bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of p-aminohippurate transport by protein kinase C in OK kidney epithelial cells
Authors: Takano M. , Nagai J. , Yasuhara M. , Inui K.
Published: December 2017

We studied the effect of phorbol 12-myristate 13-acetate (PMA), a phorbol ester which activates protein kinase C, on p-aminohippurate (PAH) transport in OK cells. PMA (10(-7) M) almost completely inhibited the transcellular transport of PAH across OK cell monolayers from the basal to the apical side, as well as the accumulation of PAH in the cells. The uptake of PAH across the basolateral membrane of OK cells was inhibited by PMA in a time-and dose-dependent fashion. Exposing the cells with other protein kinase C activators such as active phorbol esters and diacylglycerols also resulted in a significant inhibition of basolateral PAH uptake, but the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, had no effect. The inhibition of basolateral PAH uptake by PMA was blocked by staurosporine, an inhibitor of protein kinase C. Cycloheximide, actinomycin D, colchicine, and cytochalasin D did not affect the inhibitory effect of PMA on basolateral PAH uptake. These results suggested that the PAH transport system in OK cells is under the regulatory control of protein kinase C.
Sublethal heat shock and cyclosporine exposure produce tolerance against subsequent cyclosporine toxicity
Authors: Yuan C. M. , Bohen E. M. , Musio F. , Carome M. A.
Published: December 2017

Sublethal heat shock has been shown to produce tolerance in cells and tissues subsequently exposed to heat or ischemia/ATP depletion. We tested whether heating LLC-PK1 cells for 2 h at 42 degrees C induced heat shock protein-70 (HSP-70) gene expression and conferred tolerance against subsequent cyclosporine A (CyA) toxicity. HSP-70 mRNA was increased immediately after heat shock, returning to baseline by 4 h. HSP-70 protein increased by 1 h after heat shock and declined thereafter, approaching baseline after 72 h. Cells heat shocked at 4 and 24 h prior to CyA exposure were significantly more viable than controls, at CyA concentrations near the median lethal dose (LD50). Cytoprotection declined with time after heat shock, concurrent with declining HSP-70 protein levels. Sublethal CyA exposure (50 micrograms/ml) for 24 h produced upregulation of HSP-70 mRNA and protein. Pretreatment with 50 micrograms/ml CyA for 24 h followed by exposure to a toxic concentration of CyA (200 micrograms/ml) produced significant cytoprotection compared with untreated controls. In conclusion, HSP-70 protein induction by sublethal heat shock or CyA exposure was associated with tolerance against subsequent lethal CyA exposure.
Localization and regulation of the rat renal Na(+)-K(+)-2Cl- cotransporter, BSC-1
Authors: Ecelbarger C. A. , Terris J. , Hoyer J. R. , Nielsen S. , Wade J. B. , Knepper M. A.
Published: December 2017

To investigate the role of the thick ascending limb (TAL) Na(+)-K(+)-2Cl- cotransporter in regulation of water excretion, we have prepared a peptide-derived polyclonal antibody based on the cloned cDNA sequence of the rat type 1 bumetanide-sensitive cotransporter, BSC-1 (also termed "NKCC-2"). Immunoblots revealed a single broad 161-kDa band in membrane fractions of rat renal outer medulla and cortex but not from rat colon or parotid gland. A similar protein was labeled in mouse kidney. Immunoperoxidase immunohistochemistry in rat kidney revealed labeling restricted to the medullary and cortical TAL segments. Because long-term regulation of urinary concentrating ability may depend on regulation of Na(+)-K(+)-2Cl- cotransporter abundance, we used immunoblotting to evaluate the effects of several in vivo factors on expression levels of BSC-1 protein in rat kidney outer medulla. Chronic oral saline loading with 0.16 M NaCl markedly increased BSC-1 abundance. However, long-term vasopressin infusion or thirsting of rats did not affect BSC-1 abundance. Chronic furosemide infusion caused a 9-kDa upward shift in apparent molecular mass and an apparent increase in expression level. These results support the previous identification of BSC-1 as the TAL Na(+)-K(+)-2Cl- transporter and demonstrate that the expression of this transporter is regulated.
Regulation of extracellular calcium sensing in rat osteoclasts by femtomolar calcitonin concentrations
Authors: Zaidi M. , Shankar V. S. , Adebanjo O. A. , Lai F. A. , Pazianas M. , Sunavala G. , Spielman A. I. , Rifkin B. R.
Published: December 2017

Certain eukaryotic cells can sense changes in their extracellular Ca2+ concentration through molecular structures termed Ca(2+)-sensing receptors (CaRs). We have shown recently that in the bone-resorbing osteoclast, a unique cell surface-expressed ryanodine receptor (RyR), functions as the CaR. The present study demonstrates that the sensitivity of this receptor is modulated by physiological femtomolar concentrations of the bone-conserving hormone, calcitonin. Calcitonin was found to inhibit cytosolic Ca2+ responses to both Ca2+ and Ni2+. The latter inhibition was mimicked by amylin (10(-12) M), calcitonin gene-related peptide (10(-12) M), cholera toxin (5 micrograms/l) and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) (2.5 x 10(-4) or 5 x 10(-4) M) and was reversed by the protein kinase A phosphorylation inhibitor, IP-20. Finally, using a quench flow module, we showed that cellular cAMP levels rise to a peak within 25 ms of calcitonin application; this is consistent with the peptide's rapid effect on CaR activation. We conclude, therefore, that cAMP plays a critical role in the control of CaR function by calcitonin.
Site-specific regulation of organic osmolytes along the rat nephron
Authors: Schmolke M. , Bornemann A. , Guder W. G.
Published: December 2017

