bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear localization of β-catenin and loss of apical brush border actin in cystic tubules of bcl-2 −/− mice
Authors: Sorenson Christine M.
Published: December 2017

Tight regulation of the rates of cell proliferation and apoptosis is critical for normal nephrogenesis. Nephrogenesis is profoundly affected by the loss of bcl-2 expression. Bcl-2-deficient (bcl-2 −/−) mice are born with renal hypoplasia and succumb to renal failure secondary to renal multicystic disease. Cell-cell and cell-matrix interactions impact tissue architecture by modulating cell proliferation, migration, differentiation, and apoptosis. E-cadherin mediates calcium-dependent homotypic cell-cell interactions that are stabilized by its association with catenins and the actin cytoskeleton. The contribution of altered cell-cell interactions to renal cystic disease has not been delineated. Cystic kidneys from bcl-2 −/− mice displayed nuclear localization of β-catenin and loss of apical brush border actin staining. The protein levels of α-catenin, β-catenin, actin, and E-cadherin were not altered in cystic kidneys compared with normal kidneys. Therefore, an altered distribution of β-catenin and actin, in kidneys from bcl-2 −/− mice, may indicate improper cell-cell interactions interfering with renal maturation and contributing to renal cyst formation.
Rat proximal tubule cell line transformed with origin-defective SV40 DNA: autocrine ANG II feedback
Authors: Ingelfinger Julie R. , Jung Flavia , Diamant Daniel , Haveran Liam , Lee Edwin , Brem Andrew , Tang Shiow-Shih
Published: December 2017

The renal proximal tubule (PT) is a major site for a complete tissue renin-angiotensin system (RAS) and produces endogenous angiotensin II (ANG II). The present studies demonstrate autocrine RAS feedback in a line of origin-defective SV40 plasmid transformed immortalized rat PT cells (IRPTC) designated as line 93-p-2–1, which are highly differentiated and express all RAS components. Receptor competition assays and Southern blot following RT-PCR demonstrated that these IRPTC express AT1and AT2angiotensin receptor subtypes. Autocrine RAS feedback was examined following exposure to ANG II (10−8M), and it was noted that angiotensinogen mRNA increases significantly by 1 h and remains elevated through 24 h. The AT1blocker losartan prevents this increase. Moreover, ANG II upregulates expression of ANG II receptor mRNA (both AT1and AT2). Thus the present studies demonstrate positive ANG II feedback with angiotensinogen and ANG II receptors in PTC, suggesting that the main site of such intrarenal feedback in vivo is within PT. ANG II secreted by line 93-p-2–1 is increased by isoproterenol, suggesting β-adrenergic regulation in IRPTC.
Kinin influences on renal regional blood flow responses to angiotensin-converting enzyme inhibition in dogs
Authors: Omoro Sophia A. , Majid Dewan S. A. , El-Dahr Samir S. , Navar L. Gabriel
Published: December 2017

The relative roles of ANG II and bradykinin (BK) in the regulation of renal medullary circulation have remained unclear. We compared the contributions of ANG II and BK to the renal medullary blood flow (MBF) responses to angiotensin-converting enzyme (ACE) inhibition (enalaprilat, 33 μg ⋅ kg−1 ⋅ min−1) in dogs maintained on a normal-salt diet (0.63%, 3 days, n = 14; group 1) with those fed a low-salt diet (0.01%, 5 days, n = 14; group 2), which upregulates both the kallikrein-kinin and the renin-angiotensin systems. MBF responses to ACE inhibition were evaluated either before ( n = 7) or after ( n = 7) treatment with the BK B2 receptor blocker icatibant (100–300 μg) in both groups. Laser-Doppler needle flow probes were used to determine relative changes in MBF and cortical blood flow (CBF). ACE inhibition increased MBF ( group 1, 33 ± 9%, P ≤ 0.01; group 2, 24 ± 9%, P ≤ 0.005) as well as CBF ( group 1, 23 ± 2%, P ≤ 0.006; group 2, 28 ± 10%, P ≤ 0.05). These responses were prevented by prior blockade of B2receptors in group 2, but not in group 1. These data indicate that under normal sodium intake, increases in MBF and CBF caused by ACE inhibition are primarily due to reduced intrarenal ANG II levels. In contrast, the renal vasodilatory responses to ACE inhibition in dogs on low salt intake were markedly dependent on the activation of BK B2 receptors.
Developmental expression of the epithelial Na+channel in kidney and uroepithelia
Authors: Watanabe Shigeru , Matsushita Kazumichi , McCray Paul B. , Stokes John B.
Published: December 2017

The epithelial Na+channel (ENaC) plays an important role in regulating Na+balance in neonatal and adult life. Using in situ hybridization, we localized α-, β-, and γ-rat ENaC (rENaC) mRNA in developing rat kidney and uroepithelia. rENaC mRNA was first detectable on fetal day 16, and by fetal day 17, mRNA was abundant in the terminal collecting duct and uroepithelia. After birth, the intensity of the signals for all three subunits increased in the cortical collecting ducts and by 9 days after birth had diminished in the inner medullary collecting ducts. Expression in uroepithelial cells was different. mRNA for β- and γ-rENaC, but not α-rENaC, was detected in pelvis, ureters, and bladder at all stages of development beyond fetal day 16. By RNase protection assay (RPA), the greatest increase in subunit abundance in the kidney occurred before birth. Between postnatal days 9 and 30, the abundance of β- and γ-rENaC decreased relative to α-rENaC in outer and inner medulla. The urinary bladder, in contrast, demonstrated the greatest increase in β- and γ-rENaC mRNA abundance after birth. We were generally unable to detect α-rENaC by RPA in urinary bladder. Feeding weaned rats a diet of high or low NaCl did not change the abundance of any of the subunit mRNAs in bladder. These results demonstrate additional heterogeneity of developmental expression and regulation of ENaC. The differences between the collecting duct and uroepithelial cell rENaC mRNA regulation raise the possibility of significant differences in function.
Adenosine-induced renal vasoconstriction in diabetes mellitus rats: role of nitric oxide
Authors: Pflueger Axel C. , Osswald Hartmut , Knox Franklyn G.
Published: December 2017

