bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin-like growth factor I preserves renal function postoperatively
Authors: Franklin S. C. , Moulton M. , Sicard G. A. , Hammerman M. R. , Miller S. B.
Published: December 2017

Deterioration of renal function, which can lead to postoperative renal failure, is a complication of surgery involving the suprarenal aorta and surgery involving the renal arteries. Fifty-four patients who were at risk for developing this complication were enrolled in a double-blind, randomized, placebo-controlled trial of insulin-like growth factor (IGF-I) as a therapeutic agent to prevent the decline in renal function. The primary end point was the incidence of renal dysfunction, defined as a reduction of the glomerular filtration rate (creatinine clearance) at each of three measurements over 72 h. IGF-I (100 microg/kg subcutaneously every 12 h for 6 doses) or placebo was administered on admission to the intensive care unit immediately postoperatively. IGF-I- and placebo-treated groups were well matched for sex, age, type of surgery, renal ischemic time during surgery (ischemic index), baseline creatinine clearance, and baseline serum creatinine. No patient in the study developed acute renal failure postoperatively. IGF-I was well tolerated. A smaller proportion of patients in the IGF-I group had a postoperative decline in renal function (22%) than in the placebo-treated group (33%). There were no significant differences in levels of serum creatinine at time of discharge, length of hospital stay, length of intensive care unit stay, length of intubation, or incidence of dialysis or death. Our findings establish the feasibility and potential utility for the use of IGF-I to reduce the incidence of postoperative renal dysfunction in high-risk patients.
G alpha(i-2) mediates renal LLC-PK1 growth by a Raf-independent activation of p42/p44 MAP kinase
Authors: Kinane T. B. , Kang I. , Chu A. , Chin S. H. , Ercolani L.
Published: December 2017

The protooncogene G alpha(i-2) plays a pivotal role in signaling pathways that control renal cell growth and differentiation. Mitogen-activated protein kinases (MAPKs) are potential downstream effectors for G alpha(i-2) in these pathways. In predifferentiated LLC-PK1 renal cells, the temporal maximal expression of G alpha(i-2) coincided with maximal activation of MAPK(p42/p44). By contrast, pertussis toxin treatment of these cells inhibited cell growth and reduced MAPK(p42/p44) activity by 30%. These findings reflected upstream activation of MAPK kinase (MEK1), as transient transfection of cells with a plasmid encoding a constitutively active form of MEK1 increased MAPK(p42/p44) activity and cell growth, whereas treatment with PD-098059, an inhibitor of MEK1 activity, reduced MAPK(p42/p44) activity and cell growth. Expression of a guanosinetriphosphatase (GTPase)-deficient G alpha(i-2) in these cells increased MAPK(p42/p44) activity and correspondingly reduced cell doubling time from 24 to 10 h without altering the activity of Raf-1 or c-Jun/stress-activated protein kinases (SAPKs). By contrast, expression of a GTPase-deficient G alpha(i-3) in these cells reduced both their cell doubling time by 30% and MAPK(p42/p44) activity by 60%. As the known MEKK isoforms (MEKK1, -2, and -3) can also activate SAPKs, these findings suggest the GTP-charged G alpha(i-2) subunit transduces growth signals in renal cells via activation of MAPK(p42/p44) and that such activation may be linked to pathways containing novel MEKK isoforms that preferentially activate MEKs.
Low NO concentrations inhibit osteoclast formation in mouse marrow cultures by cGMP-dependent mechanism
Authors: Holliday L. S. , Dean A. D. , Lin R. H. , Greenwald J. E. , Gluck S. L.
Published: December 2017

High concentrations of nitric oxide (NO) inhibit bone resorption by mature osteoclasts. We examined the effects of low NO concentrations on osteoclast formation in mouse bone marrow cultures. The NO releasers sodium nitroprusside (SNP) and S-nitroso-N-acetyl-DL-penicillamine inhibited the formation of multinucleated cells expressing tartrate-resistant acid phosphatase (a marker for osteoclasts) when administered during the last 3 days of 6-day cultures (differentiation stage) but not during the first 3 days (proliferation stage). SNP (1 microM) completely inhibited pit formation on dentine wafers when added to cultures during osteoclast formation, but 100 microM SNP was required to inhibit pitting by mature osteoclasts. Conversely, the NO synthase inhibitors aminoguanidine and nitro-L-arginine methyl ester both increased osteoclast formation. Inhibition of osteoclast formation by NO likely was guanosine 3',5'-cyclic monophosphate (cGMP) dependent, as SNP increased cGMP in marrow cultures, and 1 mM 8-bromo-cGMP or dibutyryl-cGMP reduced osteoclast formation when administered during the differentiation stage. The cGMP-specific type V phosphodiesterase inhibitor, zaprinast (M & B 22948) also inhibited osteoclast formation (half-maximal inhibitory constant, 100 microM) only when added during the differentiation stage. We conclude that the differentiation stage of osteoclast formation is inhibited by increases in cGMP levels elicited by NO.
Immunoglobulin light chain alters mesangial cell calcium homeostasis
Authors: Zhu L. , Herrera G. A. , White C. R. , Sanders P. W.
Published: December 2017