The regulation of organic osmolytes was investigated in acute furosemide and chronic lithium diuresis along the nephron and in urinary bladder of rats. Sorbitol, myo-inositol, glycerophosphorylcholine, and betaine were measured enzymatically or by high performance liquid chromatography in homogenates and bioluminometrically in microdissected tubules. In untreated rats, all osmolytes except myo-inositol increased along the corticopapillary axis. An efflux of all osmolytes (-50%) was observed in homogenates of outer and inner medulla after acute furosemide diuresis (15 min, urinary osmolality = 329 mosmol/kgH2O) and for both polyols in microdissected tubules (30 min). In urinary bladder, only low concentrations of myo-inositol were found not to be affected by furosemide treatment. Chronic lithium treatment (7 days; urinary osmolality = 385 mosmol/kgH2O) decreased inner medullary but not outer medullary osmolyte concentrations. The results confirm a site-specific organic osmolyte pattern along the rat nephron, which is rapidly changed in a segment-specific way by different mechanisms of diuresis. The bladder epithelium does not accumulate organic osmolytes because no "osmotic gap" exists across the basolateral membrane. The osmotic difference across the apical membrane is maintained by the apical tightness of these cells.
Role of prostaglandins in renal medullary circulation: response to different vasoconstrictors
Authors: Parekh N. , Zou A. P.
Published: December 2017

This study investigated effects of renal prostaglandins and their interaction with different vasoactive agents in regulating regional renal blood flow. Using intravenous infusions, we compared effects of different pressor hormones and a nitric oxide (NO) inhibitor under control conditions and after inhibition of cyclooxygenase. Because vasodilator effects of prostanoids are considered to be mediated via opening of ATP-dependent K+ channels, we also studied effects of a prostacyclin analogue (iloprost), a channel opener (lemakalim), and a channel blocker (glibenclamide). Blood flow in renal cortex (CBF) and medulla (MBF) was determined with previously described platinum electrodes inserted into the kidney of anesthetized rats. Angiotensin II and norepinephrine reduced predominantly only CBF (-24 and -19%, respectively). After indomethacin, which selectively reduced MBF (-25%), angiotensin II and norepinephrine also reduced MBF (-45 and -35%, respectively), whereas the corresponding changes in CBF were not affected by indomethacin. Arginine vasopressin and the NO inhibitor NG-nitro-L-arginine methyl ester reduced both CBF and MBF by approximately 30% both under control conditions and after indomethacin. Iloprost and lemakalim increased selectively MBF (15 and 27%, respectively), whereas glibenclamide selectively decreased MBF (-19%). Our data indicate that renal prostaglandins are predominantly involved in regulating medullary circulation. They probably exert their dilatory action on medullary vessels via opening of ATP-dependent K+ channels and are involved in antagonizing medullary effects of pressor hormones in an agonist-specific manner.
Reduced bicarbonate secretion mediates increased distal tubule acidification induced by dietary acid
Authors: Wesson D. E.
Published: December 2017

We examined the components of net HCO3 reabsorption (H+/HCO3 secretion) in in vivo perfused distal tubules of anesthetized rats to determine the mechanisms by which dietary acid increases acidification in this nephron segment. Animals eating a minimum electrolyte diet drank either 80 mM NH4Cl or 40 mM (NH4)2SO4 for 7-10 days and were compared with controls drinking distilled H2O. Net HCO3 reabsorption in distal tubules perfused with HCO3 concentration ([HCO3]) similar to that in situ (5 mM) was higher in (NH4)2SO4 animals than in control (21.6 +/- 1.8 vs. 12.5 +/- 1.3 pmol.mm-1.min-1, respectively, P < 0.02), but that for NH4Cl (17.9 +/- 1.5 pmol.mm-1.min-1, P = 0.09 vs. control) animals was not. Calculated H+ secretion was not different among groups perfused with the 5 mM HCO3 solution, but calculated HCO3 secretion was lower in (NH4)2SO4 animals than control (-2.4 +/- 0.3 vs. -5.3 +/- 0.6 pmol.mm-1.min-1, respectively, P < 0.02), but that for NH4Cl (-7.2 +/- 0.7 pmol.mm-1.min-1, P = not significant vs. control) was not. When distal tubules were perfused with solutions containing higher [HCO3] (10 nM), both net HCO3 reabsorption and calculated H+ secretion were significantly higher than control in both acid-ingesting groups. The data show that reduced HCO3 secretion mediates the increased distal tubule acidification induced by dietary acid, particularly at the low tubule fluid [HCO3] in situ. The data also show that acid ingested as the Cl- compared with the SO4(-) salt does not reduce HCO3 secretion and less effectively increases acidification in this nephron segment.
Primary structure of rat HGF receptor and induced expression in glomerular mesangial cells
Authors: Liu Y. , Tolbert E. M. , Sun A. M. , Dworkin L. D.
Published: December 2017

The c-met protooncogene encodes a transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF). It has been widely suggested that HGF and its receptor constitute a paracrine signaling system, in which mesenchymally derived cells produce ligand that binds to the receptor predominantly expressed on cells of epithelial origin. In this study, we have isolated and completely sequenced the entire coding region of c-met cDNA from the rat kidney. The nucleotide sequence of the rat c-met cDNA revealed that the HGF receptor is encoded within single open-reading frame as 190 kDa of a transmembrane glycoprotein consisting of 1,382 amino acids. Determination of c-met mRNA levels in various tissues revealed a widespread expression of c-met with the highest levels in kidney, lung, and liver. We found simultaneous induction of both HGF and its receptor gene expression by interleukin-6 (IL-6) in primary cultured rat glomerular mesangial cells. The expression of HGF and c-met was remarkably stimulated following incubation of rat mesangial cells with IL-6, in a time- and dose-dependent manner Our data suggest that autocrine action of HGF may be achieved in vivo through simultaneous induction of both HGF and its receptor expression in renal mesenchymal cells.
Developmental regulation of CFTR expression during human nephrogenesis
Authors: Devuyst O. , Burrow C. R. , Schwiebert E. M. , Guggino W. B. , Wilson P. D.
Published: December 2017

Cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and protein are expressed in proximal and distal tubules of the human kidney, but CFTR expression pattern during human nephrogenesis is unknown. We have now studied CFTR expression in fetal kidneys by immunohistochemistry and Western blot analysis, using six antibodies against human CFTR. CFTR was expressed in 12-wk human fetal kidneys, mostly in the apical membrane region of the ureteric bud epithelial cells. By 15 wk, CFTR was also diffusely expressed throughout the cytoplasm of proximal tubules and loops of Henle. No glomerular staining was seen at any state. From 15 to 24 wk of gestation this staining pattern remained constant and also included immunoreactivity of the transitional epithelium. Western blot for CFTR was performed on membrane extracts of human fetal kidneys, using T84 cells as a positive control. A 165-kDa protein corresponding to the predicted size of CFTR was seen at 13 wk and throughout development. We also observed a 75-kDa protein that was distinctly regulated during development. This protein was detected with several antibodies against the first half of CFTR (including the regulatory "R" domain) but not with a COOH-terminal-specific antibody and had the predicted size of a functional splice variant of CFTR identified in the human kidney. These results show the complex regulation of CFTR during nephrogenesis and raise the question of the respective roles of the full-length and the splice variant CFTR proteins in the human kidney.
The beta 1- and beta 2-adrenoceptor subtypes in cultured rat inner medullary collecting duct cells
Authors: Yasuda G. , Sun L. , Lee H. C. , Umemura S. , Jeffries W. B.
Published: December 2017

We investigated beta-adrenoceptor subtype(s) expressed in cultured rat inner medullary collecting duct (IMCD) cells. In radioligand binding assay, [125I]iodocyanopindolol bound to IMCD cell membranes, representing a single class of binding sites (dissociation constant = 96.1 pM, maximum binding capacity = 18.2 fmol/mg protein, n = 8). In competition studies, ICI-89406 (beta 1-antagonist) and ICI-118551 (beta 2-antagonist) bound with high affinity, fitting a two-site model. Isoproterenol increased intracellular adenosine 3',5'-cyclic monophosphate (cAMP) accumulation (half-maximal effective concentration = 200 nM). Propranolol completely inhibited isoproterenol-induced cAMP accumulation [half-maximal inhibitory concentration (IC50) = 270 nM]. ICI-89406 and ICI-118551 inhibited cAMP accumulation by 50% (IC50 = 1.5 microM and 1.7 microM, respectively). The combined addition of ICI-89406 and ICI-118551 resulted in a curve indistinguishable from that of propranolol. The beta 1- and beta 2-adrenoceptor mRNAs have been demonstrated using reverse transcription-polymerase chain reaction. In initial and terminal IMCD cells, propranolol (3 microM) inhibited isoproterenol-stimulated cAMP accumulation by 80%, whereas ICI-89406 (3 microM) and ICI-118551 (3 microM) resulted in only partial inhibition (50%). We conclude that both beta 1- and beta 2-adrenoceptors are expressed in initial and terminal IMCD cells in primary culture.
Halide transport patterns of apical anion exchange in rabbit cortical collecting duct intercalated cells
Authors: Emmons C.
Published: December 2017

To investigate halide selectivity patterns of cortical collecting duct (CCD) intercalated cells (ICs), intracellular pH (pHi) was measured in perfused rabbit outer CCD ICs in Na(+)-free, HCO-3-buffered, 115 mM Cl solutions. Apical anion exchange activity was measured as the dpH/dt after lumen Cl- replacement with gluconate. The perfusate was randomly changed to equimolar Br-, F-, or I-. Addition of Br- to the lumen in place of gluconate caused a brisk acidification similar to that with Cl- return. The acidification rate with I- replacement was only approximately 25% of that seen with Cl- readdition, and F- substitution resulted in a small alkalinization. In a separate protocol, each halide was substituted for lumen Cl-. Replacement of lumen Cl- with Br- resulted in intracellular acidification, whereas F- substitution caused an increase in pHi similar to that with gluconate. A slower rate of alkalinization was seen with I-. Separate tubules were perfused and bathed in Cl(-)-free gluconate solutions for 10 min, and then either Cl- or Br- was returned to the lumen. Similar rates of acidification were found with either Cl- or Br- return. Taken together, the results show that, at a halide concentration of 115 mM, the halide selectivity transport pattern of the apical anion exchanger of CCD ICs is Cl- = Br- > I- > F-.
Regulation of epithelial Na+ channels from M-1 cortical collecting duct cells
Authors: Chalfant M. L. , Peterson-Yantorno K. , O'Brien T. G. , Civan M. M.
Published: December 2017

The M-1 cell line is derived from the mouse cortical collecting duct and displays the low-conductance, highly Na(+)-selective channel activity of the alpha,beta, gamma-heterotrimeric epithelial Na+ channel (ENaC). The short-circuit current (Isc) across M-1 monolayers was 89 +/- 4 microA/cm2, and the transepithelial conductance was 2.1 +/- 0.2 mS/cm2. Isc was abolished by blocking the Na+ pump with ouabain. Both Isc and transepithelial conductance (gT) were inhibited by benzamil > amiloride >> dimethylamiloride. Under our experimental conditions, vasopressin, vasopressin, forskolin, and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) had no detectable effects on Isc or gT. Increasing apical Na+ entry with nystatin increased Isc. The possible regulation of the M-1 Na+ channel by cAMP-activated protein kinase A (PKA) was further examined with excised inside-out patches. The open-time probability (Po) was not fixed, displaying substantial variance. Perfusion with ATP itself, with the catalytic subunit of PKA with ATP, or with alkaline phosphatase had no consistent effect on Po, the unitary current, or the kinetics of the M-1 Na+ channel. The data are consistent with the concept that PKA stimulates ENaCs by phosphorylating a site with access to but not within the apical membrane patch during cell-attached and excised-patch studies.
In situ localization and osmotic regulation of the Na(+)-myo-inositol cotransporter in rat brain
Authors: Ibsen L. , Strange K.
Published: December 2017

