bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular and molecular mechanisms of renal peptide transport
Authors: Daniel H. , Herget M.
Published: December 2017

Renal epithelial cells express membrane transport proteins capable of cellular uptake of a large variety of di- and tripeptides. These transporters contribute to renal amino acid homeostasis and the efficiency of conservation of amino acid nitrogen. In addition, these transporters appear to play a role in the renal handling of xenobiotics that possess a peptide backbone. Peptide carriers specialized in transport of di- and tripeptides have been identified in bacteria, fungi, plants, and epithelial cells of mammalian intestine and kidney. They appear to represent an archaic transporter family conserved throughout evolution. As a unique feature, these peptide carriers utilize a transmembrane-electrochemical proton gradient as the driving force that enables them to transport peptides against a concentration gradient. Renal peptide transporters have been characterized in terms of mechanism of transport function and substrate specificity in a number of model systems. Within the last two years, kidney peptide transporters of a variety of species have been identified by cloning techniques. In this review we discuss the physiological importance of renal peptide carriers and the transport mechanisms at the cellular level. We also present the recent advancements in functional expression of the cloned proteins that provide first insights into their molecular architecture and mode of operation.
The persistent effect of long-term enalapril on pressure natriuresis in spontaneously hypertensive rats
Authors: Dukacz S. A. , Adams M. A. , Kline R. L.
Published: December 2017

Long-term angiotensin-converting enzyme inhibitor treatment has been shown to have a persistent antihypertensive effect in spontaneously hypertensive rats (SHR) long after discontinuation of treatment. To test the hypothesis that this persistent effect involves a shift in the pressure-natriuresis relation, we performed experiments in male, anesthetized SHR at 18 wk of age with fixed neural and hormonal influences on the kidney. Renal function was assessed at various levels of arterial pressure using standard clearance techniques. Enalapril (25 mg.kg-1.day-1 in drinking water) was administered from 4 to 14 wk of age and again 3 days before renal function studies. The following four groups of SHR were studied: 1) 10-wk treatment, 2) 10-wk + 3-day treatment, 3) 3-day treatment, and 4) untreated. Groups 1 and 4 had an intact renin-angiotensin system; groups 2 and 3 had the renin-angiotensin system blocked. Mean arterial pressure (MAP, mmHg; means +/- SE) under Inactin anesthesia was 139 +/- 4 (n = 9), 109 +/- 3 (n = 8), 149 +/- 1 (n = 9), and 181 +/- 7 mmHg (n = 9) for each of the four groups, respectively. Glomerular filtration rate was similar in all groups at resting levels of MAP, whereas renal blood flow was elevated in all treatment groups when compared with that in untreated SHR. Pressure-natriuresis, pressure-diuresis, and pressure-fractional sodium excretion curves for the 10-wk treatment group and 3-day only treatment group were shifted leftward to significantly lower pressures by approximately 25 mmHg, compared with the untreated group. The curves for the treated +3-day group were shifted an additional 30 mmHg to the left. The relationship between renal artery pressure (RAP) and renal interstitial hydrostatic pressure was also shifted 25-30 mmHg but only in rats that received the long-term treatment with enalapril. Three-day enalapril had no significant effect on this relationship. These data indicate that the persistent effect of long-term enalapril treatment on arterial pressure in SHR is the result of a shift in the pressure-natriuresis relationship. The mechanism for this effect involves hemodynamic changes that act to improve transmission of RAP to the interstitium, resulting in enhanced sodium excretion for a given level of RAP.
Low permeabilities of MDCK cell monolayers: a model barrier epithelium
Authors: Lavelle J. P. , Negrete H. O. , Poland P. A. , Kinlough C. L. , Meyers S. D. , Hughey R. P. , Zeidel M. L.
Published: December 2017

Barrier epithelia such as the renal collecting duct (in the absence of antidiuretic hormone) and thick ascending limb, as well as the stomach and mammalian bladder, exhibit extremely low permeabilities to water and small nonelectrolytes. A cell culture model of such epithelia is needed to determine how the structure of barrier apical membranes reduce permeability and how such membranes may be generated and maintained. In the present studies, the transepithelial electrical resistance and isotopic water and urea fluxes were measured for Madin-Darby canine kidney (MDCK) type I and type II cells, as well as type I cells expressing the mucin protein, MUC1, in their apical membranes. Although earlier studies had found the unstirred layer effects too great to permit measurement of transepithelial permeabilities, use of ultrathin semipermeable supports in this study overcame this difficulty. Apical membrane diffusive water permeabilities were 1.8 +/- 0.4 x 10(-4) cm/s and 3.5 +/- 0.5 x 10(-4) cm/s in MDCK type I and type II cells, respectively, at 20 degrees C. Urea permeability in type I cells at the same temperature was 6.0 +/- 0.9 x 10(-6) cm/s. These values resemble those of other barrier epithelial apical membranes, either isolated or in intact epithelia, and the water permeability values are far below those of other epithelial cells in culture. Transfection of MDCK type I cells with the major human urinary epithelial mucin, MUC1, led to abundant expression of the fully glycosylated form of the protein on immunoblots, and flow cytometry revealed that virtually all the cells expressed the protein. However, MUC1 had no effect on water or urea permeabilities. In conclusion, MDCK cells grown on semipermeable supports form a model barrier epithelium. Abundant expression of mucins does not alter the permeability properties of these cells.
Inhibition of PAH transport by parathyroid hormone in OK cells: involvement of protein kinase C pathway
Authors: Nagai Junya , Yano Ikuko , Hashimoto Yukiya , Takano Mikihisa , Inui Ken-Ichi
Published: December 2017