In rats with streptozotocin (STZ)-induced diabetes, the renal vasoconstrictor effect of adenosine is enhanced. We investigated the role of nitric oxide (NO) in the renal vascular response to exogenous and endogenous adenosine in control and STZ diabetic rats. Exogenous adenosine (0.01–100 nmol) injected into the abdominal aorta decreased renal blood flow (RBF) in a dose-dependent manner to a much greater extent in STZ rats than in control rats ( P < 0.001). Inhibition of NO synthesis with Nω-nitro-l-arginine (l-NNA, 30 μmol/kg iv) and with renal perfusion pressure controlled potentiated the adenosine-induced renal vasoconstriction to a significantly greater extent in control rats than in STZ rats. In control rats,l-NNA shifted the dose-response curve of exogenous adenosine-induced RBF reductions to the left by a factor of 32 [half-maximal effective dose (ED50), from 5.5 to 0.17 nmol adenosine, n = 6] and in STZ rats only by a factor of 4.6 (ED50, from 0.32 to 0.07 nmol adenosine, n = 6). The renal response to endogenous adenosine was assessed by the magnitude of the postocclusive reduction of RBF (POR) after a 30-s renal artery occlusion. POR was markedly enhanced in STZ rats (−67.8 ± 3.8%, P < 0.001) compared with control rats (−38.8 ± 4.3%).l-NNA markedly enhanced POR in control rats but did not increase POR in STZ rats. These findings demonstrate a greater potentiation of the adenosine-induced renal vasoconstriction in the presence ofl-NNA infusion in control rats compared with STZ rats. We conclude that the increased vasoconstrictor sensitivity of the diabetic renal vasculature to adenosine is caused by a defective NO-dependent renal vasodilation of the afferent arteriole in diabetic rats.
Isoforms of the Na-K-2Cl cotransporter in murine TAL II. Functional characterization and activation by cAMP
Authors: Plata Consuelo , Mount David B. , Rubio Verena , Hebert Steven C. , Gamba Gerardo
Published: December 2017

The functional properties of alternatively spliced isoforms of the mouse apical Na+-K+-2Cl−cotransporter (mBSC1) were examined, using expression in Xenopus oocytes and measurement of22Na+or86Rb+uptake. A total of six isoforms, generated by the combinatorial association of three 5′ exon cassettes (A, B, and F) with two alternative 3′ ends, are expressed in mouse thick ascending limb (TAL) [see companion article, D. B. Mount, A. Baekgaard, A. E. Hall, C. Plata, J. Xu, D. R. Beier, G. Gamba, and S. C. Hebert. Am. J. Physiol. 276 ( Renal Physiol. 45): F347–F358, 1999]. The two 3′ ends predict COOH-terminal cytoplasmic domains of 129 amino acids (the C4 COOH terminus) and 457 amino acids (the C9 terminus). The three C9 isoforms (mBSC1-A9/F9/B9) all express Na+-K+-2Cl−cotransport activity, whereas C4 isoforms are nonfunctional in Xenopus oocytes. Activation or inhibition of protein kinase A (PKA) does not affect the activity of the C9 isoforms. The coinjection of mBSC1-A4 with mBSC1-F9 reduces tracer uptake, compared with mBSC1-F9 alone, an effect of C4 isoforms that is partially reversed by the addition of cAMP-IBMX to the uptake medium. The inhibitory effect of C4 isoforms is a dose-dependent function of the alternatively spliced COOH terminus. Isoforms with a C4 COOH terminus thus exert a dominant negative effect on Na+-K+-2Cl−cotransport, a property that is reversed by the activation of PKA. This interaction between coexpressed COOH-terminal isoforms of mBSC1 may account for the regulation of Na+-K+-2Cl−cotransport in the mouse TAL by hormones that generate cAMP.
Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney
Authors: Simon Matthias , Maresh John G. , Harris Stephen E. , Hernandez James D. , Arar Mazen , Olson Merle S. , Abboud Hanna E.
Published: December 2017

BMP-7, a member of the bone morphogenic protein subfamily (BMPs) of the transforming growth factor-β superfamily of secreted growth factors, is abundantly expressed in the fetal kidney. The precise role of this protein in renal physiology or pathology is unknown. A cDNA that encodes rat BMP-7 was cloned and used as a probe to localize BMP-7 mRNA expression by in situ hybridization in the adult rat kidney. The highest expression of BMP-7 mRNA could be seen in tubules of the outer medulla. In glomeruli, a few cells, mainly located at the periphery of the glomerular tuft, showed specific and strong signals. Also, high BMP-7 mRNA expression could be localized to the adventitia of renal arteries, as well as to the epithelial cell layer of the renal pelvis and the ureter. Preliminary evidence suggests that BMP-7 enhances recovery when infused into rats with ischemia-induced acute renal failure. We examined BMP-7 mRNA expression in kidneys with acute renal failure induced by unilateral renal artery clamping. BMP-7 mRNA abundance as analyzed by solution hybridization was reduced in ischemic kidneys after 6 and 16 h of reperfusion compared with the contralateral kidney. In situ hybridization in ischemic kidneys showed a marked decrease of BMP-7 mRNA in the outer medulla and in glomeruli. Utilizing rat metanephric mesenchymal cells in culture, we also demonstrate that BMP-7 induces epithelial cell differentiation. Taken together, these data suggest that BMP-7 is important in both stimulating and maintaining a healthy differentiated epithelial cell phenotype.
ACE inhibition and ANG II receptor blockade improve glomerular size-selectivity in IgA nephropathy
Authors: Remuzzi Andrea , Perico Norberto , Sangalli Fabio , Vendramin Giovanni , Moriggi Monica , Ruggenenti Piero , Remuzzi Giuseppe
Published: December 2017

