, glomerular thrombosis, proximal tubular vacuolization, and arteriolar thickening were more prominent. In renal cortex, eNOS mRNA expression exhibited a 2.7-fold increase in CsA, whereas, in medulla, nNOS and iNOS expression were lower in CsA than in Veh, while eNOS tended to increase. Our results support the hypothesis that NO synthesis is enhanced at cortical level during CsA nephrotoxicity, counterbalancing predominantly preglomerular vasoconstriction. Higher NO production could be the result of increased eNOS mRNA expression.