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R F Reilly

Publications and source records attributed to R F Reilly.

33 records · Page 2Linked to original sources
PubMed1993

Immunolocalization of the Na+/Ca2+ exchanger in rabbit kidney.

We recently isolated a cDNA encoding a Na+/Ca2+ exchanger from rabbit kidney that was highly similar to the canine cardiac sarcolemmal Na+/Ca2+ exchanger. In the present study, we used two different antibodies to the exchanger to identify the protein and establish its cellular and subcellular localization in the kidney. The first antibody was prepared against a fusion protein consisting of 190 amino acids of the large, presumably intracellular loop of the rabbit renal exchanger fused to the maltose-binding protein. The second was a monoclonal antibody generated against the isolated purified canine cardiac sarcolemmal exchanger. To identify the Na+/Ca2+ exchanger protein, we performed immunoblot analysis against a membrane vesicle preparation from rabbit kidney cortex. Both antibodies immunoblotted proteins of 120 and 70 kDa that are known to be associated with the exchanger. Indirect immunofluorescence revealed that both antisera labeled the basolateral surface of the majority of cells in the connecting tubule (CNT). Since the phase-dense (intercalated) cells in the CNT were not stained, this suggested that the labeled cells were CNT cells. No labeling was detected in other nephron segments with the exception of occasional faint staining of the majority cell population of the cortical collecting duct. The fact that we did not detect labeling in other nephron segments is consistent with either 1) the absence of expression of the Na+/Ca2+ exchanger in these segments, 2) the expression of the exchanger in levels below the threshold of detection of the two antibodies used in this study, or 3) the exchanger in these segments is represented by a different isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Isolation of putative voltage-gated epithelial K-channel isoforms from rabbit kidney and LLC-PK1 cells.

Epithelial voltage-gated potassium (K) channels have been well studied using electrophysiological methods, but little is known about their structures. We tested the hypothesis that some of these channels belong to the Shaker gene family, which encodes voltage-gated K channels in excitable tissues. From published sequences of Shaker proteins in Drosophila, rat, and mouse brain, we chose regions that were conserved between species. Based on these protein sequences, degenerate oligonucleotides flanking the putative voltage sensor (S4) were synthesized and used as primers for the polymerase chain reaction. Five Shaker-like cDNAs were amplified from rabbit kidney cortex and three from LLC-PK1, an epithelial cell line derived from pig kidney. Each partial-length rabbit kidney cDNA is approximately 850 base pairs (bp) long. The deduced amino acid sequences contain five putative transmembrane segments and are 79-97% identical to two Shaker isoforms expressed in rat brain (RBK1 and RBK2). Sequence similarity is greatest in the putative transmembrane segments S1-S5. Importantly, the S4 segment, the putative voltage gate is highly conserved in all 5 cDNAs. Southern analysis of rabbit genomic DNA suggests that each isoform is encoded by a different gene. The partial length LLC-PK1 cDNAs are 450-bp long, and the deduced amino acid sequences are 77-99% identical to the rabbit cDNAs. This is, to our knowledge, the first demonstration that Shaker-like genes are expressed in renal epithelial cells. These genes most likely encode voltage-gated K channels involved in renal epithelial K transport.

Animals↗
PubMed1992

cDNA cloning of a renal Na(+)-Ca2+ exchanger.

In the present study, the polymerase chain reaction (PCR) and library screening were used to clone a cDNA for a rabbit kidney Na(+)-Ca2+ exchanger on the basis of homology with the canine cardiac sarcolemmal sequence (D. A. Nicoll, S. Longoni, and K. D. Philipson. Science Wash. DC 250:562-565, 1990). There is a high degree of similarity between the two sequences, with nucleotide identities of 95, 89, and 90% in the hydrophobic membrane-associated domain, cytoplasmic domain, and 3'-untranslated region, respectively. The rabbit kidney cDNA encodes a predicted protein of 941 amino acids, 29 amino acids shorter than the canine sequence, with a relative molecular weight of 105,121. The deduced amino acid sequence is 96% identical in the membrane-associated domain and 94% identical in the cytoplasmic domain. Northern blot analysis reveals that the cDNA is expressed in the renal cortex. No expression is detected in the medulla. This result is in agreement with micropuncture studies that show Na(+)-Ca2+ exchanger activity in cortical but not medullary nephron segments.

Amino Acid Sequence↗
PubMed1992

Effects of chronic metabolic acidosis on Na(+)-H+ exchangers in LLC-PK1 renal epithelial cells.

