Biomedical subjects

Y H Lien

Publications and source records attributed to Y H Lien.

At least 19 recordsLinked to original sources

PubMed1998

Correction of renal tubular acidosis in carbonic anhydrase II-deficient mice with gene therapy.

Carbonic anhydrase II (CAII) deficiency in humans is associated with a syndrome of renal tubular acidosis, osteopetrosis, and cerebral calcification. A strain of mice of CAII deficiency due to a point mutation also manifests renal tubular acidosis. We report here that retrograde injection of cationic liposome complexed with a CAII chimeric gene, using a cytomegalovirus (CMV) promoter/enhancer as an expression cassette to drive human CAII cDNA, into the renal pelvis of CAII-deficient mice results in expression of CAII in the kidney. The levels of both the CAII gene and its corresponding mRNA were highest by day 3 after treatment, diminishing thereafter, but remaining detectable by 1 mo. After gene therapy, CAII-deficient mice restored the ability to acidify urine after oral administration of ammonium chloride. The ability to acidify urine was maintained at 3 wk after gene therapy, and was eventually lost by 6 wk. Immunohistochemistry studies using anti-CAII antibodies showed that CAII was expressed in tubular cells of the outer medulla and corticomedullary junction. The gene therapy was not associated with nephrotoxicity as assessed by blood urea nitrogen levels and renal histology. To our knowledge, this is the first successful gene therapy of a genetic renal disease. Our results demonstrate the potential of gene therapy as a novel treatment for hereditary renal tubular defects.

Acidosis, Renal Tubular↗

PubMed1998

Promoter activity of carbonic anhydrase II regulatory regions in cultured renal proximal tubular cells.

Carbonic anhydrase II (CAII) plays an important role in the acid-base homeostasis of the body and its deficiency results in renal tubular acidosis. In order to identify the regulatory regions in the CAII gene for the future development of kidney-targeted gene therapy, we investigated the 5' region of the gene for its promoter activity. Deletion constructs with various lengths of the 5' flanking region of the human CAII promoter were ligated to the CAT reporter gene and lipofected in primary cultures of mouse proximal renal tubular cells and in cells of the established porcine proximal tubular cell line, LLC-PK1. The CAT activity was measured 48 hours after gene transfection. The -12000/CAT and -1300/CAT constructs expressed the highest CAT activity in both types of renal tubular cells (143- and 180-fold increase, respectively, in mouse proximal tubular cells; 50- and 70-fold increase, respectively, in LLC-PK1 cells) but not the -420/CAT, -270/CAT, or -180/CAT constructs (9, 12, and 9% of that of -1300/CAT construct, respectively, in mouse proximal tubular cells and, 23, 9, and 8%, respectively, in LLC-PK1 cells, all p <0.01 vs. -1300/CAT construct). No cytotoxicity was detected in the transfected cells. A computer search identified multiple putative transcription factor binding elements including Ap1 and Ap2 binding elements, which are present in the -1300/CAT construct but not in the shorter constructs. In conclusion, we demonstrate that the human CAII 5' sequence of proximal 1.3 kb contains strong promoter sequence(s) for renal tubular cells.

Animals↗

PubMed1998

Respiratory acidosis in carbonic anhydrase II-deficient mice.

To investigate the role of carbonic anhydrase (CA) II on pulmonary CO2 exchange, we analyzed arterial blood gases from CA II-deficient and normal control mice. CA II-deficient mice had a low arterial blood pH (7.18 +/- 0.06) and HCO3- concentration ([HCO3-]; 17.5 +/- 1.9 meq/l) and a high Pco2 (47.4 +/- 5.3 mmHg), consistent with mixed respiratory and metabolic acidosis. To eliminate the influence of metabolic acidosis on arterial blood gases, NaHCO3 (4 mmol/kg body weight) was given intraperitoneally, and arterial blood gases were analyzed 4 h later. Normal mice had a small increase in pH and were able to maintain Pco2 and [HCO3-]. The metabolic acidosis in CA II-deficient mice was corrected ([HCO3-], 22.9 +/- 2.4 meq/l), and respiratory acidosis became more profound (Pco2, 50.4 +/- 2.4 mmHg). These results indicate that CA II-deficient mice have a partial respiratory compensation for metabolic acidosis. We conclude that CA II-deficient mice have a mixed respiratory and metabolic acidosis. It is most likely that CO2 retention in these animals is due to CA II deficiency in both red blood cells and type II pneumocytes.