Organic osmolytes accumulate in the mammalian brain when plasma osmolality is elevated chronically. An understanding of organic osmolyte regulation is important for elucidating the pathophysiology of numerous disease states, particularly those in which plasma osmolality is altered or manipulated. To this end, we have previously characterized the mechanisms of osmoregulatory myo-inositol accumulation and loss in rat brain glial cells. The validity of cell culture models of physiological functions, however, requires demonstration of similar behavior in vivo. We therefore examined the effect of serum hypertonicity and its correction on expression of the Na(+)-myo-inositol cotransporter (SMIT) in rat brain. Northern analysis revealed that chronic serum hypertonicity increased brain SMIT mRNA levels four- to sevenfold. Rapid reduction of serum osmolality caused a rapid fall in SMIT mRNA levels. In situ hybridization revealed a widespread distribution of cells expressing SMIT with striking regional variability in the number of and intensity of cells labeled. These results confirm and extend our studies of cultured glial cells and indicate that myo-inositol accumulation in vivo is due to increased expression of SMIT. Regional variability in the distribution of SMIT may be a factor in the differential vulnerability of certain brain regions to shifts in plasma osmolality.
Effect of adrenomedullin on cAMP levels along the rat nephron: comparison with CGRP
Authors: Edwards R. M. , Trizna W. , Stack E. , Aiyar N.
Published: December 2017

Adrenomedullin (ADM) is a recently discovered hypotensive peptide that has been detected in the kidney. Recent studies have shown that intrarenal administration of ADM produces natriuresis without changes in glomerular filtration rate. Although ADM stimulates adenosine 3',5'-cyclic monophosphate (cAMP) synthesis in some tissues, the site of action and signal transduction pathway in the kidney are unknown. In the present study, we examined the effects of ADM and calcitonin gene-related peptide (CGRP) on cAMP levels in glomeruli and tubules dissected from rat kidney. ADM (1 microM) stimulated cAMP formation in glomeruli (4-fold), cortical thick ascending limb (CTAL, 6-fold), and in the distal convoluted tubule (DCT, 16-fold). In glomeruli, ADM was more potent than CGRP [50% effective concentration (EC50) = 33.7 vs. 156 nM], whereas the opposite was true in the DCT (CGRP, EC50 = 55 vs. 327 nM for ADM). In both glomeruli and DCT, the responses to maximum concentrations of ADM and CGRP were not additive. In glomeruli, the CGRP antagonist, CGRP-(8-37), had no effect on ADM-induced increases in cAMP and inhibited CGRP activity only at concentrations > 1 microM. However, in the DCT and CTAL, CGRP-(8-37) inhibited both ADM- and CGRP-induced increases in cAMP. We conclude that in the kidney, as in other tissues, ADM and CGRP stimulate cAMP accumulation. Our results suggest that ADM and CGRP may be important in the regulation of glomerular and distal tubule function and are consistent with the view that glomeruli have “ADM-like” receptors that have a high affinity for ADM but a low affinity for CGRP and CGRP-(8-37), whereas the DCT has “CGRP-like” receptors with high affinity for CGRP and CGRP-(8-37) but a lower affinity for ADM.
Megalin/gp330 mediates uptake of albumin in renal proximal tubule
Authors: Cui S. , Verroust P. J. , Moestrup S. K. , Christensen E. I.
Published: December 2017

Serum albumin filtered in renal glomeruli is reabsorbed very efficiently in the proximal tubule by endocytosis. The present study was undertaken to determine whether megalin/gp330 binds and mediates endocytosis of albumin. Rat serum albumin (RSA) labeled with 125I and colloidal gold particles labeled with bovine serum albumin (BSA) were microinfused into rat surface proximal tubules in vivo, and tubular uptake was determined in the presence or absence of different substances known to interfere with ligand binding to megalin. Binding of 125I-BSA and 125I-RSA to purified megalin was also determined directly using Sepharose columns. The results revealed that the tubular uptake of 125I-labeled RSA was significantly inhibited by receptor-associated protein (RAP), which reduced the uptake by > 50% and by cold RSA. The uptake of BSA gold by the proximal tubule was very intensive. BSA gold was found in small and large endocytic vacuoles, dense apical tubules, and in lysosomes. The uptake was reduced by RAP to 17%, by EDTA to 19%, by BSA to 16%, by megalin to 35%, by cytochrome c to 49%, and, together with gentamicin, there was virtually no uptake. Megalin-Sepharose columns bound 125I-labeled BSA as well as 125I-RSA, the binding was inhibited by RAP and EDTA, and analysis of the eluate revealed the bound tracer to be albumin. In conclusion, the present study demonstrates that megalin is a mediator of albumin reabsorption in renal proximal tubules.
Localization of bumetanide- and thiazide-sensitive Na-K-Cl cotransporters along the rat nephron
Authors: Yang T. , Huang Y. G. , Singh I. , Schnermann J. , Briggs J. P.
Published: December 2017