Porcine renal epithelial cells (LLC-PK1/clone 4) have Na(+)-H+ exchangers with different kinetic properties in their apical and basolateral membranes. cDNAs encoding the basolateral Na(+)-H+ exchanger were recently cloned. To determine whether expression of the basolateral Na(+)-H+ exchanger was affected by chronic metabolic acidosis, LLC-PK1/clone 4 cells were grown on permeant supports and incubated in control medium (pH 7.4) or acid medium (pH 6.9). After 48 h, Na(+)-H+ exchanger transport activity was measured as N-ethyl-N-isopropylamiloride (EIPA)-sensitive 22Na+ influx. Acidification caused an 84% stimulation of the transport activity of the basolateral Na(+)-H+ exchanger. The apical Na(+)-H+ exchanger was stimulated 72%, and there was no change in the EIPA-insensitive 22Na+ flux across either membrane. Stimulation of Na(+)-H+ exchange was not due to differences in intracellular pH at the time transport was assayed. To determine whether there were corresponding changes in transcript levels, poly(A)+ RNA was isolated from LLC-PK1 cells and hybridized with a cDNA encoding the basolateral Na(+)-H+ exchanger. Levels of transcripts encoding the basolateral Na(+)-H+ exchanger were increased 70% after 48 h of acidification, and there were no changes in transcripts encoding cytoskeletal gamma-actin or glyceraldehyde-3-phosphate dehydrogenase. We conclude that conditions simulating chronic metabolic acidosis coordinately increase the transport activity and transcript levels of the basolateral Na(+)-H+ exchanger in porcine renal epithelial cells.

Acidosis↗
PubMed1992

Immunocytochemical characterization of Na(+)-H+ exchanger isoform NHE-1 in rabbit kidney.

We have recently isolated cDNAs encoding a Na(+)-H+ exchanger isoform, referred to as NHE-1, from rabbit kidney and LLC-PK1 cells. To identify the NHE-1 protein and to establish its cellular and subcellular localization in the rabbit kidney, we prepared antibodies to a NHE-1 fusion protein. cDNA encoding the COOH-terminal 41 amino acids of NHE-1 was subcloned into a maltose-binding protein vector and the purified fusion protein (FP347A) used to immunize guinea pigs. To identify the NHE-1 protein, we performed Western blot analysis against membrane fractions prepared from rabbit renal cortex. Anti-FP347A antibody specifically reacted with a polypeptide with an apparent molecular mass of 100-110 kDa that was enriched in basolateral membrane fractions. When indirect immunofluorescence was performed on semithin (0.5 micron) cryosections of paraformaldehyde-lysine-periodate-fixed rabbit kidney, anti-FP347A specifically stained the basolateral plasma membrane of cells of the proximal tubule, thick ascending limb, and distal convoluted tubule. Anti-FP347A similarly stained connecting tubule cells and principal cells. No staining was detected on the apical membrane of any cells of the rabbit nephron. We conclude that NHE-1 is a 100- to 110-kDa protein expressed on the basolateral membrane of multiple nephron segments.

Animals↗
PubMed1991

Cloning, sequence, and tissue distribution of a rabbit renal Na+/H+ exchanger transcript.

Rabbit kidney expresses a transcript that is similar to the human growth-factor-activatable Na+/H+ exchanger. PCR and library screening were used to clone overlapping 2.5 kb, 1.4 kb, and 1.8 kb cDNAs that together contain the entire coding region (2448 bp) and 5' untranslated region (726 bp) and part of the 3' untranslated region (128 bp) of a rabbit renal Na+/H+ exchanger transcript. The nucleotide and inferred amino acid sequences are highly conserved between rabbit and human (88% nucleotide identity, 95% amino acid identity). In rabbit, the transcript is expressed in both epithelial and non-epithelial tissues, with highest expression in stomach, brain, kidney, lung and ileum, and minimal expression in liver and skeletal muscle.

Amino Acid Sequence↗
PubMed1991

Increased Na(+)-H+ antiporter activity in apical membrane vesicles from mutant LLC-PK1 cells.