This study examined the hypothesis that certain immunoglobulin light chains directly altered mesangial cell calcium homeostasis. Intracellular Ca2+ concentration (intracellular [Ca2+]) signaling was determined in suspensions of rat mesangial cells using the acetoxymethyl ester of fura 2 with a calcium removal/replacement protocol. Pretreatment of cultured rat mesangial cells with a glomerulopathic kappa-light chain (gle) produced reversible dose- and time-dependent attenuation of ATP- and thrombin-evoked [Ca2+] transients (189 +/- 24 vs. 126 +/- 10 nM, P < 0.05 with ATP; 198 +/- 5 vs. 117 +/- 3 nM, P < 0.05 with thrombin) and capacitative calcium influx (199 +/- 14 vs. 142 +/- 17 nM, P < 0.05 for ATP; 252 +/- 19 vs. 198 +/- 18 nM, P < 0.05 for thrombin). Mesangial cells treated with gle and supplemented with myo-inositol (450 microM) did not demonstrate the attenuation of the ATP-evoked [Ca2+] transient and capacitative calcium influx. Gle also decreased mean [Ca2+] transient (80 +/- 7 vs. 56 +/- 1 nM, P < 0.05) and capacitative calcium influx (306 +/- 10 vs. 241 +/- 4 nM, P < 0.05) in response to thapsigargin, a Ca2+-adenosinetriphosphatase inhibitor. This inhibition was not reversed by exogenous myo-inositol. Another kappa-light chain (10 microg/ml) did not affect mesangial cell calcium signaling. Deranged mesangial cell calcium homeostasis by certain light chains may play a central pathogenetic role in glomerulosclerosis associated with deposition of immunoglobulin light chains.
Internephron coupling by conducted vasomotor responses in normotensive and spontaneously hypertensive rats
Authors: Wagner A. J. , Holstein-Rathlou N. H. , Marsh D. J.
Published: December 2017

We compared conducted vasomotor responses in juxtamedullary microcirculation in normotensive Sprague-Dawley (SD) and spontaneously hypertensive rats (SHR). The goals of the study were as follows: 1) decide whether internephron coupling is facilitated by conducted vasomotor responses; 2) determine whether the magnitude of induced vasoconstriction decreases with increasing distance from the stimulation site; and 3) determine whether the response is stronger in SHR than in SD rats. Microapplication of KCl to the distal afferent arteriole caused local vasoconstriction that was rapidly conducted (speed > 3.0 mm/s) into the cortical radial artery and neighboring afferent arterioles in SD and SHR. The strength of the response was significantly greater (approximately 40%, P < 0.025) in SHR than SD, and the magnitude decreased monotonically with increasing distance from the stimulation site in both strains. Mechanical length constants were similar in SD and SHR (approximately 325 microm), indicating that the signal responsible for the effect decays at the same rate in both strains. We conclude that internephron coupling strength is significantly greater in SHR and that internephron coupling is due to vascular events conducted along the preglomerular vasculature.
Epithelial cell polarity and disease
Authors: Wilson P. D.
Published: December 2017

The establishment and maintenance of epithelial polarity is essential for the integrity and function of epithelial organs and is particularly critical in the kidney, where vectorial reabsorption and secretion are effected in different segments of the nephron by the differential polarized insertion of channels, transporters, and related proteins into apical membranes lining the tubule lumen or basolateral membranes adjacent to the interstitium and blood space. Faulty intracellular delivery and polarization of membrane proteins can lead to serious diseases such as cystic fibrosis, I cell disease, and renal cystic diseases. The best understood disease of epithelial polarity is autosomal dominant polycystic kidney disease (ADPKD) caused by mutations in a >462-kDa, developmentally regulated membrane protein, “polycystin.” ADPKD cysts are characteristically lined by a single layer of structurally polarized epithelial cells with normal functional intercellular tight junctions but with aberrant polarization of some important membrane proteins. Abnormal apical membrane polarity of biochemically active, ouabain-sensitive Na-K-adenosinetriphosphatase (Na-K-ATPase) in ADPKD cyst epithelia leads to abnormal sodium ion secretion and provides a mechanism for aberrant fluid secretion. In addition, apically mislocated, functional epidermal growth factor (EGF) receptors on cyst epithelia, together with EGF synthesis and secretion into cyst lumens, provide a mechanism for autocrine regulation of increased epithelial cell proliferation in ADPKD. Underlying mechanisms for these abnormalities in polarized distribution of membrane proteins include the aberrant expression of fetal gene products, such as the beta2-subunit of Na-K-ATPase, in ADPKD kidneys. Overexpression of polycystin protein in ADPKD cyst epithelia, low levels restricted to medullary collecting tubules in normal adult kidneys, and high levels in ureteric bud-derived structures in human fetal kidneys further suggest a failure of downregulation of fetal genes as a mechanism for the polarity abnormalities that characterize ADPKD.
Insulin-like growth factor I stimulates apical sodium/hydrogen exchange in human proximal tubule cells
Authors: Johnson D. W. , Brew B. K. , Poronnik P. , Cook D. I. , Field M. J. , Gyory A. Z. , Pollock C. A.
Published: December 2017

To determine whether insulin-like growth factor I (IGF-I) stimulated apical sodium/hydrogen exchange (NHE), confluent primary human proximal tubule cells (PTC) were incubated for 48 h in serum-free media in the presence or absence of 100 ng/ml IGF-I. Cells incubated in IGF-I demonstrated significant increases in thymidine incorporation (181.2 +/- 30.3% of control values; n = 12, P = 0.01) and in resting intracellular pH (pHi) (7.52 +/- 0.08 vs. 7.30 +/- 0.06; n = 20, P < 0.05), as determined by 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein quantitative microspectrofluorometry. Following intracellular acid loading, ethylisopropylamiloride (EIPA)-inhibitable H+ efflux and 22Na+ influx after 1 min were both significantly enhanced in IGF-I-treated cells compared with controls (8.78 +/- 1.69 vs. 3.03 +/- 0.72 mM/min and 3.47 +/- 0.49 vs. 1.55 +/- 0.35 nmol x mg protein(-1) x min(-1), respectively). 22Na+ uptake studies in PTC grown on permeable supports demonstrated preferential stimulation of apical vs. basolateral NHE. The 50% inhibitory concentrations (IC50) in IGF-I-treated and control cells for EIPA (0.5 and 1.1 microM, respectively) and for HOE-694 (4.0 and 10.0 microM, respectively) were also consistent with predominant activation of apical, rather than basolateral, NHE activity. Kinetic analysis revealed an increase in maximal transport velocity (Vmax, 15.50 +/- 1.50 vs. 7.26 +/- 3.07 mM/min; n = 10, P < 0.05), without a significant change in antiporter affinity for extracellular Na+. Incubation of PTC with 100 ng/ml IGF-I produced an acute, reversible, and EIPA-inhibitable pHi increase of 0.05 +/- 0.01 pH units (n = 5, P < 0.05). The results suggest that IGF-I may contribute to the metachronous stimulation of apical NHE and PTC growth observed in many physiological and pathological conditions involving the human kidney.
Nitric oxide-induced hyperpolarization stimulates low-conductance Na+ channel of rat CCD
Authors: Lu M. , Giebisch G. , Wang W.
Published: December 2017