Protein trafficking across the glomerular capillary has a pathogenic role in subsequent renal damage. Despite evidence that angiotensin-converting enzyme (ACE) inhibitors improve glomerular size-selectivity, whether this effect is solely due to ANG II blocking or if other mediators also play a contributory role is not clear yet. We studied 20 proteinuric patients with IgA nephropathy, who received either enalapril (20 mg/day) or the ANG II receptor blocker irbesartan (100 mg/day) for 28 days in a randomized double-blind study. Measurements of blood pressure, renal hemodynamics, and fractional clearance of neutral dextran of graded sizes were performed before and after 28 days of treatment. Both enalapril and irbesartan significantly reduced blood pressure over baseline. This reduction reached the maximum effect 4–6 h after drug administration but did not last for the entire 24-h period. Despite transient antihypertensive effect, proteinuria was effectively reduced by both treatments to comparable extents. Neither enalapril nor irbesartan modified the sieving coefficients of small dextran molecules, but both effectively reduced transglomerular passage of large test macromolecules. Theoretical analysis of sieving coefficients showed that neither drug affected significantly the mean pore radius or the spread of the pore-size distribution, but both importantly and comparably reduced the importance of a nonselective shunt pathway. These data suggest that antagonism of ANG II is the key mechanism by which ACE inhibitors exert their beneficial effect on glomerular size-selective function and consequently on glomerular filtration and urinary output of plasma proteins.
Potassium supplement upregulates the expression of renal kallikrein and bradykinin B2receptor in SHR
Authors: Jin Lan , Chao Lee , Chao Julie
Published: December 2017

High potassium intake is known to attenuate hypertension, glomerular lesion, ischemic damage, and stroke-associated death. Our recent studies showed that expression of recombinant kallikrein by somatic gene delivery reduced high blood pressure, cardiac hypertrophy, and renal injury in hypertensive animal models. The aim of this study is to explore the potential role of the tissue kallikrein-kinin system in blood pressure reduction and renal protection in spontaneously hypertensive rats (SHR) on a high-potassium diet. Young SHR were given drinking water with or without 1% potassium chloride for 6 wk. Systolic blood pressure was significantly reduced beginning at 1 wk, and the effect lasted for 6 wk in the potassium-supplemented group compared with that in the control group. Potassium supplement induced 70 and 40% increases in urinary kallikrein levels and renal bradykinin B2receptor density, respectively ( P < 0.05), but did not change serum kininogen levels. Similarly, Northern blot analysis showed that renal kallikrein mRNA levels increased 2.7-fold, whereas hepatic kininogen mRNA levels remained unchanged in rats with high potassium intake. No difference was observed in β-actin mRNA levels in the kidney or liver of either group. Competitive RT-PCR showed a 1.7-fold increase in renal bradykinin B2receptor mRNA levels in rats with high potassium intake. Potassium supplement significantly increased water intake, urine excretion, urinary kinin, cAMP, and cGMP levels. This study suggests that upregulation of the tissue kallikrein-kinin system may be attributed, in part, to blood pressure-lowering and diuretic effects of high potassium intake.
Developmental expression of aquaporin 1 in the rat renal vasculature
Authors: Kim Jin , Kim Wan-Young , Han Ki-Hwan , Knepper Mark A. , Nielsen Søren , Madsen Kirsten M.
Published: December 2017

Aquaporin 1 (AQP-1) is a water channel protein that is constitutively expressed in renal proximal tubule and descending thin limb cells as well as in endothelial cells of the descending vasa recta. Studies in the developing rat kidney have demonstrated that AQP-1 is expressed in renal tubules before birth. However, nothing is known about the expression of AQP-1 in the renal vasculature during kidney development. The purpose of this study was to establish the distribution of AQP-1 in the renal vasculature of the developing rat kidney and follow the differentiation of the vascular system during kidney development. Kidneys from 16-, 17-, 18-, and 20-day-old fetuses and 1-, 4-, 7-, 14-, 21-, and 28-day-old pups were preserved and processed for immunohistochemical studies using a preembedding immunoperoxidase procedure. AQP-1 immunoreactivity was detected using affinity-purified rabbit polyclonal antibodies to AQP-1. AQP-1 was expressed throughout the arterial portion of the renal vasculature of the fetal and neonatal kidney from gestational age 17 days to 1 wk after birth. AQP-1 immunoreactivity gradually disappeared from the renal vasculature between 1 and 2 wk of age and remained only in the descending vasa recta. In contrast, AQP-1 immunoreactivity was not observed in lymphatic vessels until 3 wk of age and persisted in the adult kidney. AQP-1 was also expressed in a population of interstitial cells in the terminal part of the renal papilla at 3 wk of age as well as in the adult kidney. The transient expression of AQP-1 in the arterial portion of the renal vasculature in the developing rat kidney suggests that AQP-1 is important for fluid equilibrium and/or drainage in the developing kidney or, alternatively, plays a role in the regulation of growth and/or branching of the vascular tree during kidney development.
Partial characterization of apoptotic factor in Alzheimer plasma
Authors: Maesaka John K. , Palaia Thomas , Chowdhury Soheli A. , Shimamura Tetsuo , Fishbane Steven , Reichman William , Coyne Andrew , O’Rear Julian J. , El-Sabban Marwan E.
Published: December 2017