Acidosis, Respiratory↗

PubMed1997

Kidney-targeted liposome-mediated gene transfer in mice.

To develop gene therapy targeted to the kidney, we compared three different routes of liposome-mediated gene delivery to the kidney in mice, ie intra-renal-pelvic, intra-renal-arterial, and intra-renal-parenchymal injections. A plasmid construct, pCMV beta gal, containing a cytomegalovirus (CMV) immediate-early gene promoter and a beta-galactosidase reporter gene was mixed with a 1:1 liposome mixture of N[1-(2,3-dioleoyloxy)propyl]-N,N,trimethylammonium chloride (DOTMA)/dioleoyl phosphatidyl ethanolamine (DOPE). The pCMV beta gal-liposome complex was injected into the left kidney via three different routes. The efficacy of gene transfer was assessed using 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) staining on frozen kidney sections 3 to 42 days after injections. Cells with beta-galactosidase activity were detected in the cortex and outer medulla in both intra-renal-pelvic and intra-renal-arterial groups, but not in the intra-renal-parenchymal group or in the contralateral noninjected kidney. Evidence of gene transfer was observed only in tubular epithelial cells, but not in glomerular, vascular, or interstitial compartments. The levels of beta-galactosidase expression started to decrease 3 weeks after injection. The gene transfer in the kidney was not associated with nephrotoxicity as assessed by blood urea nitrogen levels and renal histology. We conclude that both intra-renal-pelvic and intra-renal-arterial injections provide a transient gene transfer to the renal tubular cells and are suitable routes for kidney-targeted gene therapy.

Animals↗

PubMed1997

Kinetics and osmoregulation of Na(+)-and Cl(-)-dependent betaine transporter in rat renal medulla.

Betaine is one of the major organic osmolytes that accumulate in the renal medulla in response to high extracellular tonicity. Recent studies in MDCK cells have shown that betaine is taken up by an Na(+)- and Cl(-)-dependent transporter located on the basolateral membrane. We demonstrate here the presence of Na(+)-Cl(-)-dependent betaine transporter(s) in tubule suspensions prepared from the rat outer and inner medulla. The betaine transport activity was two to three times higher in the inner medulla compared with the outer medulla. The removal of Na+ and Cl- reduced betaine uptake in the outer medullary tubules by 86% and 82%, respectively. The betaine uptake was decreased by 39% in hypotonic buffer (189 mosmol/ kgH2O) and increased by 82% in hypertonic buffer (545 mosmol/kgH2O), compared with isotonic buffer (308 mosmol/ kgH2O). Kinetic studies of Na(+)-dependent betaine uptake in the outer medullary tubules revealed both a low- and a high-affinity component as follows: low-affinity and high volume component with Michaelis constant (K(m)1) of 8.6 mM and maximal uptake rate (Vmax1) of 112 pmol.microgram protein-1.h-1; and a low-volume and high-affinity component with K(m)2 of 0.141 mM and Vmax2 of 10 pmol. microgram protein-1.h-1. To investigate whether the Na(+)-Cl(-)-dependent betaine transporter is regulated by tonicity in vivo, we quantitated its mRNA in rat renal cortex and outer and inner medulla using both canine and rat Na(+)-Cl(-)-dependent betaine transporter cDNA probes. A single band of 3.0 kb was seen in the Northern blots prepared from both outer and inner medulla, but not in the cortex. Water deprivation for 3 days increased the abundance of this mRNA in the outer and inner medulla by 140% and 170%, respectively, but did not affect its expression in the cortex. In conclusion, Na(+)-Cl(-)-dependent betaine transporter(s) is present in rat outer and inner medullary tubules, and betaine transporter mRNA abundance is regulated by the hydration state in vivo.

Animals↗

PubMed1997

Distribution of de novo synthesized betaine in rat kidney: role of renal synthesis on medullary betaine accumulation.