We used the patch-clamp technique in the split-open cortical collecting duct (CCD) to investigate the effect of nitric oxide (NO) on the low-conductance (6-pS) Na+ channel that can be blocked by 1 microM amiloride. We confirmed that the number of Na+ channels increased significantly in CCDs of rats on a low-Na+ diet (17). Application of 100 microM N(G)-nitro-L-arginine methyl ester (L-NAME), an agent that blocks endogenous NO synthase, reduced NPo [the product of channel number (N) and open probability (Po)] to 45% of the control value. The effect of L-NAME was specific, since addition of D-NAME, which does not inhibit NO synthase, did not change the activity of the Na+ channel. That the effect of L-NAME results from inhibition of NO synthase is further confirmed by experiments in which addition of an exogenous NO donor, either 10 microM S-nitroso-N-acetyl penicillamine or sodium nitroprusside (SNP), restored the Na+ channel activity when it had been blocked by L-NAME. The action of NO involves a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent pathway, since 100 microM 8-bromo-cGMP (8-BrcGMP) mimicked the effect of SNAP on K+ channels. However, 100 microM 8-BrcGMP did not alter the activity of Na+ channels in inside-out patches, suggesting an indirect action. Because the Na+ channel is activated by hyperpolarization (19) and NO stimulates basolateral K+ channels (16), we tested whether hyperpolarization mediated the effect of NO. In perforated whole cell recordings, addition of L-NAME depolarized the cell membrane from -73 to 51 mV, and application of 10 microM SNP repolarized the membrane to -68 mV. Furthermore, the L-NAME-induced decrease in NPo was effectively restored by 25 mV hyperpolarization of the patch membranes, and addition of 2 mM Ba2+ also abolished the effect of L-NAME. We concluded that the stimulatory effect of NO on the Na+ channel is an indirect effect mediated by a NO-induced increase of basolateral K+ conductance.
Ultramicrodetermination of vasopressin-regulated urea transporter protein in microdissected renal tubules
Authors: Kishore B. K. , Terris J. , Fernandez-Llama P. , Knepper M. A.
Published: December 2017

The vasopressin-regulated urea transporter (VRUT) is a 97-kDa protein (also called “UT-1”) responsible for facilitated urea transport across the apical plasma membrane of inner medullary collecting duct (IMCD) cells. To determine the abundance of VRUT protein in collecting duct cells of the rat, we designed a sensitive fluorescence-based enzyme-linked immunosorbent assay capable of detecting <5 fmol of VRUT protein. In collecting duct segments, measurable VRUT was found in microdissected IMCD segments but not in other portions of the collecting duct. In the mid-IMCD, the measured level averaged 5.3 fmol/mm tubule length, corresponding to approximately 5 million copies of VRUT per cell. Thus VRUT is extremely abundant in the IMCD, accounting, in part, for the extremely high urea permeability of this segment. Feeding a low-protein diet (8% protein) markedly decreased urea clearance but did not alter the quantity of VRUT protein in the IMCD. Thus increased urea transport across the collecting duct with dietary protein restriction is not a consequence of increased expression of VRUT. Based on urea fluxes measured in the IMCD and our measurements of the number of copies of VRUT, we estimate a turnover number of > or = 0.3-1 x 10(5) s. In view of the large magnitude of this value and previously reported biophysical properties of urea transport in collecting ducts, we hypothesize that the VRUT may function as a channel rather than a carrier.
Effect of sympathetic and angiotensin-aldosterone systems on renal salt conservation in the rat
Authors: Honrath U. , Veress A. T. , Chong C. K. , Sonnenberg H.
Published: December 2017

During dietary salt deprivation, the sympathetic nervous system and the angiotensin-aldosterone system are stimulated. Both systems are thought to be essential for maximal salt conservation by the kidney. To study their relative contributions, we produced negative salt balance in rats by intraperitoneal injection of furosemide, followed by a low-salt diet (<0.008% NaCl). In a 1-wk metabolic study, the animals were unable to replace the drug-induced salt deficit. Six groups of rats were studied. A control group established baseline function, a second group of 6-hydroxydopamine (OHDA) rats were treated with OHDA to destroy sympathetic efferent nerve terminals, and a third group (losartan) were treated with the angiotensin-receptor antagonist losartan. The influence of catecholamines and aldosterone released from the adrenal gland was studied in a further three groups. Rats were sham-adrenalectomized (sham), subjected to bilateral adrenal enucleation (Enuc) to eliminate catecholamine secretion, or were bilaterally adrenalectomized (Adx), eliminating both catecholamine and corticosteroid release. Dexamethasone was used as glucocorticoid replacement in this group. Steady-state urinary salt excretion was not different between control and OHDA rats. The losartan group showed significantly increased sodium but not chloride excretion. Surprisingly, there were no differences in salt excretion among sham, Enuc, and Adx groups. We conclude that, during a state of chronic salt depletion, renal mechanism(s) independent of neuronally released or systemically circulating catecholamines or of adrenally released aldosterone can ensure maximal salt conservation by the kidney. Although our data show that losartan increased sodium excretion under these conditions, we suggest that the losartan effect can be explained by a reduction of bicarbonate reabsorption, obligating simultaneous excretion of the cation.
Expression of the insulin-like growth factor system in the hypokalemic rat kidney
Authors: Rohan R. M. , Unterman T. G. , Liu L. , Hise M. K.
Published: December 2017