The present study was undertaken to investigate the mRNA localization of the two major kidney-specific Na-K-Cl transport proteins, the bumetanide-sensitive cotransporter (NKCC2 in rabbit and BSC1 in rat) and the thiazide-sensitive cotransporter (TSC). NKCC2 from rabbit and mouse has been shown to exist in three isoforms (designated A, B, and F) that differ only in a 96-bp region. The divergent region of each of the three NKCC2 isoforms was cloned from rat kidney by a polymerase chain reaction (PCR)-based strategy, and isoform-specific primers were chosen. RNA and cDNA were prepared from renal cortex and medulla and from microdissected nephron segments. Using reverse transcription (RT)-PCR, the B isoform was detected only in cortex and the F isoform only in medulla, whereas the A from was found in both. In dissected nephron segments, the B form was found exclusively in cortical thick ascending limb (CTAL) and macula densa-containing segment (MDCS), the F form only in medullary thick ascending limb (MTAL) and outer medullary collecting duct, and the A form in CTAL, MDCS, and MTAL. An additional isoform including both A and F exons was identified by direct sequencing of a 592-bp product from medulla. The AF product was found only in the medulla and was localized exclusively in MTAL. TSC mRNA was detected exclusively in the distal convoluted tubule. Differential nephron localization of NKCC2 isoforms suggests that Na-K-Cl cotransporters may differ in their transport characteristics to explain regulation of salt transport along the nephron.
Asymmetry of tubuloglomerular feedback effector mechanism with respect to ambient tubular flow
Authors: Thomson S. , Vallon V. , Blantz R. C.
Published: December 2017

The contribution of preglomerular resistance changes to the tubuloglomerular feedback (TGF) effector response was evaluated by micropuncture in hydropenic rats. Studies were performed in free-flowing nephrons to compare the fractional compensation (C) for perturbations in late proximal flow (VLP) with associated changes in glomerular capillary hydrostatic pressure (PGC). VLP was monitored by a noninvasive optical technique, and PGC was monitored by direct capillary micropuncture. Data were employed in conjunction with a model of glomerular filtration to analyze the TGF effector mechanism. C varied with the applied perturbation (VH), showing a single peak near the natural operating point. In contrast, the effect of TGF on PGC was asymmetric about the operating point, such that PGC was sensitive only to large positive perturbations. The model predicts that changes in preglomerular resistance account for only a small fraction of the integrated TGF response to small disturbances in the ambient state of the nephron but account for a greater share of the response to larger increments in flow. The TGF response in hydropenic rats is mediated by both pre- and postglomerular vascular elements.
Prostacyclin increases distal tubule HCO3 secretion in the rat
Authors: Wesson D. E.
Published: December 2017

We examined whether prostacyclin (PGI2), a prostaglandin synthesized in the renal cortex that increases adenosine 3',5'-cyclic monophosphate levels in distal nephron epithelia, mediates increased HCO3 secretion in in vivo perfused distal tubules of anesthetized rats given dietary HCO3. Animals eating a minimum electrolyte diet given 80 mM NaHCO3 drinking solution increased urine excretion of 6-keto-PGF1 alpha, a PGI2 metabolite, by 2.6 +/- 0.3-fold compared with those drinking distilled H2O. NaHCO3 animals infused with indomethacin to inhibit PGI2 synthesis had lower HCO3 secretion than those without indomethacin (-8.9 +/- 0.9 vs. -18.7 +/- 1.8 pmol.mm-1.min-1, P < 0.01). By contrast, NaHCO3 animals infused with both PGI2 and indomethacin had higher HCO3 secretion than those given indomethacin alone (-16.0 +/- 1.5, P < 0.02 vs. indomethacin group). HCO3 secretion was not different between controls with and without indomethacin but was higher in the PGI2 + indomethacin compared with the indomethacin alone controls (-11.2 +/- 1.2 vs. -4.5 +/- 0.5, P < 0.01). The data show that PGI2 increases distal tubule HCO3 secretion in rats and suggest that this agent contributes to the increased distal tubule HCO3 secretion induced by dietary HCO3.
The p53-independent activation of transcription of p21 WAF1/CIP1/SDI1 after acute renal failure
Authors: Megyesi J. , Udvarhelyi N. , Safirstein R. L. , Price P. M.
Published: December 2017

In three different models of acute renal failure (ischemia, ureteral obstruction, and cisplatin administration), the p21WAF1/CIP1/SDI1 gene, the protein product of which is associated with cell-cycle interruption, terminal differentiation, and cellular senescence, was activated in murine kidney cells. This transcription was localized in kidney only to cells of thick ascending limbs and distal convoluted tubules. Although the tumor suppressor protein, p53, can trans-activate the p21 gene in some cells, increased levels of nuclear p53 protein could be demonstrated only in the cisplatin model of acute renal failure. High levels of p21 mRNA were induced in kidney of p53 "null" mice, demonstrating that p21 gene activation was through a p53-independent pathway. We also present evidence that, in the cisplatin model, both p53-independent and p53-dependent induction of p21 mRNA occur simultaneously. We conclude that p21 gene activation is a general response to renal injury and could be a key determinant of cell fate in the cell in which it is expressed.
K depletion modifies the properties of Sch-28080-sensitive K-ATPase in rat collecting duct
Authors: Buffin-Meyer B. , Younes-Ibrahim M. , Barlet-Bas C. , Cheval L. , Marsy S. , Doucet A.
Published: December 2017