The trimethylamine glycine-betaine is accumulated to high concentrations in medullary cells of mammalian kidneys, whereas betaine synthesis from choline is predominant in the renal cortex. We investigated the contribution of renal betaine synthesis to medullary betaine accumulation. De novo synthesis of betaine in situ was accomplished by injecting [14C]choline into the renal artery of male Sprague-Dawley rats. [14C]betaine was measured in the renal cortex and medulla, as well as in serum and urine samples. Betaine concentration in the cortex decreased from 3.5 +/- 1.3 at 5 min to 0.4 +/- 0.2 nmol/mg protein at 60 min, but it increased from 1.4 +/- 0.1 to 2.5 +/- 0.6 nmol/mg protein in the medulla. Serum and total urine [14C]betaine increased from 2.7 +/- 1.3 and 0.9 +/- 0.1 nmol/ml at 5 min to 5.3 +/- 0.3 and 2.1 +/- 0.4 nmol/ml at 60 min, respectively. Concentrations of newly synthesized betaine were not decreased by the ligation of the hepatic artery and portal vein, suggesting that most [14C]betaine was synthesized in the kidney. Coinjection with 5 mM dimethylamino-ethanol, a choline oxidase inhibitor, and 100 mM cold betaine reduced medullary betaine accumulation by 80 and 76%, respectively. Water deprivation for 60 h increased both cortical and medullary [14C]betaine, whereas furosemide diuresis decreased the medullary [14C]betaine concentration. We concluded that betaine synthesized in the kidney can be accumulated in the medulla and that the medullary concentrations of newly synthesized betaine are closely related to the hydration state of the animal.

Animals↗

PubMed1997

Remission of posttransplant lymphoproliferative disorder after interferon alfa therapy.

Posttransplant lymphoproliferative disorder (PTLD) is one of the major complications of immunosuppressive therapy. PTLD is strongly associated with the Epstein-Barr virus (EBV). It is believed that EBV-infected B cells proliferate in an unchecked manner due to suppression of cytotoxic T cells and elevation of B cell-promoting cytokines. There is no consensus on the treatment of PTLD other than reduction of immunosuppressive therapy. We report a case of PTLD with monoclonal B cells confined to the lymph nodes. The patient did not initially respond to reduction of immunosuppression, oral acyclovir, and intravenous immunoglobulin injection. She subsequently responded when subcutaneous injections of interferon alfa (5 million U) were given three times a week. The patient received a 3-mo course of interferon and remained in remission 12 mo after treatment. Her graft function was well maintained, and cyclosporin A was restarted 2 mo after achieving remission. The clinical manifestations, risk factors, pathogenesis, and treatment of PTLD, as well as 12 previously reported cases of PTLD treated with interferon, were reviewed. On the basis of the results presented here, it appears that interferon alfa may be useful in treating PTLD and that there is a need for further clinical trials.

Aged↗

PubMed1997

Gene therapy for renal diseases.

Gene therapy is a promising therapeutic approach for a variety of renal diseases including both inherited and acquired diseases. In vivo gene transfer in the kidney using viral or non-viral vectors have been reported. These approaches have been tested in a few animal models of renal diseases, including experimental glomerulonephritis, ischemic renal failure, and carbonic anhydrase II deficiency. Selection of vectors, routes, and therapeutic genes is critical to the success of gene therapy targeted to the specific compartment of the kidney. Limitations of gene therapy for renal diseases exist and consist of: duration of transgene expression is short, transfection efficiency is not adequate, immune reactions are induced by adenoviral vector, and insertional mutagenesis may be caused by retroviral and adeno-associated viral vectors. Further studies are needed for improvement of gene delivery, minimization of side effects and development of cell-specific and long-term regulated gene expression.

Carbonic Anhydrases↗

PubMed1996

Hemodialysis increases apparent diffusion coefficient of brain water in nephrectomized rats measured by isotropic diffusion-weighted magnetic resonance imaging.