We studied the renal expression of the insulin-like growth factor (IGF) system to gain a better perspective of its potential role in the hyperplastic adaptation of the distal nephron to potassium deficiency. Rats were pair fed 1% or 0.002% potassium diets for periods up to 10 days. IGF-I mRNA was diminished in potassium-deficient rats within 4 days, whereas mRNA for IGF binding protein-1 (IGFBP-1), a collecting duct-associated protein, was increased by day 7. At day 10 mRNA for IGFBP-1 in potassium-deficient animals averaged 2.07 +/- 0.53 (mean +/- SD, relative densitometry units) compared with 0.89 +/- 0.26 in control rats (n = 4, P = 0.002). Conversely, IGFBP-3, a binding protein whose mRNA has been localized to the interstitial compartment, averaged 2.40 +/- 0.02 in potassium-deficient rats and 4.77 +/- 0.05 in controls (n = 4, P < 0.03) at day 10 of treatment. Immunohistochemistry performed using a specific IGFBP-1 antibody revealed hyperplasia of distal nephron segments along with an increase in IGFBP-1 in potassium-depleted rats. These data suggest that IGFBP-1 may play an important role in the control of cellular adaptations in the hypokalemic rat kidney either directly by influencing cell migration or indirectly by localizing IGF-I to the distal nephron.
Hydrogen peroxide downregulates IL-1-driven mesangial iNOS activity: implications for glomerulonephritis
Authors: Jaimes E. A. , Nath K. A. , Raij L.
Published: December 2017

In glomerulonephritides, autacoids such as nitric oxide (NO), reactive oxygen species, and prostanoids are produced in increased amounts in response to cytokines such as interleukin-1 (IL-1). These autacoids influence the expression of glomerular injury by their direct as well as interactive actions. We studied the effect of hydrogen peroxide (H2O2) on NO production in rat mesangial cells. We demonstrate that transient exposure of mesangial cells to H2O2 prior to sustained exposure to IL-1 decreased extracellular accumulation of NO2/NO3 and cellular guanosine 3,'5'-cyclic monophosphate (cGMP) content. H2O2 markedly impaired inducible nitric oxide synthase (iNOS) activity induced by IL-1 directly measured by the conversion of L-[14C]arginine to L-[14C]citrulline. Such impairment in iNOS activity was accompanied by a parallel reduction in iNOS protein abundance but not by a reduced expression of iNOS mRNA. The inhibitory effect of H2O2 on NOS activity was further supported by peroxide-induced impairment in IL-1-driven, NO-dependent synthesis of prostaglandin E2. Our studies thus provide the first direct evidence of a posttranscriptional inhibitory effect of H2O2 on iNOS activity. Additionally, our studies uncover the existence of a previously unrecognized effect of H2O2 on the production of NO that may exert influence on the severity of glomerular injury during glomerular inflammation.
Expression of PTHrP, PTH/PTHrP receptor, and Ca(2+)-sensing receptor mRNAs along the rat nephron
Authors: Yang T. , Hassan S. , Huang Y. G. , Smart A. M. , Briggs J. P. , Schnermann J. B.
Published: December 2017

To provide a frame of reference for studies of renal divalent cation and phosphate metabolism, we assessed the cellular localization of kidney calcium receptor (RaKCaR), parathyroid hormone-related protein (PTHrP), and parathyroid hormone/ parathyroid hormone-related protein (PTH/PTHrP) receptor mRNA. The studies used using reverse transcription-polymerase chain reaction (RT-PCR) applied to cDNA prepared from dissected rat nephron segments and from primary cultures of mouse juxtaglomerular granular cells. With species-specific primers, PCR products of expected size were obtained for RaKCaR (967 bp), PTHrP (420 bp), and PTH/PTHrP receptor (817 bp), with product identity being confirmed by restriction digestion. RaKCaR mRNA was found in medullary and cortical thick ascending limbs (MTAL and CTAL, respectively), the macula densa-containing segment, distal convoluted tubules (DCT), and, to a lesser extent, in cortical collecting ducts (CCD). It was not found in glomeruli, proximal convoluted and straight tubules (PCT and PST, respectively), outer and inner medullary collecting ducts (OMCD and IMCD, respectively), or in juxtaglomerular granular cell isolates. PTHrP mRNA was predominantly expressed in glomeruli and at lower levels in PCT and the macula densacontaining segment but was not detectable in CTAL, MTAL, DCT, and CD segments. Presence of PTH/PTHrP receptor mRNA was demonstrated in glomeruli, PCT, PST, CTAL, MTAL, and DCT but not in CD segments. These results suggest that the function of TAL and DCT cells, in addition to being affected by PTH, may be directly altered by extracellular divalent cations through RaKCaR and that PTHrP may act in the glomerulus and proximal tubule as an autocrine or paracrine regulator of hemodynamics and phosphate transport.
ANG II-dependent HCO3- reabsorption in surviving rat distal tubules: expression/activation of H(+)-ATPase
Authors: Levine D. Z. , Iacovitti M. , Buckman S. , Hincke M. T. , Luck B. , Fryer J. N.
Published: December 2017