Two distinct Sch-28080-sensitive K-adenosine triphosphatases (K-ATPases) were previously described in the rat nephron: a ouabain-resistant K-ATPase (type I) present in collecting ducts (CD) and a ouabain-sensitive from (type II) located in proximal tubules (PT) and thick ascending limbs (TAL). In K-depleted rats, K-ATPase activity is increased in CD, whereas it is reduced in PT and TAL. Because expression of colonic H-K-ATPase is restricted to the CD of K-depleted rats, we hypothesized that K-ATPase from the CD of K-depleted rats might be different from types I and II. Indeed, type III K-ATPase displays higher sensitivities to ouabain and to Sch-28080 than type II, a lower sensitivity to Sch-28080 than type I, and, conversely to types I and II, it can be stimulated by Na+. Pharmacological differences between types II and III K-ATPases were confirmed by [3H]ouabain binding experiments. Thus the rat kidney expresses three K-ATPases that differ by their pharmacological and kinetic properties, their distribution profile along the nephron and their behavior during K depletion.
Amylin stimulates proximal tubular sodium transport and cell proliferation in the rat kidney
Authors: Harris P. J. , Cooper M. E. , Hiranyachattada S. , Berka J. L. , Kelly D. J. , Nobes M. , Wookey P. J.
Published: December 2017

In autoradiographic studies in anesthetized rats, 125I-labeled amylin binding was associated with proximal convoluted tubules but not distal tubules, interstitium, or glomeruli in the renal cortex. Split-drop micropuncture experiments showed that perfusion of the peritubular capillaries with amylin (10(-9) M) stimulated proximal tubular fluid absorption by 28%. This effect was inhibited by luminal addition of ethylisopropylamiloride, indicating mediation by a brush-border Na+/H+ exchanger. Intravenous infusion of an amylin binding antagonist, AC-187, reduced proximal fluid reabsorption (22%) in anesthetized rats, indicating a role for endogenous amylin in salt homeostasis. In primary cultures of rat proximal tubule cells, amylin (10(-7) M) stimulated proliferation with a potency equal to epidermal growth factor. Peptide antagonists (AC-187, AC-413, and AC-512) of the amylin binding sites in the renal cortex blocked the mitogenic action of amylin. We conclude that amylin acts on renal proximal tubules to promote sodium and water reabsorption and cell proliferation. These novel actions may have implications for the development of hypertension for example in non-insulin-dependent diabetes mellitus and obesity in which hyperamylinemia has been observed.
Transforming growth factor-beta selectively inhibits branching morphogenesis but not tubulogenesis
Authors: Sakurai H. , Nigam S. K.
Published: December 2017

When cultured in type I collagen gels, two kidney-derived cell lines, Madin-Darby canine kidney (MDCK) cells and murine inner medullary collecting duct (mIMCD3) cells, from branching tubular structures in the presence of Swiss 3T3 conditioned medium, in which hepatocyte growth factor (HGF) is the major branching tubule inducing factor. However, upon incubation with transforming growth factor-beta (TGF-beta) in the presence of 3T3 conditioned medium, MDCK tubulogenesis and branching was markedly inhibited. In contrast, mIMCD3 cells, which are much less susceptible to growth and tubulogenesis inhibition by TGF-beta, formed long straight tubulelike structures in presence of TGF-beta, suggesting a dissociation between tubulogenesis and branching morphogenesis. Interestingly, those long tubules that did branch often superficially resembled the early branching ureteric bud in embryonic kidneys. Quantitation of branching events revealed a selective branch-inhibiting effect of TGF-beta on mIMCD3 cells at concentrations between 0.02 and 2 ng/ml. There was no qualitative or quantitative difference among TGF-beta 1, -beta 2, and -beta 3 on inhibition of branching events, suggesting existence of potentially redundant mechanisms for modulating branching morphogenesis. Concentrations of TGF-beta that resulted in long nonbranching tubules also altered the profile of extracellular matrix-degrading proteases and their inhibitors expressed by developing tubules. Ratios of urokinase type plasminogen activator (u-PA) to plasminogen activator inhibitor (PAI-l) and matrix metalloprotease (MMP)-1 to tissue inhibitor of metalloprotease (TIMP)-1 were both markedly decreased. In addition, apart from a direct effect on epithelial cell branching morphogenesis, TGF-beta downregulated the expression of HGF mRNA in Swiss 3T3 cells. Thus TGF-beta exerts at least three distinct effects relevant to tubulogenesis and branching morphogenesis inhibition of branching morphogenesis alone (mIMCD3 cells), inhibition of both tubulogenesis and branching morphogenesis (MDCK cells), and inhibition of the expression of growth factor which induce tubulogenesis and branching morphogenesis (3T3 cells). In the context of epithelial tissue development, which requires tightly regulated branching tubulogenesis of epithelial cells, the data suggest a model where branching patterns are regulated by a precise temporal and spatial balance between branching morphogens such as HGF and inhibitory morphogens such as members of the TGF-beta superfamily [e.g., TGF-beta isoforms, certain bone morphogenetic proteins].
Molecular physiology of urinary concentrating mechanism: regulation of aquaporin water channels by vasopressin
Authors: Knepper M. A.
Published: December 2017

The purpose of this review is to illustrate the application of molecular methodologies to the investigation of a fundamentally integrative problem in renal physiology, namely, the mechanism of regulation of water excretion by the kidney and the concomitant concentration of solutes in the urine. A new revolution in renal physiology is occurring as new research tools have become available as a result of the cloning of cDNAs for many of the major transporters and receptors in the renal medulla. Among the important renal medullary transporters are the aquaporin water channels, which mediate the osmotic water transport across renal medullary epithelia. One of these water channels, aquaporin-2, has been shown to be the target for short-term regulation of collecting duct water permeability by vasopressin. In addition, two collecting duct water channels, aquaporin-2 and aquaporin-3, are targets for long-term regulation by vasopressin through effects on the absolute expression levels of the water channel proteins. This review focuses on the mechanisms of both short- and long-term regulation of these water channels by vasopressin.
Arginine vasopressin interacts with thromboxane in hydronephrosis
Authors: Takenaka T. , Forster H.
Published: December 2017