We have previously shown that the p-aminohippurate (PAH) transport system in OK kidney epithelial cell line is under the regulatory control of protein kinase C. Parathyroid hormone (PTH) could activate protein kinase C, as well as protein kinase A, in OK cells. In the present study, the effect of PTH on PAH transport was studied in OK cells. PTH inhibited the transcellular transport of PAH from the basal to the apical side, as well as the accumulation of PAH in OK cells. Basolateral PAH uptake was inhibited by PTH in a dose- and time-dependent manner. Protein kinase A activators did not affect the transcellular transport or the accumulation of PAH. The PTH-induced inhibition of the accumulation of PAH was blocked by a protein kinase C inhibitor staurosporine. These results suggest that PTH inhibits the PAH transport in OK cells and that the messenger system mediated by protein kinase C, not protein kinase A, plays an important role in the regulation of PAH transport by PTH.
Bradykinin-stimulated cPLA2phosphorylation is protein kinase C dependent in rabbit CCD cells
Authors: Lal Mark A. , Kennedy Chris R. J. , Proulx Pierre R. , Hébert Richard L.
Published: December 2017

We have used an established cell line of rabbit cortical collecting duct (RCCD) epithelial cells representing a mixed population of principal and intercalated cell types to determine which phospholipase A2(PLA2) enzyme therein is responsible for bradykinin (BK)-stimulated arachidonic acid (AA) release and how its activation is regulated. BK-stimulated AA release was reduced 92% by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA2(cPLA2). Examination of PLA2activity in vitro demonstrated that BK stimulation resulted in a greater than twofold increase in PLA2activity and that this activity was dithiothreitol insensitive and was inhibited by an antibody directed against cPLA2. To determine a possible role for protein kinase C (PKC) in the BK-mediated activation of cPLA2, we used the PKC-specific inhibitor Ro31-8220 and examined its effects on AA release, cPLA2activity, and phosphorylation. Ro31-8220 reduced BK-stimulated AA release and cPLA2activity by 51 and 58%, respectively. cPLA2activity stimulated by phorbol ester [phorbol 12-myristate 13-acetate (PMA)] displayed a similar degree of activation and was associated with an increase in serine phosphorylation identical to that caused by BK. The phosphorylation-induced activation of this enzyme was confirmed by the phosphatase-mediated reversal of both BK- and PMA-stimulated cPLA2activity. In addition, we have also found that PMA stimulation did not cause a synergistic potentiation of BK-stimulated AA release as did calcium ionophore. This occurred despite membrane PKC activity increasing 93% in response to PMA vs. 42% in response to BK. These data, taken together, indicate that cPLA2is the enzyme responsible for BK-mediated AA release, and, moreover, they indicate that PKC is involved in the onset responses of cPLA2to BK.
Cyclooxygenase-2 in rat nephron development
Authors: Zhang Ming-Zhi , Wang Jun-Ling , Cheng H.-F. , Harris Raymond C. , McKanna James A.
Published: December 2017

The inducible second isoform of cyclooxygenase (COX-2) that mediates inflammation also is expressed at low levels in normal adult rat kidneys and is upregulated in response to noninflammatory stimuli (R. C. Harris, J. A. McKanna, Y. Akai, H. R. Jacobson, R. N. DuBois, and M. D. Breyer. J. Clin. Invest. 94: 2504–2510, 1994). Roles in morphogenesis are indicated by reported teratogenicity of COX inhibitors and renal dysgenesis in COX-2 knockout mice (J. E. Dinchuk, B. D. Car, R. J. Focht, J. J. Johnston, B. D. Jaffee, M. B. Covington, N. R. Contel, V. M. Eng, R. J. Collins, P. M. Czerniak, A. G. Stewart, and J. M. Trzaskos. Nature 378: 406–409, 1995; S. G. Morham, R. Lagenbach, C. D. Loftin, H. F. Tiano, N. Vouloumanos, J. C. Jennette, J. F. Mahler, K. D. Kluckman, A. Ledford, C. A. Lee, and O. Smithies. Cell 83: 473–482, 1995). Blots from developing rat kidneys demonstrated that COX-2 mRNA and immunoreactive protein were present in neonates, peaked in the 2nd and 3rd postnatal weeks and declined to adult levels by the 3rd month. Immunolocalization and in situ hybridization detected intense COX-2 immunoreactivity and mRNA in a subset of thick ascending limb epithelial cells near the macula densa in each developing nephron; after 2 wk the COX-2 gradually waned. These data demonstrate that COX-2 expression is subject to normal developmental regulation and can be sustained over extended periods; they also support the conclusion that metabolites of COX-2 play important roles in the differentiation and early functions of mammalian nephrons.
Mechanism and regulation of riboflavin uptake by human renal proximal tubule epithelial cell line HK-2
Authors: Kumar Chandira K. , Yanagawa Norimoto , Ortiz Alvaro , Said Hamid M.
Published: December 2017