We have previously demonstrated that a plasma natriuretic factor is present in Alzheimer’s disease (AD), but not in multi-infarct dementia (MID) or normal controls (C). We postulated that the natriuretic factor might induce the increased cytosolic calcium reported in AD by inhibiting the sodium-calcium antiporter, thereby activating the apoptotic pathway. To test for a factor in AD plasma that induces apoptosis, we exposed nonconfluent cultured LLC-PK1cells to plasma from AD, MID, and C for 2 h and performed a terminal transferase-dUTP-nick-end labeling (TUNEL) assay. The plasma from AD increased apoptosis nearly fourfold compared with MID and C. The effect was dose dependent and the peak effect was attained after a 2-h exposure. Additionally, apoptotic morphology was detected by electron microscopy, and internucleosomal DNA cleavage was found. We inhibited apoptosis by removing calcium from the medium, inhibiting protein synthesis with cycloheximide, alternately boiling or freezing and thawing the plasma, and digesting a partially purified fraction with trypsin. Heating AD plasma to 56°C did not deactivate the apoptotic factor. These results demonstrate the presence of an apoptotic factor in the plasma of patients with AD.
Vasopressin regulates apical targeting of aquaporin-2 but not of UT1 urea transporter in renal collecting duct
Authors: Inoue Takeaki , Terris James , Ecelbarger Carolyn A. , Chou Chung-Lin , Nielsen Soren , Knepper Mark A.
Published: December 2017

In the renal inner medullary collecting duct (IMCD), vasopressin regulates two key transporters, namely aquaporin-2 (AQP2) and the vasopressin-regulated urea transporter (VRUT). Both are present in intracellular vesicles as well as the apical plasma membrane. Short-term regulation of AQP2 has been demonstrated to occur by vasopressin-induced trafficking of AQP2-containing vesicles to the apical plasma membrane. Here, we have carried out studies to determine whether short-term regulation of VRUT occurs by a similar process. Cell surface labeling with NHS-LC-biotin in rat IMCD suspensions revealed that vasopressin causes a dose-dependent increase in the amount of AQP2 labeled at the cell surface, whereas VRUT labeled at the cell surface did not increase in response to vasopressin. Immunoperoxidase labeling of inner medullary thin sections from Brattleboro rats treated with 1-desamino-8-d-arginine vasopressin (DDAVP) for 20 min revealed dramatic translocation of AQP2 to the apical region of the cell, with no change in the cellular distribution of VRUT. In addition, differential centrifugation of inner medullary homogenates from Brattleboro rats treated with DDAVP for 60 min revealed a marked depletion of AQP2 from the low-density membrane fraction (enriched in intracellular vesicles) but did not alter the quantity of VRUT in this fraction. Finally, AQP2-containing vesicles immunoisolated from a low-density membrane fraction from renal inner medulla did not contain immunoreactive VRUT. Thus vasopressin-mediated regulation of AQP2, but not of VRUT, depends on regulated vesicular trafficking to the plasma membrane.
Altered protein kinase C activation of Na+/Ca2+exchange in mesangial cells from salt-sensitive rats
Authors: Mashburn Nicole A. , Unlap M. Tino , Runquist Jeanette , Alderman Amy , Johnson Gail V. W. , Bell P. Darwin
Published: December 2017

The purpose of these studies was to determine whether there is a defect in protein kinase C (PKC) regulation of the Na+/Ca2+exchanger in cultured mesangial cells (MC) from Dahl/Rapp salt-sensitive (S) and salt-resistant (R) rats. R and S MCs were cultured, grown on coverslips, and loaded with fura 2 for measurement of single cell cytosolic calcium concentration ([Ca2+]i) in a microscope-based photometry system. Studies were performed in cells that were exposed to serum (serum fed) and in cells that were serum deprived for 24 h. Baseline [Ca2+]ivalues measured in a Ringer solution containing 150 mM NaCl were similar between R and S MCs in both serum-fed and serum-deprived groups, although baseline [Ca2+]ivalues were uniformly higher in the serum-deprived groups. Exchanger activity was assessed by reducing extracellular Na (Nae) from 150 to 2 mM, which resulted in movement of Na+out of and Ca2+into these cells (reverse-mode Na+/Ca2+exchange). PKC was activated in these cells with 15-min exposure to 100 nM phorbol 12-myristate 13-acetate (PMA). In the absence of PMA, the change in [Ca2+]i(Δ[Ca2+]i) with reduction in Naewas similar between R and S MCs in both serum-fed and serum-deprived groups, although the magnitude of Δ[Ca2+]iwas enhanced by serum deprivation. In both serum-fed and serum-deprived groups, PMA significantly increased Δ[Ca2+]iin R but not S MCs. Upregulation of exchanger activity in R MCs could be abolished by prior 24-h exposure to PMA, a maneuver that downregulates PKC activity. Other studies were performed to evaluate exchanger protein expression using monoclonal and polyclonal antibodies. Immunoblots of PMA-treated cells revealed an increase in the levels of 70- and 120-kDa proteins in the crude membrane fraction of R but not S MCs, an increase which was abrogated by prior 24-h PMA pretreatment and corresponded to reduction in the 70-kDa protein in the crude cytosolic fraction. These data demonstrate that PKC enhances Na+/Ca2+exchange activity in MCs from R but not from S rats, suggesting that there may be a defect in the PKC-Na+/Ca2+exchange regulation pathway in MCs of S rats.
Transport characteristics of the apical anion exchanger of rabbit cortical collecting duct β-cells
Authors: Emmons Cheryl
Published: December 2017