Distal tubules (DT) from sham or five-sixths nephrectomized (Nx) rats were perfused in vivo to evaluate the hypothesis that, after Nx, endogenous angiotensin II (ANG II) modulates DT in vivo bicarbonate reabsorption (JtCO2) via H(+)-adenosinetriphosphatase (H(+)-ATPase) and Na+/H+ exchange. In Nx rats JtCO2 was increased (65 +/- 7 vs. -24 +/- 21 pmol.min-1.mm-1, P < 0.01). Both luminal and intravenous AT1-receptor blockade by losartan reduced Nx DT JtCO2 (41 +/- 6 and 34 +/- 4 pmol.min-1.min-1, respectively, P < 0.05), whereas neither 10(-9) M nor 10(-11) M ANG II luminal perfusion increased JtCO2, suggesting preexisting high endogenous ANG II levels. The Na+/H+ antiporter inhibitors 5-(N-ethyl-N-isopropyl)-amiloride and 5-(N,N-dimethyl)-amiloride were without effect. Luminal perfusion of 5 nM concanamycin A, a V-type H(+)-ATPase inhibitor, reduced Nx DT JtCO2 (45 +/- 8 pmol.min-1.mm-1, P < 0.05). In Nx A-type intercalated cells, we demonstrated cellular hypertrophy, elaboration of apical microplicae, and enhanced expression/apical polarization of H(+)-ATPase. Thus ANG II is an important determinant in sustaining brisk DT JtCO2 following Nx and is associated with enhanced expression and A-type intercalated cell apical polarization of H(+)-ATPase.
Cysteine S-conjugates activate transcription factor NF-kappa B in cultured renal epithelial cells
Authors: Otieno M. A. , Anders M. W.
Published: December 2017

Activation of NF-kappa B by the nephrotoxic and cytotoxic cysteine S-conjugate S-(1,2-dichlorovinyl)-L-cysteine (DCVC) was investigated in porcine kidney-derived LLC-PK1 cells. DCVC induced binding of nuclear proteins to an NF-kappa B consensus oligonucleotide from the immunoglobulin kappa-light chain enhancer region, as determined by electrophoretic mobility shift assays, and the activated proteins were identified as the p50/RelA heterodimeric complex of NF-kappa B. Transient transfection experiments with a kappa B-controlled luciferase reporter construct showed that the NF-kappa B complex activated by DCVC was transcriptionally active. NF-kappa B transactivation was blocked by inhibition of DCVC bioactivation with the cysteine conjugate beta-lyase inhibitor (aminooxy)acetic acid, by the antioxidants N,N'-diphenyl-p-phenylenediamine and N-acetyl-L-cysteine, and by the protein kinase inhibitor staurosporine. The cysteine S-conjugates S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine also activated NF-kappa B in LLC-PK1 cells. These results demonstrate that the NF-kappa B pathway is present in LLC-PK1 cells and is induced by cysteine S-conjugates. Inhibition of DCVC-induced transactivation of NF-kappa B by staurosporine and by antioxidants indicate roles for protein kinases and oxidative stress in the NF-kappa B pathway.
Cloning, expression, and regulation of rabbit cyclooxygenase-2 in renal medullary interstitial cells
Authors: Guan Y. , Chang M. , Cho W. , Zhang Y. , Redha R. , Davis L. , Chang S. , DuBois R. N. , Hao C. M. , Breyer M.
Published: December 2017

Prostaglandin synthesis requires cyclooxygenase-1 (COX1) or -2 (COX2), which mediate the conversion of arachidonate to prostaglandin H2. COX1 is the predominant constitutive isoform, whereas COX2 expression is typically low. In the present studies we cloned rabbit COX2 and determined its distribution in unstimulated tissues. Screening rabbit eye and uterine libraries yielded two cDNAs containing identical inserts with a 1,812-nucleotide open-reading frame. This encoded a 604-amino acid polypeptide, 90% identical to human, rat, and mouse COX2. Expression of the rabbit COX2 in HEK-293 cells enhanced prostanoid synthesis. Constitutive COX2 mRNA expression was highest in kidney and urinary bladder. COX2 expression was primarily in renal outer medullary interstitial cells with cortical expression in macula densa. In cultured medullary interstitial cells, COX2 mRNA predominated, with little COX1 expression. Interstitial cell COX2 mRNA but not COX1 was induced by phorbol ester and epidermal growth factor but suppressed by dexamethasone. Phorbol ester also upregulated immunoreactive COX2. Constitutive COX2 in these tissues has important implications for side effects of COX2-selective inhibitors.
Recombinant insulin-like growth factor I normalizes expression of renal glucose transporters in diabetic rats
Authors: Asada T. , Ogawa T. , Iwai M. , Shimomura K. , Kobayashi M.
Published: December 2017

By immunocytochemistry we have studied the effect of recombinant human insulin-like growth factor I (rhIGF-I) on expression of renal GLUT-1, -2, and -5 in rats with streptozotocin (STZ)-induced diabetes. In the renal tubules of these rats, expression of GLUT-1 was reduced and that of GLUT-2 was increased. GLUT-1 expression was restored, and GLUT-2 expression was normalized by 2-wk administration of rhIGF-I. We have shown that GLUT-5 was expressed at the brush-border membrane of the proximal convoluted tubules (PCT) of the cortex and at the glomerular mesangial cells (GMC) in normal rat kidney. In the diabetic rats, GLUT-5 expression was increased at both sites, along with an increase of GLUT-2 expression at the basolateral membrane of PCT, and was decreased to normal level at both sites by treatment with rhIGF-I. Thus, like GLUT-2, GLUT-5 is suggested to regulate glucose reabsorption in PCT. The relationship between overexpression of GLUT-5 in GMC and accumulation of sorbitol and advanced glycosylation end products are discussed. Regulation of GLUT expression may play an important role on renal glucose homeostasis.
Interactions of ultrapure bovine hemoglobin with renal epithelial cells in vivo and in vitro
Authors: Somers M. , Piqueras A. I. , Strange K. , Zeidel M. L. , Pfaller W. , Gawryl M. , Harris H. W.
Published: December 2017