Riboflavin (RF), a water-soluble vitamin, is essential for normal cellular functions, growth, and development. Normal RF body homeostasis depends on intestinal absorption and recovery of the filtered vitamin in renal tubules. The mechanism and cellular regulation of the RF renal reabsorption process, especially in the human situation, are poorly understood. The aim of this study was therefore to address these issues, using a recently established human normal renal epithelial cell line, HK-2, as a model. Uptake of RF by HK-2 cells was found to be 1) linear with time for 5 min of incubation and occurring with minimal metabolic alterations, 2) temperature dependent, 3) Na+independent, 4) saturable as a function of concentration [apparent Michaelis constant ( Km) of 0.67 ± 0.21 μM and maximal velocity ( Vmax) of 10.05 ± 0.87 pmol ⋅ mg protein−1⋅ 3 min−1], 5) inhibited by structural analogs and anion transport inhibitors, and 6) energy dependent. Protein kinase C-, protein kinase A-, and protein tyrosine kinase-mediated pathways were found to have no role in regulating RF uptake. On the other hand, a Ca2+/calmodulin-mediated pathway appeared to play a role in the regulation of RF uptake by HK-2 cells via an effect on the Vmax, as well as on the apparent Kmof the RF uptake process. The uptake process of RF was also found to be adaptively regulated by the level of the substrate in the growth medium, with the effect being mediated through changes in the apparent Kmand the Vmaxof the uptake process. These results demonstrate that RF uptake by the human-derived renal epithelial cell line HK-2 is via a carrier-mediated system that is temperature and energy dependent and appears to be under the regulation of a Ca2+/calmodulin-mediated pathways and substrate level in the growth medium.
Effects of sulfate and chloride on three separate oxalate transporters reconstituted from rabbit renal cortex
Authors: Karniski Lawrence P.
Published: December 2017

Understanding the mechanism of sulfate-dependent, oxalate-stimulated chloride reabsorption in the mammalian proximal tubule is complicated by the presence of multiple oxalate and sulfate transport pathways. Accordingly, we developed a method of reconstituting functional oxalate transport from the rabbit renal cortex so that the individual transporters might be examined. Solubilized microvillus membrane proteins were separated by hydroxyapatite chromatography and then reconstituted into proteoliposomes. Two peaks of oxalate/oxalate exchange activity were observed. Sulfate (10 mM) cis-inhibits oxalate transport in the early peak by 93% and in the later peak by 41%. In contrast, 20 mM chloride inhibits oxalate/oxalate exchange by only 32% in the early peak but inhibits oxalate exchange by 70% in the later peak. Oxalate-stimulated sulfate uptake was observed in the early fractions but not in the later fractions. These data are consistent with the recovery of the sulfate/oxalate exchanger in the early hydroxyapatite fractions and the chloride/oxalate exchanger in the later fractions. The basolateral membrane sulfate/oxalate exchanger was also reconstituted. The reconstituted basolateral and apical membrane sulfate/oxalate exchangers demonstrate nearly identical patterns of substrate specificities. However, 98% of apical sulfate/oxalate exchange activity is lost following exposure to octylglucoside at room temperature, whereas the basolateral sulfate/oxalate exchange activity was reduced 67% ( P < 0.05). In conclusion, functional reconstitution of solubilized membrane proteins demonstrates that apical membrane chloride/oxalate exchange and sulfate/oxalate exchange are mediated by different transport proteins. Apical and basolateral sulfate/oxalate exchange may also represent transport on two separate exchangers.
Characterization of the 5′-flanking region of OK cell type II Na-Picotransporter gene
Authors: Hilfiker Helene , Hartmann Claudia M. , Stange Gerti , Murer Heini
Published: December 2017

The renal type II Na-Picotransport is the rate-limiting step in proximal tubular phosphate (Pi) reabsorption. Among the different “proximal tubular” cell lines, this transporter seem only to be expressed in opossum kidney cells (OK cells). We have isolated the 5′-flanking region of the ok-Npt2 gene (OK cell type II Na-Picotransporter) including exons 1–3 and containing a TFIID site (TATA box), a GCCAAT site, an AP1 site, and two microsatellite GGAA repeats. Major transcription initiation sites were determined by primer extension and rapid amplification of 5′ cDNA ends (5′-RACE). A 327-bp fragment containing the TFIID and GCAAT element was driving the downstream luciferase reporter gene in homologous transfection assays. Slightly reduced promoter activity was observed with a 198-bp fragment containing the GCAAT element; shorter fragments were without activity. Promoter activity (327-bp fragment) could also be observed in transfections into HeLa cells but not in U937 human macrophage cells, MCT mouse kidney cortex cells, and MDCK cells. Different “physiological” stimuli known to be associated with altered proximal tubular Na-Picotransport activity are without effect on transcriptional activity in above homologous transfection experiments.
Detection of dopamine receptor D1Asubtype-specific mRNA in rat kidney by in situ amplification
Authors: O’Connell Damian P. , Aherne Anna M. , Lane Eamon , Felder Robin A. , Carey Robert M.
Published: December 2017

In recent years, both molecular biological and immunohistochemical techniques, utilizing receptor subtype-specific probes and antibodies to cloned central nervous system dopamine receptors, have revealed their presence in a number of peripheral organs and tissues. Molecular techniques have been hindered by the low abundance of receptor mRNA in these sites, and reverse transcription-polymerase chain reaction (RT-PCR) has been utilized to address this problem. However, RT-PCR is most often employed on either isolated mRNA or microdissected tissue samples, thereby limiting interpretation of whole tissue distribution. The present paper describes the use of a novel self-sustained sequence replication system (3SR) to amplify a target mRNA sequence in situ within the tissue or cell of interest that is then detected with the use of an internal labeled probe, using standard nonisotopic in situ hybridization. Specifically, D1Areceptor mRNA was amplified and detected in kidney sections of Wistar-Kyoto rats (WKY). The amplified D1Areceptor mRNA was localized to renal arterioles, juxtaglomerular apparatus, and both proximal and distal tubules. mRNA was colocalized to regions shown also to contain D1Areceptor protein. D1Areceptor mRNA was predominantly localized in the cortex. Specificity of D1Areceptor mRNA detection was confirmed by appropriate localization in rat brain sections known to express D1Areceptor mRNA. In addition, we confirmed the presence of renal D1Areceptor mRNA by RT-PCR. We conclude that D1Areceptor mRNA is expressed in a site-specific manner in the WKY kidney. The use of 3SR in situ permits elucidation of site specific mRNA localization in a manner not reported previously.
Recovery of cellular functions following oxidant injury
Authors: Nowak Graz˙yna , Aleo Michael D. , Morgan Jan A. , Schnellmann Rick G.
Published: December 2017