To functionally characterize transport properties of the apical anion exchanger of rabbit β-intercalated cells, the mean change in anion exchange activity, dpHi/d t(where pHiis intracellular pH), was measured in response to lumen Cl−replacement with gluconate in perfused cortical collecting ducts (CCDs). β-Cell apical anion exchange was not affected by 15-min exposure to 0.2 mM lumen DIDS in the presence of 115 mM Cl−. In contrast, apical anion exchange was significantly inhibited by 0.1 mM lumen DIDS in the absence of Cl−. β-Cell apical anion exchange was unchanged by 15 mM maleic anhydride, 10 mM phenylglyoxal, 0.2 mM niflumic acid, 1 mM edecrin, 1 mM furosemide, 1 mM probenecid, or 0.1 mM diphenylamine-2-carboxylate. However, β-cell apical anion exchange was inhibited by α-cyano-4-hydroxycinnamic acid, with an IC50of 2.4 mM. Substitution of either sulfate or gluconate for lumen Cl−resulted in a similar rate of alkalinization. Conversely, pHiwas unchanged by substitution of sulfate for lumen gluconate, confirming the lack of transport of sulfate on the β-cell apical anion exchanger. Taken together, the results demonstrate a distinct “fingerprint” of the rabbit CCD β-cell apical anion exchanger that is unlike that of other known anion exchangers.
A role for T-type Ca2+channels in the synergistic control of aldosterone production by ANG II and K+
Authors: Chen Xiao-Liang , Bayliss Douglas A. , Fern Robert J. , Barrett Paula Q.
Published: December 2017

Independently, plasma K+and ANG II stimulate aldosterone secretion from adrenal glomerulosa (AG) cells, but together they synergistically control production. We studied mechanisms to mediate this synergy using bovine AG cells studied under physiological conditions (in 1.25 mM Ca2+at 37°C). Increasing K+from 2 to 5 mM caused a potentiation of ANG II-induced aldosterone secretion and a substantial membrane depolarization (∼21 mV). ANG II inhibited a K+-selective conductance in both 2 and 5 mM K+but caused only a slight depolarization because, under both conditions, membrane potential was close to the reversal potential of the ANG II-induced current. ANG II activated calcium/calmodulin-dependent protein kinase II (CaMKII) equivalently in 2 and 5 mM K+. However, CaMKII activation caused a hyperpolarizing shift in the activation of T-type Ca2+channels, such that substantially more current was elicited at membrane potentials established by 5 mM K+. We propose that synergy in aldosterone secretion results from K+-induced depolarization and ANG II-induced modulation of T-type channel activation, such that together they promote enhanced steady-state Ca2+flux.
Glycated albumin stimulation of PKC-β activity is linked to increased collagen IV in mesangial cells
Authors: Cohen Margo P. , Ziyadeh Fuad N. , Lautenslager Gregory T. , Cohen Jonathan A. , Shearman Clyde W.
Published: December 2017

Albumin modified by Amadori-glucose adducts induces coordinate increases in the expression of extracellular matrix proteins, transforming growth factor (TGF)-β1, and the TGF-β type II receptor in glomerular mesangial cells. Because activation of protein kinase C (PKC) accompanies the increased mesangial cell expression of matrix proteins and TGF-β1 induced by high ambient glucose, we postulated that glycated albumin (GA) modulates PKC activity and that PKC participates in mediating the GA-induced stimulation of matrix production. To test this hypothesis, we examined the effects of PKC inhibitors on collagen type IV production by mouse or rat mesangial cells incubated with GA, and the influence of GA on PKC activity in these cells. Increased collagen type IV production evoked by GA in 5.5 and 25 mM glucose in mouse mesangial cells was prevented by both general (GF-109203X) and β-specific (LY-379196) PKC inhibitors. Total PKC activity, measured by phosphorylation of a PKC-specific substrate, increased with time after exposure of rat mesangial cells to GA compared with the nonglycated, glucose-free counterpart. GA caused an increase in PKC-β1 membrane-bound fraction and in total PKC activity in media containing physiological (5.5 mM) glucose concentrations in rat mesangial cells, confirming that the glucose-modified protein, and not a “hyperglycemic” milieu, was responsible. The findings indicate that Amadori-modified albumin stimulates mesangial cell PKC activity, and that activation of the PKC-β isoform is linked to the stimulation of collagen type IV production.
Regulation of angiotensin II receptors and PKC isoforms by glucose in rat mesangial cells
Authors: Amiri Farhad , Garcia Raul
Published: December 2017

It has been shown that glomerular angiotensin II (ANG II) receptors are downregulated and protein kinase C (PKC) is activated under diabetic conditions. We, therefore, investigated ANG II receptor and PKC isoform regulation in glomerular mesangial cells (MCs) under normal and elevated glucose concentrations. MCs were isolated from collagenase-treated rat glomeruli and cultured in medium containing normal or high glucose concentrations (5.5 and 25.0 mM, respectively). Competitive binding experiments were performed using the ANG II antagonists losartan and PD-123319, and PKC analysis was conducted by Western blotting. Competitive binding studies showed that the AT1receptor was the only ANG II receptor detected on MCs grown to either subconfluence or confluence under either glucose concentration. AT1receptor density was significantly downregulated in cells grown to confluence in high-glucose medium. Furthermore, elevated glucose concentration enhanced the presence of all MC PKC isoforms. In addition, PKCβ, PKCγ and PKCε were translocated only in cells cultured in elevated glucose concentrations following 1-min stimulation by ANG II, whereas PKCα, PKCθ, and PKCλ were translocated by ANG II only in cells grown in normal glucose. Moreover, no changes in the translocation of PKCδ, PKCι, PKCζ, and PKCμ were detected in response to ANG II stimulation under euglycemic conditions. We conclude that MCs grown in high glucose concentration show altered ANG II receptor regulation as well as PKC isoform translocation compared with cells grown in normal glucose concentration.
Angiotensin II inhibits HCO 3 − absorption via a cytochromeP-450-dependent pathway in MTAL
Authors: Good David W. , George Thampi , Wang Donna H.
Published: December 2017