The influence of hydronephrosis (6-10 wk) on the renal vascular response to arginine vasopressin (AVP) was assessed, using isolated perfused normal and hydronephrotic rat kidneys. In normal kidneys, AVP (0.3 nM) reduced renal perfusate flow (RPF) by 55 +/- 7% (P < 0.01). AVP-induced decrements in RPF were reversed partially by diltiazem (10 microM) and completely by 10 nM of an AVP (V1)-receptor antagonist (AVPX). In hydronephrotic kidneys, AVP reduced RPF by 81 +/- 2% (P < 0.01) and constricted afferent (AA) and efferent arterioles (EA) by 33 +/- 3 (P < 0.01) and 33 +/- 5% (P < 0.01), respectively. The addition of diltiazem altered neither RPF nor vessel diameters. Administration of AVPX recovered RPF, AA, and EA diameters. When hydronephrotic kidneys were pretreated with thromboxane (Tx) inhibitors, AVP reduced RPF by 62 +/- 5% (P < 0.01) and constricted AAs and EAs by 26 +/- 2 (P < 0.01) and 17 +/- 3% (P < 0.05), respectively. Under Tx blockade, diltiazem partially reversed the AVP-induced reduction in RPF and restored the decrements in AA diameter. Subsequent addition of AVPX returned RPF and EA diameter. Our data indicate that AVP elicits substantial renal microvascular constriction and suggest that AVP stimulates Tx production in hydronephrotic kidneys, thereby altering renal vascular responsiveness to this peptide.
Internalization and apical-to-basolateral transport of folate in rat kidney proximal tubule
Authors: Birn H. , Nielsen S. , Christensen E. I.
Published: December 2017

Folate derivatives are filtered in the glomeruli and reabsorbed within the nephron. The amount filtered largely exceeds the minimum daily requirements. Thus folate reabsorbed within the kidney must be returned to the circulation. To establish whether renal proximal tubule can accomplish this by transport, of [3H]folate across the cell, microperfusion of rabbit, proximal tubule with [3H]folate and [14C]inulin was performed. Transtubular transport of [3H]folate was 5 +/- 1% (0.25 +/- 0.07 fmol/min) of perfused amount/mm tubule and remained constant during a 2-h perfusion period. An accumulation of 15 +/- 4% (0.8 +/- 0.3 fmol/min) of perfused amount/mm tubule was observed during the same period. Furthermore, to determine whether endocytosis may be involved in the initial process of folate uptake in proximal tubule cells, we performed light microscopy autoradiography on cryosections of rat kidney cortex incubated with [3H]folate. Folate binding sites were located apically as well as intracellularly similar to the location of [3H]folate when injected into the abdominal aorta and visualized by light microscopy autoradiography. Thus folate binding sites as well as internalized folate is localized both apically and intracellularly. Micropuncture of rat proximal tubules with folate-coupled collodial gold particles showed significantly increased endocytosis of folate gold when evaluated quantitatively and compared with controls injected with noncoupled gold particles (0.22 +/- 0.08 vs. 0.03 +/- 0.01 gold particles/micron 2 tubule cell). The results show that kidney proximal tubule cells are capable of transcellular transport of [3H]folate with limited capacity. Folate gold particle uptake suggests that folate can be internalized by endocytosis.
H-K-ATPase activity in PNA-binding intercalated cells of newborn rabbit cortical collecting duct
Authors: Constantinescu A. , Silver R. B. , Satlin L. M.
Published: December 2017

Functional and immunocytochemical studies indicate that intercalated cells in the adult rabbit cortical collecting duct (CCD) possess an H-K-adenosinetriphosphatase (H-K-ATPase). Because growing subjects must retain K+ and excrete H+, we sought to determine whether H-K-ATPase is present in the CCD early in life and, if so, to assess its activity and polarity. H-K-ATPase activity was defined as the initial rate of Sch-28080-inhibitable K+-dependent cell pH (pHi) recovery observed, in the absence of Na+, in response to an in vitro acid load. Transporter activity was assayed in intercalated cells labeled with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and apical cell surface marker rhodamine peanut lectin (PNA) in split-open CCDs isolated from neonatal and adult New Zealand White rabbits. In Na+-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions (nominal absence of CO2/HCO3-), the rate of K+-dependent pH(i) recovery from a NH4Cl-induced acid load was similar in newborn (0.056 +/- 0.015 pH U/min, n = 9) and adult (0.060 +/- 0.019 pH U/min; n = 9, P = not significant) cells. This rate of K+-dependent pH(i) recovery was significantly reduced by 10-20 pM Sch-28080, an inhibitor of gastric H-K-ATPase, in both newborns (0.009 +/- 0.003 pH U/min, n = 7) and adults (0.013 +/- 0.007 pH U/min, n = 9) (P < 0.05 compared with rates in absence of inhibitor). To determine whether the location of the transporter is consistent with a role in K+ absorption and H+ secretion, pH(i) recovery of acutely acid-loaded intercalated cells in neonatal CCDs (n = 7) microperfused and bathed in the absence of Na+ and K+ was monitored after selective addition of K+ to either the luminal or basolateral membrane. Addition of 5 mM K+ led to a significantly greater rate of pH(i) recovery when it was added to the luminal rather than the peritubular solution (0.049 +/- 0.005 vs. 0.018 +/- 0.005 pH U/min, P < 0.05). We conclude that PNA-binding intercalated cells of the neonatal CCD possess H-K-ATPase activity, predominantly located in the apical membrane. This provides a mechanism for H secretion and K+ retention, processes required for growth.
Effect of nitric oxide synthase inhibition on renal hemodynamics in humans: reversal by L-arginine
Authors: Wolzt M. , Schmetterer L. , Ferber W. , Artner E. , Mensik C. , Eichler H. G. , Krejcy K.
Published: December 2017