This study investigated the recovery of renal proximal tubule cellular (RPTC) functions following oxidant-induced sublethal injury. tert-Butylhydroperoxide (TBHP) treatment resulted in 24% cell death and loss 4 h following the exposure. The remaining sublethally injured RPTC proliferated, and monolayer DNA content returned to control values on day 4 following TBHP exposure. Basal oxygen consumption (Qo2) and ATP content in sublethally injured RPTC were decreased 64 and 63%, respectively, at 4 h and returned to control values on day 6. Net lactate consumption decreased 71% at 4 h and returned to control values on day 4. In contrast, net glutamine consumption increased 2.7-fold at 4 h and returned to control values on day 6. Ouabain-sensitive Qo2, Na+-K+-adenosinetriphosphatase (Na+-K+-ATPase) activity, and Na+-coupled glucose transport were inhibited 77, 88, and 83%, respectively, at 4 h and recovered to control values on day 6. These data show that 1) mitochondrial function, Na+-K+-ATPase activity, active Na+transport, and Na+-coupled glucose transport are decreased in sublethally injured RPTC following oxidant exposure and are repaired over time; 2) monolayer regeneration precedes the recovery of mitochondrial and transport functions, and 3) sublethal injury and subsequent regeneration are associated with alterations in metabolic substrate utilization. These results suggest that oxidant-induced sublethal injury to RPTC may contribute to renal dysfunction and that RPTC can repair and regain cellular functions following oxidant injury.
Dissociation of K channel density and ROMK mRNA in rat cortical collecting tubule during K adaptation
Authors: Frindt Gustavo , Zhou Hao , Sackin Henry , Palmer Lawrence G.
Published: December 2017

The density of conducting K channels in the apical membrane of the rat cortical collecting tubule (CCT) is increased by a high-K diet. To see whether this involved increased abundance of mRNA coding for K channel protein, we measured the relative amounts of mRNA for ROMK, the clone of the gene thought to encode the secretory K channel in the CCT. Tubules were isolated and fixed for in situ hybridization with a probe based on the ROMK sequence. Radiolabeled probe associated with the tubule was quantified using densitometric analysis of the autoradiographic images of the tubules. The densitometry signal was shown to be proportional to the amount of radioactive probe in the sample and to the time of exposure of the film. The technique was able to detect an approximately twofold increase in the abundance of mRNA coding for the water channel aquaporin 3 (AQP3), in response to a 30-h dehydration period. Tubules from rats fed a normal diet or a high-K (10% KCl) diet had equal amounts of ROMK mRNA. This suggests that an increase in the abundance of mRNA does not underlie the increase in channel density observed under these conditions.
A decrease in renal medullary tonicity stimulates anion transport in Henle’s loop of rat kidneys
Authors: Capasso Giovambattista , Saviano Caterina , Ciani Francesca , Lang Florian , Russo Ferdinando , De Santo Natale G.
Published: December 2017

To investigate the effect of reduction in renal medulla osmolality on loop of Henle (LOH) net bicarbonate reabsorption, clearance and microperfusion experiments were performed on Sprague-Dawley rats. The decrease of renal medulla osmolality was induced by intravenous infusion of either a large dose of mannitol (mannitol protocol) or a hypotonic solution (hypotonic protocol) delivered at a rate to match the sodium and bicarbonate load of the control period. During the mannitol protocol, clearance data demonstrated a rise in glomerular filtration rate (GFR), renal plasma flow, urine pH, and fractional bicarbonate excretion. On the contrary, microperfusion experiments, performed in the absence of mannitol in the tubular perfusate, revealed a significant increase both in the absolute and fractional LOH bicarbonate transport. During the hypotonic protocol, there was a decrease in GFR, associated with an increase in fractional excretion of bicarbonate. In the microperfusion experiments, hypotonic saline, similar to mannitol, stimulated absolute and fractional LOH bicarbonate transport. Net reabsorption of chloride, measured under the same experimental conditions, was also found to be activated. Therefore, the intravenous infusion of hypotonic solution affected the LOH transepithelial net reabsorption of both bicarbonate and chloride. We hypothesize that the increase in the transport rate of these two anions, along the same segment and in similar experimental conditions, may be mediated, at least in part, by decreased medullary tonicity, which is one factor common both to hypertonic mannitol and hypotonic saline infusion.
Copper metabolism in the kidney of rats administered copper and copper-metallothionein
Authors: Kurasaki Masaaki , Okabe Masashi , Saito Shigeru , Suzuki-Kurasaki Mika
Published: December 2017