Using an ultrapurified hemoglobin (Hb) solution, we investigated the physiological effects and cellular processing of Hb in rat kidneys and in cultured opossum kidney (OK) cells. Rats infused with < 5.0 g/kg Hb showed no change in baseline serum creatinine (SCr) values (0.58 +/- 0.05 mg/dl) over 48 h, whereas transient acute renal failure followed infusion of 7.5 g/kg Hb (SCr 3.4 +/- 1.02 mg/dl, P = 0.02). Histology of Hb-infused kidneys demonstrated tubular epithelial cell injury. Renal injury was not caused by volume or oncotic load, cardiovascular effect, or ATP depletion. After Hb infusion, heme oxygenase, the rate-limiting enzyme in Hb catabolism, was induced in an organ-specific fashion. Inhibiting heme oxygenase activity with cimetidine did not alter Hb renal injury. Using OK cells, we determined that renal epithelia process Hb by fluid-phase endocytosis. Proton permeability of fluorescein Hb endosomes was unaltered compared with fluorescein dextran controls, demonstrating that Hb does not alter endosomal membrane integrity. These data suggest that Hb renal injury in rats occurs following large doses of ultrapure Hb, does not alter early steps in Hb endosomal processing by renal epithelia, and involves a mechanism that is not heme oxygenase dependent.
Mechanisms of chloride transport in the proximal tubule
Authors: Aronson P. S. , Giebisch G.
Published: December 2017

The major fraction of filtered Cl- is reabsorbed in the proximal tubule. An important component of Cl- reabsorption is passive and paracellular, driven by the lumen-negative potential difference in the early proximal tubule and the outwardly directed concentration gradient for Cl- in the later proximal tubule. Evidence suggests that a significant additional component of NaCl reabsorption in the proximal tubule is active and transcellular. Cl-/formate and Cl-/oxalate exchangers have been identified as mechanisms of uphill Cl- entry across the apical membrane. For steady-state Cl- absorption to occur by these mechanisms, formate and oxalate must recycle from lumen to cell. Recent studies indicate that recycling of formate occurs by H(+)-coupled formate transport in parallel with Na+/H+ exchange, whereas oxalate recycling takes place by oxalate/sulfate exchange in parallel with Na(+)-sulfate cotransport. The predominant route for Cl- exit across the basolateral membrane is via Cl- channels. Unresolved issues include the adequacy of formate recycling to sustain Cl- absorption by Cl-/formate exchange, the existence and contributions of additional mechanisms for apical Cl entry and basolateral Cl- exit, and the relative magnitudes of transcellular and paracellular transport under physiological conditions. In addition, the molecular identification and mechanisms of regulation of the Cl-/formate and Cl-/oxalate exchangers remain to be defined.
Posttranscriptional upregulation of Na(+)-K(+)-ATPase activity in newborn guinea pig renal cortex
Authors: Guillery E. N. , Huss D. J. , McDonough A. A. , Klein L. C.
Published: December 2017

We measured Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity and subunit abundance in renal cortical homogenates and basolateral membranes (BLM) from fetal, newborn, and adult guinea pigs. Pump specific activity increased four- to fivefold in cortical homogenates and BLM during the transition from fetus to newborn. Immunoblots of BLM showed that alpha- and beta-subunit abundance increased four- to seven- and fourfold, respectively, during the transition from fetus to newborn. Immunoblots of cortical homogenates revealed similar developmental patterns, with newborns having 3.5-fold (alpha) and 2.3-fold (beta) greater subunit abundances than fetuses. Therefore, the bulk of the postnatal increase in BLM-Na(+)-K(+)-ATPase abundance resulted from increased pump production or decreased pump degradation, rather than from redistribution of pumps from intracellular pools. Despite the developmental increase in alpha- and beta-subunit protein levels, newborn guinea pig kidneys had only 1.4- to 2.1-fold greater alpha 1-subunit mRNA abundance and only a 1.5-fold greater beta 1-subunit mRNA abundance than fetal kidneys. These results demonstrate large increases in renal cortical Na(+)-K(+)-ATPase specific activity and protein abundance immediately after birth. These increases, which appear to result largely from posttranscriptional upregulation, may play an important role in mediating the rapid postnatal increase in tubular NaCl reabsorption.
Cloning of rat and mouse aquaporin-2 gene promoters and identification of a negative cis-regulatory element
Authors: Rai T. , Uchida S. , Marumo F. , Sasaki S.
Published: December 2017

The promoters of rat and mouse aquaporin-2 (AQP-2) genes were cloned and compared with that of human genes. Nucleotide identity up to -593 bp was 62%, and consensus sequences such as TATA box and adenosine 3',5'-cyclic monophosphate responsive element were conserved. Deoxyribonuclease I footprint assay revealed a footprinted region at -210 to -184 bp in rat AQP-2 gene promoter produced by nuclear extract from nonexpressing (liver) tissue. The sequence of this region included a GATA motif but otherwise showed no homology with any other previously known cis-elements. Electromobility shift assay and ultraviolet cross-linking analysis confirmed that specific binding proteins to this element were present in kidney, spleen, and liver and that these proteins were distinct from GATA factors. Both deletion and mutation of this cis-element abolished the protein DNA binding and increased promoter activity in in vitro reporter gene assay using rat cultured hepatocyte Ac2F cells, suggesting the negative regulatory role of this cis-element. These results indicate that tissue-specific expression of AQP-2 gene may in part be regulated by this novel negative acting cis-element.
Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney
Authors: Biemesderfer D. , Rutherford P. A. , Nagy T. , Pizzonia J. H. , Abu-Alfa A. K. , Aronson P. S.
Published: December 2017

Previous immunochemical studies have shown that NHE3 is an apical Na+/H+ exchanger in some renal epithelia. The purpose of the present study was to develop high-affinity, isoform-specific monoclonal antibodies (MAbs) that would be useful for carrying out high-resolution immunocytochemical studies of NHE3 in the adult and neonatal mammalian kidney. Three MAbs were developed to a fusion protein containing amino acids 702-832 of rabbit NHE3. Specificity was established by immunoblotting membranes from NHE-deficient LAP cells that had been transfected with either NHE1,-2, -3, or -4. With the use of high-resolution immunocytochemical techniques, NHE3 was found in vesicles in the apical cytoplasm of proximal tubule cells, as well as in the apical plasma membrane of the proximal tubule, and in both the thin and thick limbs of the loop of Henle. When localized in the 1-day-old rat kidney, NHE3 was first detected in the late stages of the S-shaped body. In later stages of nephron development, the pattern of NHE3 staining was similar to that seen in the adult. This study demonstrates 1) the specificity of three MAbs for Na+/H+ exchanger isoform NHE3; 2) NHE3 is present in an intracellular vesicular compartment in cells of the proximal tubule, consistent with possible regulation by membrane recycling; and 3) NHE3 is expressed on the apical membrane in early stages of the developing nephron.
Inducible expression of bcl-2 by the lac operator/repressor system in MDCK cells
Authors: Lin H. H. , Yang T. P. , Jiang S. T. , Liu H. S. , Tang M. J.
Published: December 2017