The nature of brain edema in dialysis disequilibrium syndrome (DDS) was investigated by diffusion-weighted magnetic resonance imaging (DWI). DWI was performed on normal or bilaterally nephrectomized rats before, and immediately after, hemodialysis. Hemodialysis was performed with a custom-made dialyzer (surface area 150 cm2) against a bicarbonate-buffered bath for 90 min with or without 70 mM urea. Hemodialysis with non-urea bath decreased plasma urea by 21 mM, and plasma osmolality by 22 mosmol/kg H2O, and increased brain water content by 8.0% (all < 0.05), while hemodialysis with urea bath did not affect plasma urea, osmolality, or brain water content. Three sets of axial DWI images of the brain were obtained at different gradient weighing factors with an in-plane resolution of 0.39 mm2. The apparent diffusion coefficient (Dapp) of the brain water was not affected by bilateral nephrectomy, or by hemodialysis in normal rats. In nephrectomized rats, brain Dapp was significantly increased after dialysis with non-urea bath (1.15 +/- 0.08 vs 0.89 +/- 0.07 x 10(-9)m2/sec, P < 0.01). No significant changes of brain water Dapp could be observed after dialysis with urea bath. The increased Dapp associated with DDS indicates that brain extracellular water increases and/or intracellular water decreases after hemodialysis. Our results strongly suggest that the brain edema induced by hemodialysis in uremic rats is due to interstitial edema rather than cytotoxic edema. Furthermore, our results support a primary role for the "reverse urea effect" in the pathogenesis of brain edema in DDS.DWI may be a useful diagnostic tool for DDS in patients with end-stage renal disease.

Animals↗

PubMed1996

Acute massive gentamicin intoxication in a patient with end-stage renal disease.

A 65-year-old man with end-stage renal disease on continuous ambulatory peritoneal dialysis accidentally received an acute massive overdose of gentamicin as a treatment of peritonitis. The patient developed acute vestibular dysfunction and hearing loss following the overdose. His serum gentamicin had reached the extremely toxic level of 220 microg/mL. To remove the gentamicin, the patient received hemodialysis and hemoperfusion immediately. This was followed by two more courses of hemodialysis during the following 2 days. The gentamicin level was brought down to 10 microg/mL after the third hemodialysis. Moderate and persistent high-frequency hearing loss was documented with serial audiograms. The patient made a gradual but incomplete recovery from the vestibular dysfunction. The complications of gentamicin toxicity and its management are discussed with respect to our patient.

Aged↗

PubMed1996

Low dose megestrol increases serum albumin in malnourished dialysis patients.

To evaluate the efficacy of low dose megestrol on malnourished dialysis patients we treated 16 dialysis patients with persistent hypoalbuminemia ( < 3.5 gm/dl for 2 consecutive months) and adequate dialysis at a dose of 20 mg orally twice daily. Twelve patients on peritoneal dialysis and 4 on hemodialysis were followed for 4.3 +/- 0.6 m (2-11 m). Within one month serum albumin rose from 2.7 +/- 0.1 to 3.0 +/- 0.2 gm/dl (p < 0.05) and remained elevated at the end of follow-up (3.1 +/- 0.2, p < 0.05 vs. pre-treatment levels). In the 12 responders (increase of albumin > 0.3 gm/dl), all of whom reported improved appetite, the maximal increase of serum albumin in 2 months was 0.8 +/- 0.1 gm/dl (range: 0.3-1.2). Four patients did not respond (change of albumin: -0.05 +/- 0.18, range: -0.6-0.2) because of encephalopathy, amyloidosis, depression or noncompliance. One patient stopped megestrol because of vaginal bleeding from uterine leiomyoma. Three patients died from causes unrelated to the megestrol. Our preliminary study suggests that low dose megestrol (40 mg per day) increases serum albumin levels in 75% of dialysis patients with malnutrition. It is well tolerated but may cause vaginal bleeding from uterine tumors.

Administration, Oral↗

PubMed1995

Modulation of intracellular Ca2+ by glucose in MDCK cells: role of endoplasmic reticulum Ca(2+)-ATPase.