To gain a greater understanding of the mechanism of Cu metabolism in kidneys of rats, using autofluorescence of Cu-metallothioneins (Cu-MTs) we revealed the behavior of Cu-MT in the kidneys of rats administered Cu-MT. Yellow and orange fluorescent signals of Cu-MT were observed in the cortex. By microscopic studies, Cu-MT was dominant in the proximal convolute tubular cells of the cortex. A high concentration of Cu-MT presented in the lysosome-like organelles of the proximal convolute tubular adjacent to the glomeruli. During the time course after the injection, the orange signal in lysosome-like organelles gradually converted to a yellow signal, indicating that the Cu-MT was involved in a degradation process in lysosomes by oxidation, and the MT mRNA increased in the cortex, although the immunoreactivity of MT was almost constant in the same region. These results suggested that Cu bound to the injected MT was released in lysosomes and became a new inducer of MT biosynthesis in the cortex. In conclusion, the biosynthesis and degradation of Cu-MT occur repeatedly in the proximal convolute tubular cells.
A mathematical model of the inner medullary collecting duct of the rat: acid/base transport
Authors: Weinstein Alan M.
Published: December 2017

A mathematical model of the inner medullary collecting duct (IMCD) of the rat has been developed that is suitable for simulating luminal buffer titration and ammonia secretion by this nephron segment. Luminal proton secretion has been assigned to an H-K-ATPase, which has been represented by adapting the kinetic model of the gastric enzyme by Brzezinski et al. (P. Brzezinski, B. G. Malmstrom, P. Lorentzon, and B. Wallmark. Biochim. Biophys. Acta 942: 215–219, 1988). In shifting to a 2 H+:1 ATP stoichiometry, the model enzyme can acidify the tubule lumen ∼3 pH units below that of the cytosol, when luminal K+is in abundance. Peritubular base exit is a combination of ammonia recycling and[Formula: see text] flux (either via[Formula: see text] exchange or via a Cl−channel). Ammonia recycling involves[Formula: see text] uptake on the Na-K-ATPase followed by diffusive NH3exit [S. M. Wall. Am. J. Physiol. 270 ( Renal Physiol. 39): F432–F439, 1996]; model calculations suggest that this is the principal mode of base exit. By virtue of this mechanism, the model also suggests that realistic elevations in peritubular K+concentration will compromise IMCD acid secretion. Although ammonia recycling is insensitive to carbonic anhydrase (CA) inhibition, the base exit linked to [Formula: see text] flux provides a CA-sensitive component to acid secretion. In model simulations, it is observed that increased luminal NaCl entry increases ammonia cycling but decreases peritubular [Formula: see text]exchange (due to increased cell Cl−). This parallel system of peritubular base exit stabilizes acid secretion in the face of variable Na+reabsorption.
Direct visualization of renal vascular morphogenesis inFlk1heterozygous mutant mice
Authors: Robert Barry , St. John Patricia L. , Abrahamson Dale R.
Published: December 2017

Flk1, a receptor tyrosine kinase for vascular endothelial growth factor (VEGF), is the earliest known marker for endothelial precursors (angioblasts). We examined heterozygous mice in which the Flk1gene was partially replaced by a promoter-less LacZ insert and used β-galactosidase histochemistry to view cells transcribing Flk1. In day 10 ( E10) embryos, a Flk1-positive network surrounded the metanephric blastema, and, at E11, a vessel entered the metanephros from its ventral aspect alongside the ingrowing ureteric bud. However, aortic branches did not engage embryonic kidneys at these time points. In newborns, β-galactosidase was localized exclusively and intensely to endothelial cells of all vessels and glomeruli. In contrast, when E12 kidneys grown in organ culture for 6 days were examined, only scattered Flk1-positive cells were seen, glomeruli were unlabeled, and vessels were absent. When organ-cultured kidneys were then grafted into wild-type anterior eye chambers, numerous Flk1-positive endothelial cells in vessels and glomeruli were found, all stemming from the graft. Image analysis showed that grafts with the most abundant glomerulo- and tubulogenesis were also those with the richest expression of Flk1. We conclude that 1) kidney microvessels precede renal artery development, 2) angioblast differentiation is arrested in organ culture but released on grafting when vasculogenesis resumes, and 3) nephrogenesis and microvessel assembly are tightly coupled in vivo.
Evidence that calgranulin is produced by kidney cells and is an inhibitor of calcium oxalate crystallization
Authors: Pillay Sokalingum N. , Asplin John R. , Coe Fredric L.
Published: December 2017

Urine produced by normal human kidneys is almost always supersaturated with respect to calcium oxalate (CaOx), the most common constituent of human kidney stones. Crystallization, with risk of renal damage and kidney stones, appears to be affected by molecules in urine that retard nucleation, growth, aggregation, and renal cell adherence of CaOx. The repertoire of such molecules is incompletely known. We have purified a 28-kDa protein from urine using salt precipitation, preparative isoelectric focusing, and sizing chromatography. Amino acid composition and NH2-terminal amino sequence analysis showed complete homology to calgranulin. Calgranulin was found to be a potent inhibitor of CaOx crystal growth (44% of control) and aggregation (50% of control) in the nanomolar range. Calgranulin cDNA was cloned from a human kidney expression library. Western analysis of human and rat kidney homogenates and mRNA temporal expression from two independent renal epithelial cell lines showed that calgranulin is produced in the kidney. Given its urinary abundance and potency, calgranulin may contribute importantly to the normal urinary inhibition of crystal growth and aggregation and therefore to the renal defense against clinical stone disease.
Identification of a novelcis-acting element for fibroblast-specific transcription of theFSP1gene
Authors: Okada Hirokazu , Danoff Theodore M. , Fischer Andreas , Lopez-Guisa Jesus M. , Strutz Frank , Neilson Eric G.
Published: December 2017