The lac operator/repressor-inducible system was utilized to dissect the biological consequences of human bcl-2 gene expression in Madin-Darby canine kidney (MDCK) cells. Cells were made transgenic for a constitutively expressed lacI gene, encoding lac repressor, and the bcl-2 gene that had been inserted downstream of a simian virus 40 (SV40) promoter containing the lac operator sequence. The expression of the bcl-2 gene could therefore be repressed to basal level by binding of lac repressor to the lac operator sequence in proximity to this SV40 regulatory region and be specifically activated by administration of the lactose analog isopropyl-beta-D-thiogalactoside (IPTG). We showed that expression of bcl-2 gene could be induced by 0.01 mM IPTG, and the maximal induction was obtained at 1 mM. With the treatment of IPTG, the Bcl-2 protein could be induced within 6 h. Moreover, the IPTG-inducible expression of Bcl-2 protein is a reversible process. Finally, functional assays revealed that IPTG-induced expression of bcl-2 gene conferred partial or complete resistance to homeless cell death or confluent cell death, respectively. The inducible expression system should be particularly useful for dissecting the effect of bcl-2 in phenotypic or morphological changes of MDCK cells.
Membrane potential measurements in renal afferent and efferent arterioles: actions of angiotensin II
Authors: Loutzenhiser R. , Chilton L. , Trottier G.
Published: December 2017

An adaptation of the in vitro perfused hydronephrotic rat kidney model allowing in situ measurement of arteriolar membrane potentials is described. At a renal perfusion pressure of 80 mmHg, resting membrane potentials of interlobular arteries (22 +/- 2 microns) and afferent (14 +/- 1 microns) and efferent arterioles (12 +/- 1 microns) were -40 +/- 2 (n = 8), -40 +/- 1 (n = 45), and -38 +/- 2 mV (n = 22), respectively (P = 0.75). Using a dual-pipette system to stabilize the impalement site, we measured afferent and efferent arteriolar membrane potentials during angiotensin II (ANG II)-induced vasoconstriction. ANG II (0.1 nM) reduced afferent arteriolar diameters from 13 +/- 1 to 8 +/- 1 microns (n = 8, P = 0.005) and membrane potentials from -40 +/- 2 to -29 +/- mV (P = 0.012). ANG II elicited a similar vasoconstriction in efferent arterioles, decreasing diameters from 13 +/- 1 to 8 +/- 1 microns (n = 8, P = 0.004), but failed to elicit a significant depolarization (-39 +/- 2 for control; -36 +/- 3 mV for ANG II; P = 0.27). Our findings thus indicate that resting membrane potentials of pre- and postglomerular arterioles are similar and lie near the threshold activation potential for L-type Ca channels. ANG II-induced vasoconstriction appears to be closely coupled to membrane depolarization in the afferent arteriole, whereas mechanical and electrical responses appear to be dissociated in the efferent arteriole.
Absence of tubuloglomerular feedback responses in AT1A receptor-deficient mice
Authors: Schnermann J. B. , Traynor T. , Yang T. , Huang Y. G. , Oliverio M. I. , Coffman T. , Briggs J. P.
Published: December 2017

Experiments were performed in a recently generated strain of mice with an angiotensin II AT1A-receptor null mutation (M. Ito, M. I. Oliverio, P. J. Mannon, C. F. Best, N. Maeda, O. Smithies, and T. M. coffman. Proc. Natl. Acad. Sci. USA 92: 3521-3525, 1995) to examine the effects of chronic AT1A receptor deficiency on tubuloglomerular feeback (TGF) responses. All animals were genotyped by polymerase chain reaction using primers designed to amplify sequences from the deleted AT1A gene and from the neomycin resistance gene. Normal mice (AT1A +/+) and mice heterozygous (AT1A +/-) and homozygous (AT1A -/-) for the gene disruption were anesthetized, and stop-flow pressures (PSF) were determined during changes in loop perfusion rate with previously established micropuncture methods. In five AT1A +/+ mice (26 tubules) mean PSF at zero loop flow was 37.2 +/- 1.5 mmHg, falling to 28.2 +/- 1.9 mmHg at a flow of 45 nl/min (P < 0.0001). Flow rate causing the half-maximum response (V1/2) was 8.7 +/- 0.4 nl/min. In four AT1A +/- animals (19 tubules) mean PSF at zero flow was 39.9 +/- 2.4 mmHg, falling to 34.8 +/- 2.7 mmHg at 45 nl/min (mean V1/2 8.6 +/- 1.04 nl/min). In five AT1A -/- mice (24 tubules) PSF was not significantly affected by loop flow with PSF averaging 33.9 +/- 1.7 mmHg at zero flow and 33.2 +/- 1.6 mmHg at 45 nl/min (not significant). Mean arterial blood pressures in the anesthetized and laparotomized mice were 91.8 +/- 2.2, 97.1 +/- 3, and 80.7 +/- 3.2 mmHg in the AT1A +/+, AT1A +/-, and AT1A -/- animals, respectively. Blood pressure responses to exogenous angiotensin II were greatly blunted in the AT1A -/- mice. We conclude that AT1A receptor-mediated effects of angiotensin II are in essential component of TGF responsiveness under chronic conditions. Our studies show the feasibility of using complex micropuncture methods in mice, an approach that widens the potential of genetically altered mouse strains as experimental models.
High glucose stimulates expression of p27Kip1 in cultured mouse mesangial cells: relationship to hypertrophy
Authors: Wolf G. , Schroeder R. , Ziyadeh F. N. , Thaiss F. , Zahner G. , Stahl R. A.
Published: December 2017