Intracellular free calcium ([Ca2+]i) has multiple functional roles in renal epithelia, including mediating ligand- and volume-activated K+ and Cl- channels, modulating the permeability of apical membrane to Na+, and regulating tubuloglomerular feedback. We investigated glucose effects on intracellular pH (pHi) and [Ca2+]i in Madin-Darby canine kidney (MDCK) cells using fluorescent probes, SNARF-1 and fura 2, respectively. The addition of glucose decreased both pHi and [Ca2+]i in a dose-dependent fashion. Thapsigargin (TG) and cyclopiazonic acid (CPA), well-known endoplasmic reticulum (ER) Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase) inhibitors, abolished the glucose-induced [Ca2+]i decrease. Without glucose, 1 microM TG induced a sustained elevation in [Ca2+]i, which increased further with glucose addition, whereas 15 microM CPA induced a transient increase in [Ca2+]i that was not affected by further addition of glucose. The sustained elevation in [Ca2+]i induced by TG was dependent on extracellular Ca2+. TG-induced [Ca2+]i increase was modulated by glucose, i.e., at higher glucose concentrations, TG induced a larger and more rapid rise in [Ca2+]i. We conclude that glucose has dual effects on [Ca2+]i regulation. Glucose alone reduces [Ca2+]i by activating ER-type Ca(2+)-ATPase, since this phenomenon is TG and CPA sensitive. In the presence of TG, glucose increases [Ca2+]i probably by increasing Ca2+ entry. Our data suggest a model in which TG activates capacitative Ca2+ entry by depletion of the ER Ca2+ pool. Glucose increases TG-induced [Ca2+]i elevation by further enhancing capacitative Ca2+ entry.

Animals↗

PubMed1995

Role of organic osmolytes in myelinolysis. A topographic study in rats after rapid correction of hyponatremia.

Organic osmolytes have been implicated in the pathogenesis of myelinolysis because some of them are accumulated slowly during correction of chronic hyponatremia. I investigated whether there was a topographic correlation between demyelinative lesions and the regional changes of organic osmolytes after rapid correction of chronic hyponatremia. In normal female Sprague-Dawley rats, concentrations of glutamate, glutamine, taurine, and betaine were highest in the cerebral cortex and decreased toward the brain stem. Conversely, glycine level was highest in the brainstem, and decreased toward the cortex. Myoinositol, glycerophosphorylcholine, glycerophosphorylethanolamine, and creatine were distributed more evenly. In chronic hyponatremic rats (plasma Na 110 +/- 4 meq/liter), organic osmolytes decreased globally with the total loss ranging from 13 (medulla) to 24 (cerebellum) mmol/kg H2O. After rapid correction with intraperitoneal injection of hypertonic saline, the recovery of the loss of organic osmolytes was 48% in the cerebral cortex, cerebellum, and medulla oblongata, 44% in pons, but only 17% in midbrain and 36% in striatum. Histopathology of the brain was examined in nine rats 2-7 d after correction of hyponatremia. Large demyelinative lesions were seen persistently in the midbrain and striatum, and smaller lesions in cerebrum, cerebellum, and pons were found less frequently. This is the first report of regional distribution of brain organic osmolytes. After rapid correction of chronic hyponatremia, a topographic correlation between demyelination lesions and delayed accumulation of organic osmolytes exists.

Animals↗

PubMed1995

Acute renal failure in membranous glomerulonephropathy: a result of superimposed crescentic glomerulonephritis.

A patient who presented with acute renal failure and anasarca secondary to crescentic glomerulonephritis superimposed on existing membranous glomerulonephropathy of 15 years' duration is described. The patient responded to an initial course of prednisone but failed to respond to a second course after relapse. The differential diagnosis of acute renal failure in the setting of nephrotic syndrome is discussed. Eighteen cases of crescentic glomerulonephritis superimposed on membranous glomerulonephropathy are reviewed. The clinical setting is heterogeneous with variable presentation and outcome. It appears that patients without antiglomerular basement membrane antibodies have a better prognosis than those who have antibodies. Patients with membranous glomerulonephropathy who develop unexplained acute renal failure should undergo early renal biopsy in order to rule out unexpected pathologic complications.

Acute Kidney Injury↗

PubMed1994

Scyllo-inositol depletion in hepatic encephalopathy.