The FSP1 gene encodes a filament-binding S100 protein with paired EF hands that is specifically expressed in fibroblasts. This led us to look for cis-acting elements in the FSP1 promoter that might engage nuclear transcription factors unique to fibroblasts. The first exon of FSP1 is noncoding, therefore, a series of luciferase reporter minigenes were created containing varying lengths of 5′-flanking sequence, the first intron, and the noncoding region of the second exon. A position and promoter-dependent proximal element between −187 and −88 bp was shown to be active in fibroblasts but not in epithelium. Sequence in the first intron from +777 to +964 had an enhancing effect that was not cell type specific. Hsv TK reporter constructs driven by this promoter/intron cassette in transgenic mice were coexpressed appropriately with FSP1 in tissue fibroblasts. Gel mobility shift competitor assays identified a novel domain, FTS-1 (fibroblast transcription site-1; TTGAT from −177 to −173 bp), that specifically interacts with nuclear extracts from fibroblasts. The necessity of this binding site was confirmed by site-specific mutagenesis. Database searches also turned up putative FTS-1 sites in the early promoter regions of other fibroblast expressed proteins, including the α1and α2(I), and α1(III) collagens and the αSM-actin gene. We hypothesize that the selective engagement of FTS-1 elements may contribute to the mesenchymal phenotype of fibroblasts and perhaps other dedifferentiated cells.
PMA and staurosporine affect expression of the PCK gene in LLC-PK1-F+cells
Authors: Liu Wenlin , Feifel Elisabeth , Holcomb Thomas , Liu Xiangdong , Spitaler Nikolaus , Gstraunthaler Gerhard , Curthoys Norman P.
Published: December 2017

The addition of phorbol 12-myristate 13-acetate (PMA) to renal LLC-PK1-F+cells caused a rapid decrease in the level of phospho enolpyruvate carboxykinase (PCK) mRNA and reversed the stimulatory effects of exposure to acidic medium (pH 6.9, 10 mM [Formula: see text]) or cAMP. In contrast, prolonged treatment with PMA increased the levels of PCK mRNA. The two effects correlated with the membrane translocation and downregulation of the α-isozyme of protein kinase C and were blocked by pretreatment with specific inhibitors of protein kinase C. The rapid decrease in PCK mRNA caused by PMA occurred with a half-life ( t½= 1 h) that is significantly faster than that measured during recovery from acid medium or following inhibition of transcription ( t½= 4 h). The effect of PMA was reversed by staurosporine, which apparently acts by inhibiting a signaling pathway other than protein kinase C. Staurosporine had no effect on the half-life of the PCK mRNA, but it stimulated the activity of a chloramphenicol acetyltransferase gene that was driven by the initial 490 base pairs of the PCK promoter and transiently transfected into LLC-PK1-F+cells. This effect was additive to that of cAMP, and neither stimulation was reversed by PMA. The stimulatory effect of staurosporine was mapped to the cAMP response element (CRE-1) and P3(II) element of the PCK promoter. The data indicate that, in LLC-PK1-F+cells, activation of protein kinase C decreases the stability of the PCK mRNA, whereas transcription of the PCK gene may be suppressed by a kinase that is inhibited by staurosporine.
Inducible transcriptional activity of bcn-1 element from laminin γ1-chain gene promoter in renal and nonrenal cells
Authors: Suzuki Hideaki , Denisenko Oleg N. , Suzuki Yu , Schullery Daniel S. , Bomsztyk Karol
Published: December 2017

Laminin is a major component of the extracellular matrix whose expression is regulated by growth factors. The laminin γ1-chain promoter contains a newly identified transcriptional element denoted bcn-1 that is both active and inducible in mesangial cells. In this study, we explored activation of the bcn-1 element in other renal and nonrenal cells. Treatment of rat glomerular epithelial cells (GEC) with phorbol 12-myristate 13-acetate (PMA) increased activity of the bcn-1 transcriptional element, within the context of the native laminin γ1-chain promoter or when cloned upstream of a heterologous promoter. Treatment of GEC with PMA induced nuclear DNA-binding activity, BCN-1, which was recognized by the bcn-1 motif in a gel shift assay. These results provide evidence that the bcn-1 motif and its cognate BCN-1 factor(s) may regulate transcription of the laminin γ1-chain in GEC. The bcn-1 element and its cognate BCN-1 DNA-binding activity were also inducible in monkey kidney COS-7 and in human T cell Jurkat lines. SDS-PAGE of in situ ultraviolet cross-linked nucleoproteins from GEC, COS, and Jurkat cells revealed one major 110–115 kDa adduct in all three cell lines. These results demonstrate that the bcn-1 element is active in renal and nonrenal cells from different mammalian species where the same protein contributes to the inducible BCN-1 DNA-binding activity.
Chloride currents in primary cultures of rabbit proximal and distal convoluted tubules
Authors: Rubera Isabelle , Tauc Michel , Bidet Michel , Poujeol Chantal , Cuiller Béatrice , Watrin Annette , Touret Nicolas , Poujeol Philippe
Published: December 2017