The role of ANG II in the regulation of ion reabsorption by the renal thick ascending limb is poorly understood. Here, we demonstrate that ANG II (10−8M in the bath) inhibits [Formula: see text] absorption by 40% in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. The inhibition by ANG II was abolished by pretreatment with eicosatetraynoic acid (10 μM), a general inhibitor of arachidonic acid metabolism, or 17-octadecynoic acid (10 μM), a highly selective inhibitor of cytochrome P-450 pathways. Bath addition of 20-hydroxyeicosatetraenoic acid (20-HETE; 10−8M), the major P-450 metabolite in the MTAL, inhibited [Formula: see text] absorption, whereas pretreatment with 20-HETE prevented the inhibition by ANG II. The addition of 15-HETE (10−8M) to the bath had no effect on [Formula: see text]absorption. The inhibition of [Formula: see text]absorption by ANG II was reduced by >50% in the presence of the tyrosine kinase inhibitors genistein (7 μM) or herbimycin A (1 μM). We found no role for cAMP, protein kinase C, or NO in the inhibition by ANG II. However, addition of the exogenous NO donor S-nitroso- N-acetylpenicillamine (SNAP; 10 μM) or the NO synthase (NOS) substratel-arginine (1 mM) to the bath stimulated [Formula: see text] absorption by 35%, suggesting that NO directly regulates MTAL[Formula: see text] absorption. Addition of 10−11to 10−10M ANG II to the bath did not affect [Formula: see text] absorption. We conclude that ANG II inhibits [Formula: see text]absorption in the MTAL via a cytochrome P-450-dependent signaling pathway, most likely involving the production of 20-HETE. Tyrosine kinase pathways also appear to play a role in the ANG II-induced transport inhibition. The inhibition of [Formula: see text]absorption by ANG II in the MTAL may play a key role in the ability of the kidney to regulate sodium balance and extracellular fluid volume independently of acid-base balance.
Nitric oxide production is low in end-stage renal disease patients on peritoneal dialysis
Authors: Schmidt Rebecca J. , Yokota Stanley , Tracy Timothy S. , Sorkin Michael I. , Baylis Chris
Published: December 2017

To test the hypothesis that nitric oxide (NO) deficiency occurs in end-stage renal disease (ESRD), NO oxidation products (NO2+ NO3= NOx) and cGMP were measured in blood, urine, and dialysate effluent of peritoneal dialysis (PD) patients and compared with blood and urine of healthy subjects. All subjects were on a controlled low-nitrate diet (∼330 μmol/day). NOxand cGMP outputs were significantly reduced in PD patients (334 ± 50 μmol/24 h and 55 ± 13 nmol/24 h, respectively) vs. controls (823 ± 101 μmol/24 h and 149 ± 46 nmol/24 h). Plasma arginine was borderline low, plasma citrulline was elevated and plasma levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine were approximately five time higher in PD patients (2.2 ± 0.3 μM) vs. controls (0.4 ± 0.1 μM). Although blood pressure (BP) was not different between groups at the time of study, 10 of 11 PD patients were on medication for hypertension. These studies demonstrate that total NO production is low in ESRD, and with appropriate caution, we conclude that this NO deficiency may contribute to the increased BP that occurs in ESRD.
Developmental expression of ROMK in rat kidney
Authors: Zolotnitskaya Anna , Satlin Lisa M.
Published: December 2017

The apical secretory K+(SK) channel in the principal cell represents the rate-limiting step for K+secretion across the cortical collecting duct (CCD). Patch clamp analysis of maturing rabbit principal cells identifies an increase in number of conducting SK channels after the 2nd week of life [L. M. Satlin and L. G. Palmer. Am. J. Physiol. 272 ( Renal Physiol. 41): F397–F404, 1997], ∼1 wk after an increase in activity of the amiloride-sensitive epithelial Na+channel (ENaC) is detected. To correlate the postnatal increase in channel activity with developmental expression of ROMK, the molecular correlate of the SK channel, we used gene-specific probes to show a developmental increase in abundance of renal ROMK mRNA and a ROMK-specific antibody to examine the nephron distribution, localization, and abundance of this protein in developing rat kidney. Using antibodies directed against aquaporin-3 (AQP-3) and Tamm-Horsfall protein (THP), we confirmed that ROMK was expressed along the apical membranes of principal cells and thick ascending limbs of Henle (TALH) in adult kidney. Within the midcortex of the neonatal kidney, ROMK-positive segments revealed weak coincident staining with the TALH-specific antibody but did not colabel with an antibody directed against distal and connecting tubule (CNT)-specific kallikrein or the lectin Dolichos biflorus agglutinin (DBA), which labels proximal tubules and collecting ducts. In inner cortex and outer medulla of kidneys from 1-wk-old animals, ROMK protein was identified in medullary TALH (MTALH) and DBA-positive collecting ducts. By 3 wk of age, coincident ROMK and DBA expression was detected in midcortical and outer cortical CNTs and CCDs. Immunoblot analysis of plasma membrane-enriched fractions of maturing rat kidney revealed a developmental increase in a ∼40-kDa band, the expected size for ROMK. Immunolocalization of α-ENaC showed apical staining of a majority of cells in distal nephron segments after the 1st week of postnatal life. The β- and γ-ENaC subunit expression was routinely detected in a mostly cytoplasmic distribution immediately after birth, albeit in low abundance; γ-ENaC showed some apical polarization. These results suggest that the postnatal increases in a principal cell apical SK and Na+channel activity are mediated, at least in part, by increases in abundance of ROMK message and protein and ENaC subunit proteins.
Partial ATP depletion induces Fas- and caspase-mediated apoptosis in MDCK cells
Authors: Feldenberg L. Richard , Thevananther Sundararajah , del Rio Marcela , de Leon Maryely , Devarajan Prasad
Published: December 2017