Hypertrophy of mesangial cells is an early hallmark of diabetic nephropathy. We have previously shown that murine mesangial cells (MMC), cultured in high-glucose medium, are arrested in the G1 phase of the cell cycle and undergo hypertrophy. This study was undertaken to test whether high glucose-containing medium influences the expression of p27Kip1, an inhibitor of G1 phase active cyclin-dependent kinases (CDK). Incubation of MMC, in the absence of other factors for 48-96 h, in medium containing high D-glucose (450 mg/dl), stimulated p27Kip1 protein expression but failed to influence mRNA abundance. These effects were independent of the osmolarity of the medium. High glucose-stimulated expression of p27Kip1 involved activation of protein kinase C and was partly dependent on induction of transforming growth factor-beta (TGF-beta). Immunoprecipitation experiments revealed that only small amounts of p27Kip1 protein from MMC grown in high-glucose medium preferentially associates with CDK2 but not with CDK4. The p27Kip1 antisense, but not missense, oligonucleotides inhibited high glucose-stimulated total protein synthesis and facilitated G1 phase exit. Our data showed for the first time that expression of p27Kip1 protein is pivotal in mesangial cell hypertrophy induced by high ambient glucose. These findings may be important in the deciphering of molecular processes causing diabetic glomerular hypertrophy.
Differentiation and cell polarity during renal cyst formation in the Han:SPRD (cy/+) rat, a model for ADPKD
Authors: Obermuller N. , Gretz N. , Kriz W. , van der Woude F. J. , Reilly R. F. , Murer H. , Biber J. , Witzgall R.
Published: December 2017

Despite the recent positional cloning of genes responsible for autosomal dominant polycystic kidney disease (ADPKD), the exact pathogenetic mechanisms underlying this disorder are still unclear. To learn more about cyst formation, we investigated cell differentiation and cell polarity in the Han:SPRD (cy/+) rat between 21 days and 60 wk of age. At early stages of cyst development, alkaline phosphatase, aquaporin-1, NaSi-1 cotransporter, and Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) were expressed normally. Clusterin mRNA was only sparsely expressed at the onset of cystic degeneration and increased thereafter, being highest in noncystic nephron segments. In cyst wall cells, clusterin on the one hand and alkaline phosphatase, aquaporin-1, NaSi-1-cotransporter, and Na(+)-K(+)-ATPase on the other were expressed in a mutually exclusive fashion. No change in cell polarity could be observed at any stage. Our data therefore argue against a change in cell polarity and against an early arrest in normal tubular development during cyst formation in the Han:SPRD (cy/+) rat model of ADPKD but favor the hypothesis that tubular epithelia develop in an orderly fashion and degenerate thereafter.
Experimental bilateral urinary obstruction in fetal sheep: transforming growth factor-beta 1 expression
Authors: Medjebeur A. A. , Bussieres L. , Gasser B. , Gimonet V. , Laborde K.
Published: December 2017

To gain insight into the role of transforming growth factor-beta 1 (TGF-beta 1) in the development of kidney pathology following fetal obstruction, we measured TGF-beta 1 gene expression, the active peptide, and the urinary concentration in a model of fetal bilateral urinary obstruction (BUO) in sheep. Fetal lambs underwent BUO at 60 (FO-60) or 80 days (FO-80) of gestation and were studied at 120 days. Independently of the onset or duration of obstruction, all fetuses developed type IV dysplasia (IV) associated with an arrest in the nephrogenesis or hydronephrosis. Fetal glomerular filtration rate was not significantly modified, whereas sodium tubular reabsorption was significantly decreased, and urinary TGF-beta 1 concentration was elevated in hydronephrosis but not in IV. Levels of TGF-beta 1 mRNA were increased in hydronephrosis compared with normal kidneys, and active TGF-beta 1 immunoreactivity was increased in both hydronephrotic and IV kidneys. In summary, TGF-beta 1 may play a role in the development of hydronephrosis and dysplasia in kidneys following fetal BUO. Its role in the arrest of nephrogenesis observed in the IV kidneys remains to be proved.
Differential expression of transforming growth factor-beta receptors in rat kidney development
Authors: Choi M. E. , Liu A. , Ballermann B. J.
Published: December 2017

Transforming growth factor-beta 1 (TGF-beta 1) is strongly expressed during embryogenesis and in sites undergoing intense development and morphogenesis. Two receptor serine/threonine kinases (types I and II) have been identified as signal-transducing TGF-beta receptors. This study was undertaken to further explore the role of the distinct TGF-beta receptors during kidney development. The species-specific sequence information for the two T beta R-I, namely, activin receptor-like kinase-5 (ALK-5) and Tsk7L, in the rat was sought. Two full-length T beta R-I cDNAs were cloned from a neonatal rat kidney and lung libraries, and sequencing revealed that they were the rat homologs of human ALK-5 and murine Tsk7L. Both types I and II TGF-beta receptors are expressed in the kidney as determined by Northern blot analysis. T beta R-II mRNA abundance was significantly greater in the neonatal rat kidney compared with the adult rat kidney. Similarly, ALK-5 mRNA was more highly expressed in the fetal and neonatal rat kidney than the adult rat kidney. In contrast, there was no significant difference in Tsk7L mRNA abundance among the fetal, neonatal, and adult rat kidney. Thus, based on these findings, both T beta R-II and ALK-5 are developmentally regulated in the kidney. Increased expression of T beta R-II and ALK-5 proteins in the developing kidney was confirmed by immunohistochemistry. Interestingly, the two TGF-beta receptors did not entirely colocalize, raising the intriguing possibility that other TGF-beta signaling receptors may be involved.