Cl−conductances were studied in cultured rabbit proximal convoluted tubule (PCT) epithelial cells and compared with those measured in cultured distal bright convoluted tubule (DCTb) epithelial cells. Using the whole cell patch-clamp technique, three types of Cl−conductances were identified in DCTb cultured cells. These consisted of volume-sensitive, Ca2+-activated, and forskolin-activated Cl−currents. In PCT cultured cells, only volume-sensitive and Ca2+-activated Cl−currents were recorded. The characteristics of Ca2+-activated currents in PCT cells closely resembled those in DCTb cells. Volume-sensitive Cl−currents could be elicited both in PCT and in DCTb cells by hypotonic stress. The pharmacological profile of this conductance was established for both cell types. Forskolin activated a linear Cl−current in DCTb cells but not in PCT cells. This conductance was insensitive to DIDS and corresponds to cystic fibrosis transmembrane conductance regulator (CFTR)-like channels. Quantitative measurements of SPQ fluorescence showed that only the apical membrane of DCTb cells possessed a Cl−pathway that was sensitive to forskolin. RT-PCR experiments showed the presence of CFTR mRNA in both cultures, whereas immunostaining experiments revealed the expression of CFTR in DCTb cells only. The physiological role of the different types of channels is discussed.
Isolation of rat fibrillin-1 cDNA and its relevance in metanephric development
Authors: Kanwar Yashpal S. , Ota Kosuke , Yang Qiwei , Kumar Anil , Wada Jun , Kashihara Naoki , Peterson Darryl R.
Published: December 2017

The role of fibrillin-1 in metanephrogenesis was investigated. Fibrillin-1 cDNA was isolated from the rat kidney cDNA library and sequenced, and its spatiotemporal expression was studied. It had ∼88% homology with human fibrillin-1 and had Ca2+binding epidermal growth factor-like domains, transforming growth factor-β binding protein motifs, and an RGD binding site. Northern blot analysis revealed an ∼10-kb transcript, and fibrillin-1 expression was developmentally regulated. In situ hybridization and immunofluorescence studies indicated that at day 15 of gestation, fibrillin-1 is expressed in the metanephric mesenchyme. At day 18, its expression was confined to nascent blood vessels and glomeruli, and it increased in the newborn and neonatal kidneys. Immunoprecipitation revealed an ∼300-kDa band by SDS-PAGE. Treatment with fibrillin-1 antisense oligodeoxynucleotide induced marked dysmorphogenesis of the embryonic metanephroi. Concomitantly, the fibrillin-1 mRNA, antibody reactivity in the metanephroi, and fibrillin-1-specific radioincorporation were reduced. These data indicate that, like αvβ3integrin, a known morphogen and a putative receptor of fibrillin-1, the fibrillin-1 modulates events related to early organogenesis and possibly also the vascularization of the rat kidney.
Adaptation of the outer medullary collecting duct to metabolic acidosis in vitro
Authors: Tsuruoka Shuichi , Schwartz George J.
Published: December 2017

Metabolic acidosis in vivo, as well as in vitro (1 h at pH 6.8 followed by 2 h at pH 7.4) stimulates H+-ATPase-dependent H+ secretion in outer medullary collecting ducts from the inner stripe (OMCDi) (S. Tsuruoka and G. J. Schwartz. J. Clin. Invest. 99: 1420–1431, 1997). Another group has shown that the adaptation to metabolic acidosis in vivo is mediated by an apical polarization of H+ pumps without an increase in total H+pump mRNA or protein (B. Bastani, H. Purcell, P. Hemken, D. Trigg, and S. Gluck. J. Clin. Invest. 88: 126–136, 1991). To further address the mechanism of adaptation, we measured net [Formula: see text] absorption before and after applying protein/RNA synthesis and signal transduction inhibitors during the 1 h of low pH and a cytoskeletal inhibitor during the entire 3-h incubation. Net [Formula: see text]transport, measured by microcalorimetry, increased ∼33% after in vitro acidosis. This increase was prevented by application during the first hour of anisomycin (10 μM) or actinomycin D (4 μM), but not by anisomycin applied during the 2-h incubation at pH 7.4. Similar results were obtained with the cell calcium chelator, 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM, 20 μM), the calmodulin antagonist, calmidazolium (30 nM), the endoplasmic reticulum Ca-ATPase inhibitor, thapsigargin (100 nM), and the protein kinase C (PKC) inhibitor, staurosporine (100 nM), applied during the 1 h at pH 6.8, but not with BAPTA-AM or thapsigargin used during the 2-h incubation at pH 7.4. Colchicine (10 μM) applied during the entire 3-h incubation also prevented this adaptive increase in H+ secretion, whereas lumicolchicine (10 μM, the inactive congener) did not. Colchicine also reversibly prevented any adaptive increases in transepithelial positive voltage. Thus the adaptation to acidosis in vitro required RNA and protein synthesis, changes in intracellular calcium and PKC activity, and intact microtubules. Time was required for the adaptation to occur, as the increase in [Formula: see text]transport was small after <3-h incubation. Protein synthesis and changes in cell calcium were critical during the initial period of low pH but not once the acid stimulus had been removed. Exocytosis of H+ pumps appears to occur continually during the entire 3-h incubation. These data would suggest that the synthesis and regulation of proteins involved in shuttling H+ pumps in cytoplasmic vesicles to the apical membrane via exocytosis are important for the OMCDi to adapt to low pH in vitro and probably to metabolic acidosis in vivo.
Gene transfer in the kidney
Authors: Kelley Vicki Rubin , Sukhatme Vikas P.
Published: December 2017