Brief periods of in vitro hypoxia/ischemia induce apoptosis of cultured renal epithelial cells, but the underlying mechanisms remain unknown. We show that partial ATP depletion (≈10–65% of control) results in a duration-dependent induction of apoptosis in Madin-Darby canine kidney (MDCK) cells, as evidenced by internucleosomal DNA cleavage (DNA laddering and in situ nick end labeling), morphological changes (cell shrinkage), and plasma membrane alterations (externalization of phosphatidylserine). The ATP-depleted cells display a significant upregulation of Fas, Fas ligand, and the Fas-associating protein with death domain (FADD). Exogenous application of stimulatory Fas monoclonal antibodies also induces apoptosis in nonischemic MDCK cells, indicating that they retain Fas-dependent pathways of programmed cell death. Furthermore, cleavage of poly(ADP)ribose polymerase (PARP) is evident after ATP depletion, indicating activation of caspases. Indeed, the apoptotic cells display a significant increase in caspase-8 (FLICE) activity. Finally, apoptosis induced by ATP depletion is ameliorated by pretreatment with inhibitors of caspase-8 (IETD), caspase-1 (YVAD), or caspase-3 (DEVD) but is not affected by inhibitors of serine proteases (TPCK). Our results indicate that partial ATP depletion of MDCK cells results in apoptosis and that Fas- and caspase-mediated pathways may play a critical role.
d-Serine is reabsorbed in rat renal pars recta
Authors: Silbernagl Stefan , Völker Katharina , Dantzler William H.
Published: December 2017

d-Serine normally contributes up to 3% to total plasma serine and up to 23% in chronic renal failure. d-Serine is metabolized by tubular d-amino acid oxidase (D-AAO), and highd-serine plasma levels are nephrotoxic; both events are localized in the straight part of the proximal tubule. We therefore investigated if and howd-serine is reabsorbed there. We microinfused14C-labeledd- or -l-serine + [3H]inulin into early proximal (EP), late proximal (LP), or early distal (ED) tubule sections of superficial nephrons and into long loops of Henle (LLH) of rats in vivo and in situ. The fractional reabsorption (FR) of the14C label was determined from the14C:3H ratio in the final urine. At 0.36 mM, FR ofd-[14C]serine was 86% (EP), 90% (LP), and ≈0 (ED, LLH). FR ofd-serine could be saturated and inhibited by l-serine (and vice versa). d-methionine, but notd-glutamate ord-arginine, blocked FR ofd-serine (LP). We conlude that filtered d-serine is able to enter the pars recta cells, thereby getting access to D-AAO. The uptake carrier has a very low stereospecificity and is, therefore, different from that in the proximal convolution. The colocalization of exclusive reabsorption and metabolism makes the pars recta the tubule site for the recycling of the carbon structure of d-amino acids and, at the same time, the target ofd-serine nephrotoxicity.
Quantification of nitric oxide synthase activity in microdissected segments of the rat kidney
Authors: Wu Feng , Park Frank , Cowley Allen W. , Mattson David L.
Published: December 2017

This study was designed to quantify nitric oxide synthase (NOS) activity in microdissected glomeruli (Glm), pars convoluta, pars recta, cortical collecting duct, cortical thick ascending limb, outer medullary collecting duct, medullary thick ascending limb and thin limb, inner medullary collecting duct (IMCD) and thin limb, and vasa recta (VR). Total protein from microdissected segments was incubated withl-[3H]arginine and appropriate cofactors, and thel-arginine and convertedl-citrulline were separated by reverse-phase HPLC and radiochemically quantitated. NOS activity was found to be greatest in IMCD (11.5 ± 1.0 fmol citrulline ⋅ mm−1⋅ h−1) and moderate in Glm (1.9 ± 0.3 fmol ⋅ glomerulus−1⋅ h−1) and VR (3.2 ± 0.8 fmol ⋅ mm−1⋅ h−1). All other renal structures studied exhibited significantly less NOS activity. The mRNA for NOS isoforms in the NOS activity-positive segments was then identified by RT-PCR. The IMCD contained mRNA for neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS), but Glm and VR only expressed the mRNA for nNOS and eNOS. These experiments demonstrate that the greatest enzymatic activity for NO production in the kidney is in the IMCD, three- to sixfold less activity is present in the Glm and VR, and minimal NOS activity is found in other segments studied.
Serotonin 5-HT2Areceptor induces TGF-β1 expression in mesangial cells via ERK: proliferative and fibrotic signals
Authors: Grewal Jasjit S. , Mukhin Yurii V. , Garnovskaya Maria N. , Raymond John R. , Greene Eddie L.
Published: December 2017

We examined the links between fibrotic and proliferative pathways for the 5-HT2Areceptor in rat mesangial cells. Serotonin (5-hydroxytryptamine, 5-HT) induced transforming growth factor-β1 (TGF-β1) mRNA in a concentration-dependent (peak at 30 nM 5-HT) and time-dependent fashion. For 10 nM 5-HT, the effect was noticeable at 1 h and maximal by 6 h. Inhibition of 1) protein kinase C (PKC), 2) mitogen- and extracellular signal-regulated kinase kinase (MEK1) with 2′-amino-3′-methoxyflavone (PD-90859), and 3) extracellular signal-regulated kinase (ERK) with apigenin attenuated this effect. The effect was blocked by antioxidants, N-acetyl-l-cysteine (NAC) and α-lipoic acid, and mimicked by direct application of H2O2. TGF-β1 mRNA induction was also blocked by diphenyleneiodonium and 4-(2-aminoethyl)-benzenesulfonyl fluoride, which inhibit NAD(P)H oxidase, a source of oxidants. 5-HT increased the amount of TGF-β1 protein, validating the mRNA studies and demonstrating that 5-HT potently activates ERK and induces TGF-β1 mRNA and protein in mesangial cells. Mapping studies strongly supported relative positions of the components of the signaling cascade as follow: 5-HT2Areceptor → PKC → NAD(P)H oxidase/reactive oxygen species → MEK → ERK → TGF-β1 mRNA. These studies demonstrate that mitogenic signaling components (PKC, MEK, and oxidants) are directly linked to the regulation of TGF-β1, a key mediator of fibrosis. Thus a single stimulus can direct both proliferative and fibrotic signals in renal mesangial cells.
Nitric oxide synthase in the JGA of the SHR: expression and role in tubuloglomerular feedback
Authors: Welch William J. , Tojo Akihiro , Lee Jong-Un , Kang Dae Gil , Schnackenberg Christine G. , Wilcox Christopher S.
Published: December 2017