In whole cell experiments, the PKE20 mutant of the renal epithelial cell line LLC-PK1 displays a severalfold elevation of Na(+)-H+ antiporter activity at the apical surface (J.G. Haggerty, N. Agarwal, R.F. Reilly, E. A. Adelberg, and C.W. Slayman. Proc. Natl. Acad. Sci. USA 85: 6797-6801, 1988). The present study was undertaken to explore the properties of the mutant at the membrane level. Apical membrane vesicles were prepared by the magnesium-aggregation technique, with a similar enrichment (ca. 10-fold) of the marker enzyme gamma-glutamyltranspeptidase in vesicles from parent and mutant cell lines. In both cases, 22Na influx was stimulated by an inside-acid pH gradient, inhibited by ethylisopropylamiloride (EIPA), and unaffected by valinomycin, indicating that it was mediated by Na(+)-H+ antiport. Quantitatively, PKE20 vesicles showed a 4.2-fold increase in the maximal velocity of Na(+)-H+ antiporter activity compared with the parent, with only minor increases in the activity of two other Na(+)-dependent transporters (14-56% for alpha-methylglucoside and L-glutamate). Dose-response curves for EIPA indicated that the increased Na(+)-H+ antiport activity in PKE20 vesicles was due to an increased activity of the relatively amiloride-resistant form of the Na(+)-H+ antiporter with little or no change in the amiloride-sensitive form. No differences in polypeptide composition of the two vesicle preparations could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Taken together, the results indicate that the mutation in PKE20 is expressed at the membrane level and is specific for the relatively amiloride-resistant Na(+)-H+ antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1991

cDNA cloning and immunolocalization of a Na(+)-H+ exchanger in LLC-PK1 renal epithelial cells.

LLC-PK1 cells, an established line from pig kidney, express basolateral and apical Na(+)-H+ exchangers that can be distinguished by their different sensitivities to the amiloride analogue, N-ethyl-N-isopropylamiloride. In the present study, the polymerase chain reaction (PCR) and library screening were used to clone a cDNA for one of the exchangers, based on homology with the recently isolated cDNA for a human growth factor-activatable Na(+)-H+ exchanger. There proved to be significant homology between the LLC-PK1 and human sequences, with nucleotide identities of 75, 93, and 85% in the 5'-untranslated, coding, and 3'-untranslated regions, respectively. The LLC-PK1 cDNA encodes a predicted protein of 818 amino acids with a relative molecular mass of 90,999, consisting of an amino-terminal hydrophobic region and a carboxy-terminal hydrophilic region; its deduced amino acid sequence shows 95% identity with that of the human protein. To investigate the localization of the encoded protein, antisera were generated against a synthetic oligopeptide from the hydrophobic region and a fusion protein from the carboxy-terminal hydrophilic domain. Indirect immunofluorescence and confocal microscopy revealed that the antisera labeled the basolateral but not the apical membrane of confluent LLC-PK1 cells. Labeling by the antipeptide antibody was specifically blocked by preincubation with the synthetic peptide and coincided exactly with the pattern produced by a monoclonal antibody against Na(+)-K(+)-ATPase. Thus, the LLC-PK1 cDNA encodes the basolateral Na(+)-H+ exchanger, which must differ structurally from the apical form, at least in the region of the oligopeptide and the fusion protein.

Amino Acid Sequence↗
PubMed1991

Molecular biology of renal Na(+)-H+ exchangers.

Na(+)-H+ exchangers are plasma membrane proteins that are responsible for Cl- and HCO3- reabsorption and regulation of intracellular pH in several nephron segments. Recently, a cDNA encoding a human Na(+)-H+ exchanger of unknown tissue origin was cloned, and Northern blot analysis revealed that structurally similar transcripts were expressed in rabbit kidney and porcine renal epithelial cells (LLC-PK1). To clone these renal transcripts we employed cDNA library screening and the polymerase chain reaction (PCR). We obtained 2.5 kb and 1.4 kb cDNAs corresponding to the 5' untranslated region and the membrane-associated domain of a rabbit renal Na(+)-H+ exchanger. From LLC-PK1 cells we obtained 1.5 kb and 1.3 kb cDNAs encoding the membrane-associated and cytoplasmic domains. The sequences of cDNAs from these three species were very similar and suggested a high degree of evolutionary conservation. Immunolocalization of synthetic oligopeptides derived from the deduced amino acid sequences indicated that the cloned cDNAs encoded the amiloride-sensitive form of the Na(+)-H+ exchanger present in basolateral membranes of epithelia. cDNAs were also used to study regulation of Na(+)-H+ exchanger gene expression in the kidney, and we found that metabolic acidosis stimulated both the transport rate and steady-state transcript levels of the basolateral Na(+)-H+ exchanger in LLC-PK1 cells.

Acidosis↗
PubMed1990

The valuation of a medical practice.

The valuation of a medical practice is important to the practice owners, employees, bankers, and others for many reasons. These reasons include taxation, transaction pricing, financing, litigation, and management planning. This article presents the three most common approaches to practice valuation: discounted net cash flow, market data comparable, and asset accumulation. Comprehensive illustrative examples of all three approaches are presented.

Accounting↗
PubMed1988

Pharmacologically different Na/H antiporters on the apical and basolateral surfaces of cultured porcine kidney cells (LLC-PK1).