Cerebral myo-inositol depletion is found in patients with hepatic encephalopathy and can be implicated in the pathogenesis of hepatic encephalopathy. We measured scyllo-inositol, a stereoisomer of myo-inositol, in brain extracts from patients dying in hepatic coma using HPLC and high resolution 1H MRS. The cerebral scyllo-inositol concentration, determined by both methods, in patients without hepatic encephalopathy was 0.41 +/- 0.11 mmol/kg wet weight. It decreased by 73% and 76%, respectively, as measured by HPLC and 1H MRS, in patients with hepatic encephalopathy. These findings indicate that myo-inositol depletion in patients with hepatic encephalopathy is not due to enhanced conversion of myo-inositol to scyllo-inositol or inhibition of myo-inositol transport by scyllo-inositol, but rather to the reduced biosynthesis or transport of both inositols.

Autopsy↗

PubMed1994

Bicarbonate dependency of betaine synthesis in cultured LLC-PK1 cells.

Betaine, one of the major renal organic osmolytes, is synthesized from choline by choline dehydrogenase (EC 1.1.99.1) and betaine-aldehyde dehydrogenase (EC 1.2.1.8) in the kidney. A recent in vitro study has shown that betaine synthesis by renal cortical homogenate is dependent on millimolar amounts of bicarbonate. The present study was aimed to investigate the bicarbonate dependency of betaine formation in cultured LLC-PK1 cells. The data show that betaine formation increases in accordance with a rise in extracellular bicarbonate levels. The measured quantities of [14C]betaine synthesis ranged from 13.4 +/- 1.5 (4.6 mM HCO3-) to 38.0 +/- 1.4 pmol.micrograms protein-1.h-1 (24 mM HCO3-). The carbonic anhydrase inhibitor acetazolamide, added to the incubation medium to block bicarbonate transport, reduced betaine synthesis from choline by 41-49%. We conclude that betaine synthesis in LLC-PK1 cells is dependent on extracellular bicarbonate levels and is reduced by the inhibition of carbonic anhydrase. Because betaine accumulates in renal medulla during antidiuresis, our observations suggest a possible link between acid-base homeostasis and concentration mechanisms in the kidney.

Acetazolamide↗

PubMed1993

Decrease in cerebral inositols in rats and humans.

Rats with portacaval shunts and humans with hepatic encephalopathy show severe myo-inositol depletion in the brain. The portacaval-shunted rat may therefore be a useful model for the investigation of neurochemical pathways containing myoinositol, which are modulated not only in hepatic encephalopathy but also in diabetes mellitus and Alzheimer's disease.

Animals↗

PubMed1993

Dimethyl methylphosphonate (DMMP): a 31P nuclear magnetic resonance spectroscopic probe of intracellular volume in mammalian cell cultures.

Dimethyl methylphosphonate (DMMP), when added to a suspension of erythrocytes, has been reported to have a lower frequency chemical shift inside of cells than outside. This work further investigates the same phenomenon in hollow-fiber bioreactor cultures of six mammalian cell lines and describes the application of DMMP as a measure of intra- versus extracellular volumes in mammalian cell cultures. No toxic effects of the DMMP were observed at the concentrations used here. The dependence of the shift of intracellular DMMP on intracellular protein content was shown to be similar for cultured mammalian and red blood cells. Also consistent with shifts in erythrocytes, an increase in the intracellular protein concentration due to a reduction in cultured cell volume increased the magnitude of the shift to lower frequency. Longitudinal relaxation (T1) values for intra- and extracellular DMMP were measured so that partially saturated DMMP peaks in 31P NMR spectra of mammalian cell cultures can be corrected to give the relative volumes of the intra- and extracellular compartments; this information provides a relative measure of culture growth. Intracellular volume measured by this method can also be used to quantify intracellular metabolites such as ATP during the growth of the culture. To explore the mechanism behind the intracellular shift, we have also addressed the three possible contributions to the chemical shift of DMMP: hydrogen-bonding interactions, magnetic susceptibility, and ionic strength. Data is presented which eliminates the latter two mechanisms and strongly supports the hypothesis that the observed intracellular shift is due to a reduction in hydrogen bonding between water and DMMP in the cytoplasm.

Animals↗