Animal experiments indicate that inhibition of nitric oxide synthase (NOS) influences renal hemodynamics and that this effect can be reversed by L-arginine, the precursor of NO synthesis. We have therefore studied the effects of an inhibitor of NOS, N(G)-monomethyl-L-arginine (L-NMMA), and a subsequent coinfusion with L-arginine on renal hemodynamics. In a double-blind, randomized crossover design, eight healthy volunteers (means +/- 1SD, 25.6 +/- 3.1 yr) received a primed constant infusion of L-NMMA (3 mg/kg bolus infusion over 5 min, followed by 50 microg x kg(-1) x min(-1) over 120 min) with subsequent coinfusion of L-arginine (17 mg x kg(-1) x min(-1) over 30 min). In the absence of a hypertensive response, L-NMMA decreased renal plasma flow to 79% of baseline (P < 0.005); this effect was abrogated by L-arginine. Glomerular filtration rate was not affected, NO exhalation was reduced to 30% of baseline (P < 0.005) by L-NMMA, and this effect was attenuated by L-arginine. Our results demonstrate that basal NO production maintains renal blood flow in vivo in humans. In addition, the renal vasculature is particularly sensitive to inhibition of NOS, and these pharmacodynamic effects can be reversed by excess doses of L-arginine.
Expression of AQP family in rat kidneys during development and maturation
Authors: Yamamoto T. , Sasaki S. , Fushimi K. , Ishibashi K. , Yaoita E. , Kawasaki K. , Fujinaka H. , Marumo F. , Kihara I.
Published: December 2017

The mRNA expression and localization of the aquaporin (AQP) family in rat kidney were examined by ribonuclease protection assay and immunohistochemistry. AQP1, AQP2, AQP3, and AQP4 mRNA were hardly detectable in 16-day gestation fetuses. AQP1 mRNA was explosively expressed at 1 wk, keeping the level throughout life. AQP2 mRNA expression was apparently noticed in 18-day fetuses and was enhanced gradually with age to reach a plateau at 4 wk. AQP3 and AQP4 mRNA expression was significantly found at birth but was not changed remarkably thereafter. AQP2 protein appeared first at the apical side of collecting duct cells in 18-day fetuses. The staining intensity at the site increased with age, and basolateral staining was added in adult rats. AQP3 was distinctly demonstrated at the basolateral side of collecting duct cells after birth, and the staining intensity was almost stable throughout life. The progressive induction of AQP2 expression in the first 4 wk after birth is presumed to contribute to the maturation of urinary concentrating capacity during the kidney development.
Taurine reabsorption by a carrier interacting with furosemide in short and long Henle's loops of rat nephrons
Authors: Silbernagl S. , Volker K. , Lang H. J. , Dantzler W. H.
Published: December 2017

Taurine is net reabsorbed in the proximal convolution by Cl- -stimulated Na+ symport specific for beta-amino acids but not in later nephron segments. However, large unidirectional taurine transport takes place there. To investigate unidirectional taurine reabsorption, we comicroinfused [3H]taurine and [14C]inulin into late proximal (LP), early distal (ED), and late distal (LD) tubule segments, as well as into the tips of long loops of Henle (LLH) of rats in vivo, and determined fractional reabsorption of [3H]taurine (FR) in the ipsilateral urine. FR (9 micromol/l taurine) was 80-93 (LP), 16 (ED), and 8% (LD). At 26 mmol/l taurine, FR decreased to 13 (LP) and 6% (ED). FR also decreased when Na+ or Cl- was absent or furosemide (5 x 10(-5) mol/l) was added. Bumetanide (5 x 10(-5) mol/l) had no effect, whereas aniline-2-sulfonic acid (ASA) inhibited. During LLH microinfusion, FR was 55% at 66 micromol/l and 17% at 228 micromol/l and was again inhibited by furosemide and ASA but not by bumetanide. [14C]taurine reabsorption from microperfused proximal convolutions was not influenced by furosemide. Chronic water diuresis did not affect taurine reabsorption in short Henle's loops. We conclude that taurine can enter cells of the distal nephron from the lumen by an Na+- and partly Cl- -dependent carrier with which C alpha,beta-substituted taurine (ASA) and C alpha,beta- and N-substituted beta-alanine (furosemide) directly interact. Thus proximal and distal taurine carriers seem to be different.
Expression of a mutant myosin light chain that cannot be phosphorylated increases paracellular permeability
Authors: Gandhi S. , Lorimer D. D. , de Lanerolle P.
Published: December 2017

A murine leukemia retroviral vector was engineered to contain the DNA encoding either the wild-type, rat aorta 20-kDa myosin light chain (MLC20) or a mutant form of MLC20 in which Thr18 and Ser19 were mutated into alanines. These mutations result in a MLC20 that cannot be phosphorylated by myosin light chain kinase. An 11-amino acid epitope from c-myc was added to both MLC20 sequences to facilitate identification of these proteins. Madin-Darby canine kidney cells were stably transduced, and MLC20 expression was demonstrated by Western blot analysis using a myc-specific antibody. MLC20 exchange was demonstrated by purifying myosin from the transduced cells and repeating the Western blot analysis. Actin-activated adenosinetriphosphatase assays on the purified myosins demonstrated approximately 50% decrease in the rate of ATP hydrolysis by the myosin containing the mutant MLC20. Transepithelial electrical resistance was decreased and mannitol flux was increased across monolayers of cells expressing mutant MLC20. These data demonstrate that MLC20 phosphorylation is involved in regulating paracellular permeability and epithelial barrier function.