Proximal tubule cells of the kidney contain, on their apical surface, an amiloride-sensitive Na/H antiporter that functions in Na reabsorption and proton secretion. We have investigated the localization of the antiporter in a cloned cell line of porcine renal origin, LLC-PK1/Cl4, which is often considered to be a useful model of the proximal tubule. Transport measurements were performed with differentiated monolayers grown on Nuclepore filters, permitting independent access to the apical and basolateral cell surfaces. In control experiments with LLC-PK1/Cl4 monolayers, three marker transport systems showed the expected polarity: 87% of ouabain-sensitive Rb uptake was at the basolateral surface, and 99% of Na-dependent alpha-methylglucoside transport and 93% of Na-dependent D-aspartate (L-glutamate) transport were at the apical surface. By contrast, the monolayers displayed significant Na/H antiporter activity (assayed as ethylisopropylamiloride-sensitive 22Na uptake) at both cell surfaces, with an apical uptake rate amounting to 44% and a basolateral rate amounting to 56% of the total. Significantly, the apical and basolateral antiporters could readily be distinguished from one another on the basis of ethylispropylamiloride sensitivity. The apical system had an IC50 of 13 microM, close to that reported for kidney brush border vesicle preparations, whereas the basolateral system had an IC50 of 44 nM, similar to values seen in undifferentiated LLC-PK1 cells and other cultured cell lines. The PKE20 mutant, previously selected from LLC-PK1/Cl4 on the basis of resistance to ethylisopropylamiloride, was found to overexpress the more resistant antiporter both during rapid growth and on its apical cell surface at confluence; normal amounts of the more sensitive antiporter were seen on the basolateral surface of confluent PKE20 cells. Taken together, these results suggest that there are two distinct forms of the Na/H antiporter, which are under separate genetic control.

Animals↗
PubMed1987

Aspergillus myositis in a patient with a myelodysplastic syndrome.

We present the case of an elderly man who, while being treated with corticosteroids for a myelodysplastic syndrome, developed myositis of the calf due to Aspergillus fumigatus. Despite therapy with amphotericin B the myositis failed to resolve and he died. At autopsy, a localized necrotizing myositis of the right calf was found with no evidence of disseminated Aspergillus infection. Myositis in the setting of disseminated candidiasis or cryptococcosis has been previously reported. This case is unique in that it is the first reported case of localized fungal myositis and of myositis caused by Aspergillus.

Adrenal Cortex Hormones↗
PubMedDate not supplied

A case review: anticoagulation in hemodialysis patients with heparin-induced thrombocytopenia.

We describe a patient with acute renal failure who subsequently developed heparin-induced thrombocytopenia (HIT) while on hemodialysis. Heparin was immediately discontinued and alternative modes of anticoagulation were considered as further hemodialysis was indicated. With several options available, a review of the current literature was performed to aid in the selection of the most appropriate method. We conclude that there is currently no ideal hemodialysis anticoagulation agent for a patient with HIT. Among the currently available alternatives, no anticoagulation or regional citrate infusion during hemodialysis appears to be the most reliable and safest option for these patients. Based upon its safety, reversibility, low cost and availability, a trial of warfarin also may be attempted for hemodialysis anticoagulation. Both danapranoid and lepirudin have been used effectively in hemodialysis patients with HIT, but have the disadvantage of prolonged half-lives in patients with renal failure and relatively high cost.

Acute Kidney Injury↗
PubMedDate not supplied

Glomerular lesions in HIV-infected patients: a Yale University Department of Medicine Residency Peer-Teaching Conference.

HIV-associated nephropathy (HIVAN) is a clinicopathologic entity characterized by heavy proteinuria, absence of edema and an irreversible decline in renal function. Findings on renal biopsy include: collapsed glomerular capillaries; visceral glomerular epitheliosis; microcystic tubules; mesangial prominence; and endothelial tubuloreticular inclusions. Early in the AIDS epidemic, HIVAN was the predominant glomerular lesion observed in HIV-infected patients. It is being increasingly recognized, especially in Caucasian populations, that a variety of immune complex-mediated lesions such as membranoproliferative glomerulonephritis, proliferative glomerulonephritis and IgA nephropathy are associated with HIV infection. In this review we present two cases: one patient whose first presentation of AIDS was end-stage renal disease, who on biopsy was found to have HIVAN, and the second, who was infected with HIV, and on biopsy was found to have hepatitis C-related hepatitis C related membranoproliferative glomerulonephritis. We also review the current literature on HIVAN and HIV-associated immune complex diseases (HIVICDs). Each case illustrates an important clinical point. The first that renal disease can be the first manifestation of HIV infection and the second that HIV-infected patients may develop immune complex related renal diseases, some of which may be potentially treatable.

AIDS-Associated Nephropathy↗