The spontaneously hypertensive rat (SHR) has an enhanced tubuloglomerular feedback (TGF) and a diminished buffering by juxtaglomerular apparatus (JGA)-derived NO. We examined the hypothesis that these effects are due to decreases in nitric oxide synthase (NOS) expression or limited availability of l-arginine or tetrahydrobiopterin (BH4). SHR had significantly ( P < 0.05) greater mRNA abundance (by RT-PCR) or protein (by Western analysis) for neuronal NOS (nNOS, or type I) and endothelial cell NOS (ecNOS, or type III) in renal cortex or isolated glomeruli, respectively. There was prominent expression of ecNOS in glomerular endothelium and nNOS in macula densa. Maximal TGF responses, assessed from changes in proximal stop-flow pressure during orthograde loop of Henle (LH) perfusion, were greater in SHR [Wistar-Kyoto (WKY), 8.1 ± 0.3 ( n = 46) vs. SHR, 10.3 ± 0.3 mmHg ( n = 57); P < 0.001]. Unlike WKY, TGF responses of SHR were unresponsive to microperfusion of the nNOS inhibitor, 7-nitroindazole (7-NI, 10−4M) [WKY, 9.5 ± 0.5 to 13.2 ± 0.7 ( n = 13, P < 0.001) vs. SHR, 11.8 ± 0.7 to 12.5 ± 0.6 mmHg ( n = 19, not significant)], or tol-arginine (10−3M) [WKY, 7.7 ± 0.8 to 6.3 ± 0.4 ( n = 10, P < 0.05) vs. SHR, 10.4 ± 0.7 to 10.6 ± 0.7 mmHg ( n = 10, not significant)]. Neither BH4(10−4M) nor sepiapterin (10−4M), its stable precursor, modified TGF responses in WKY or in SHR, nor did they restore a response to microperfusion of 7-NI in SHR. In conclusion, there is a diminished role for NO from nNOS in blunting of TGF in SHR which cannot be ascribed to limited NOS expression or availability of substrate or BH4.
Puromycin aminonucleoside nephrosis results in a marked increase in fractional clearance of albumin
Authors: Osicka Tanya M. , Hankin Alexandra R. , Comper Wayne D.
Published: December 2017

Puromycin aminonucleoside nephrosis (PAN) results in a marked increase in the fractional clearance of albumin. The increase in the fractional clearance of [3H]albumin to ∼0.045, as measured both in vivo and in the isolated perfused rat kidney (IPK) with PAN, occurs without an accompanying equivalent increase in glomerular capillary wall size selectivity as previously measured with dextrans. This is very similar to the marked increase in albuminuria seen with kidneys treated with inhibitors of endocytosis by the tubular epithelium, particularly lysine (T. M. Osicka, L. M. Pratt, and W. D. Comper. Nephrology 2: 199–212, 1996). The similarity is further established that, like in the presence of lysine, [3H]albumin excreted in urine from rats with PAN is essentially intact whereas, in both in vivo and IPK control experiments, excreted [3H]albumin is heavily degraded. The same observations have also been made for3H-labeled anionic horseradish peroxidase. These observations suggest that the significant albuminuria that occurs in PAN is primarily post-glomerular basement membrane in origin.
Immunolocalization of Ksp-cadherin in the adult and developing rabbit kidney
Authors: Thomson R. Brent , Aronson Peter S.
Published: December 2017

The potential for Ksp-cadherin involvement in either the development or maintenance of the metanephric kidney was assessed by immunocytochemical localization of a monoclonal antibody directed against the rabbit isoform of Ksp-cadherin in both neonatal and adult rabbit kidneys. In the adult kidney Ksp-cadherin expression was detected on the basolateral membrane of all cell types in both the tubular nephron and the collecting system. Immunoelectron microscopy indicated that Ksp-cadherin was expressed at uniform levels along the entire length of both the lateral membranes and the basal infoldings of all tubular epithelial cell types. In the nephrogenic zone of the neonatal rabbit kidney Ksp-cadherin expression was detected exclusively on the basolateral membranes of epithelial cells in the more highly differentiated regions of the expanding ureteric duct. In the highly differentiated corticomedullary and medullary regions of the neonatal kidney, distinct basolateral staining was observed in all segments of the tubular nephron and the collecting system. The relatively late appearance of Ksp-cadherin expression in the developing metanephros indicates that Ksp-cadherin probably does not participate in the direction of renal morphogenesis. However, the high levels of Ksp-cadherin expression observed in all segments of the tubular nephron and the collecting system in the adult kidney suggests that it may play a role in the maintenance of the terminally differentiated tubular epithelial phenotype.
TGF-β1-mediated hypertrophy involves inhibiting pRB phosphorylation by blocking activation of cyclin E kinase
Authors: Liu Baolian , Preisig Patricia
Published: December 2017

When renal epithelial cells are exposed to epidermal growth factor-transforming growth factor-β1 (EGF-TGF-β1) the typical EGF-mediated hyperplastic growth response is converted to a hypertrophic growth response. Hypertrophy in this setting involves cell entrance into G1, but arrest of cell cycle progression at the G1/S interface. Late G1arrest is mediated by retaining retinoblastoma protein (pRB) in its active, hypophosphorylated state. The present studies examine the mechanism by which pRB is retained in its active state. The results demonstrate that TGF-β1-mediated conversion of hyperplasia to hypertrophy involves preventing activation of cdk2/cyclin E kinase but has no effect on cdk4(6)/cyclin D kinase activity. Preventing activation of cyclin E kinase is associated with 1) decreased abundance of cdk2/cyclin E complexes and 2) retention of p57Kip2in formed cdk2/cyclin E complexes. The development of hypertrophy does not involve regulation of either cdk2, cyclin E, or cdc25A protein abundances, or the abundance of p27Kip1or p21 in formed complexes.