Gene transfer approaches offer the promise of revolutionizing medicine. In this review, we focus on the current and future prospects of somatic gene transfer into the kidney. The advantages and disadvantages of current vector systems are described, and the ex vivo and in vitro approaches applicable to the kidney are reviewed. We discuss uses of gene transfer approaches to dissect the pathogenesis of kidney disease and the future directions and applications of gene transfer to combat kidney destruction.
Lysophosphatidic acid-induced calcium mobilization and proliferation in kidney proximal tubular cells
Authors: Dixon Richard J. , Young Ken , Brunskill Nigel J.
Published: December 2017

Patients with proteinuria tend to develop progressive renal disease with proximal tubular cell atrophy and interstitial scarring. It has been suggested that the nephrotoxicity of albuminuric states may be due to the protein molecule itself or by lipids, such as lysophosphatidic acid (LPA), that albumin carries. LPA was found to cause a transient increase in intracytoplasmic free Ca2+([Ca2+]i) in opossum kidney proximal tubule cells (OK) that was maximal at 100 μM LPA and was dose dependent with an EC50of 2.6 × 10−6M. This Ca2+mobilization was from both internal stores and across the plasma membrane and was pertussis toxin (PTX) insensitive. Treatment of OK cells with 100 μM LPA for 5 min was found to cause a twofold increase in [3H]thymidine incorporation and a three- to fivefold increase over control after 24 h. This was highly PTX sensitive and insensitive to pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A. These findings may be of significance in the progression of renal disease and indicate the potential importance of lipids in modulating proximal tubule cell function and growth.
Differential effects of EGF on repair of cellular functions after dichlorovinyl-l-cysteine-induced injury
Authors: Nowak Graz˙yna , Keasler Kenneth B. , McKeller Douglas E. , Schnellmann Rick G.
Published: December 2017

This study examined the repair of renal proximal tubule cellular (RPTC) functions following sublethal injury induced by the nephrotoxicant S-(1,2-dichlorovinyl)-l-cysteine (DCVC). DCVC exposure resulted in 31% cell death and loss 24 h following the treatment. Monolayer confluence recovered through migration/spreading but not proliferation after 6 days. Basal, uncoupled, and ouabain-sensitive oxygen consumption (Qo2) decreased 47, 76, and 62%, respectively, 24 h after DCVC exposure. Na+-K+-ATPase activity and Na+-dependent glucose uptake were inhibited 80 and 68%, respectively, 24 h after DCVC exposure. None of these functions recovered over time. Addition of epidermal growth factor (EGF) following DCVC exposure did not prevent decreases in basal, uncoupled, and ouabain-sensitive Qo2values and Na+-K+-ATPase activity but promoted their recovery over 4–6 days. In contrast, no recovery of Na+-dependent glucose uptake occurred in the presence of EGF. These data show that: 1) DCVC exposure decreases mitochondrial function, Na+-K+-ATPase activity, active Na+transport, and Na+-dependent glucose uptake in sublethally injured RPTC; 2) DCVC-treated RPTC do not proliferate nor regain their physiological functions in this model; and 3) EGF promotes recovery of mitochondrial function and active Na+transport but not Na+-dependent glucose uptake. These results suggest that cysteine conjugates may cause renal dysfunction, in part, by decreasing RPTC functions and inhibiting their repair.
ROS stimulate reorganization of mesangial cell-collagen gels by tyrosine kinase signaling
Authors: Zent Roy , Ailenberg Menachem , Downey Gregory P. , Silverman Melvin
Published: December 2017

Reactive oxygen species (ROS) initiate multiple pathological and physiological cellular responses, including tyrosine phosphorylation of proteins. In this study, we investigated the effects of ROS on cell-extracellular matrix interactions utilizing the floating three-dimensional collagen gel assay. Exposure of mesangial cells grown in three-dimensional culture to H2O2, 3-amino-1,2,4-triazole (a catalase inhibitor), or puromycin is associated with gel reorganization accompanied by tyrosine phosphorylation of multiple proteins, including focal adhesion kinase (FAK). Neutrophils cocultured with mesangial cells in three-dimensional culture also induce mesangial cell-collagen gel reorganization and initiate tyrosine phosphorylation of a similar set of proteins. Collectively, these results show that ROS of either endogenous or exogenous origin can modulate mesangial cell-extracellular matrix interactions through initiation of a phosphotyrosine kinase signaling cascade. Consequently, ROS may play a role as signaling molecules that regulate mesangial cell-extracellular matrix interactions in both physiological and pathological conditions.
Long-term regulation of aquaporins in the kidney
Authors: Marples David , Frøkiaer Jørgen , Nielsen Søren
Published: December 2017

The discovery of the aquaporin family of water channels has greatly improved our understanding of how water crosses epithelial cells, particularly in the kidney. The study of the mechanisms involved in the regulation of collecting duct water permeability, in particular, has advanced very rapidly since the identification and characterization of aquaporin-2 (AQP2) in 1993. One of the more surprising findings has been the dramatic long-term changes that are seen in the abundance of this protein, as well as the recognition that these changes represent a way of modulating the acute antidiuretic effects of vasopressin. Furthermore, such changes seem to be of etiological and pathological significance in a number of clinical disorders of water balance. This review focuses on the various conditions in which AQP2 expression is altered (either increased or decreased) and on what this can tell us about the signals and mechanisms controlling these changes. Ultimately, this may be of great value in the clinical management of water balance disorders. Evidence is also now beginning to emerge that there are similar changes in the expression of other renal aquaporins, which had previously been thought to provide an essentially constitutive water permeability pathway, suggesting that they too should be considered as regulatory factors in the control of body water balance.