Hypomethylation of CTCFL promoters as a noninvasive biomarker in plasma from patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third deadliest cancer in the world with high morbidity and poor prognosis. CTCFL (CCCTC-binding factor like) is a member of the cancer testis antigen (CTA) family with oncogenic properties. To demonstrate whether the hypomethylation of CTCFL promoters in plasma could be used as a noninvasive biomarker to predict poor prognosis of HCC, we extracted cell-free DNA from the plasma and detected the methylation status of CTCFL in 43 HCC, 5 liver cirrhosis and 6 benign lesion samples using methylation specific PCR (MSP). Our study indicated that the hypomethylation of CTCFL promoters in HCC plasma samples (60.4%) was significantly different from that in benign lesion plasma samples (16.7%) with a p-value of 0.043. Analysis of clinicopathological data showed that the methylation status of CTCFL promoters was significantly correlated with microvascular involvement (MVI) (p=0.001) and postoperative recurrence (p=0.031). Furthermore, clinical prognosis data of 347 HCC patients from The Cancer Genome Atlas (TCGA) database displayed that the hypomethylated group had worse overall survival than the hypermethylated group (p=0.0056). In conclusion, we provide evidence that the hypomethylation of CTCFL promoters in cell-free DNA is a biomarker for monitoring HCC patients, which can be used as a noninvasive prediction index for tumor recurrence and provide the individualized decision-making for clinicians.

Combination of LINE-1 hypomethylation and RASSF1A promoter hypermethylation in serum DNA is a non-invasion prognostic biomarker for early recurrence of hepatocellular carcinoma after curative resection.

Hepatocarcinogenesis, a multistep process, involves not only genetic mutations but also epigenetic alterations. Widespread of global DNA hypomethylation is accompanied with specific regional hypermethylation especially at tumor suppressor genes' promoters. The aim of this study is to determine the efficacy of combined DNA methylation analysis of a global DNA methylation marker - LINE-1 and a tumor suppressor gene highly associated with the malignancy of HCC- RASSF1A in serum as a novel prognostic marker for diagnosis of early recurrence after curative resection.LINE-1 was hypomethylated in 66.7% (70/105) and RASSF1A promoter was hypermethylated in 73.3% (77/105) of HCC serum DNA samples by methylation specific PCR, but in none of the healthy controls: LINE-1 hypometylation (0/50) and RASSF1A hypermethylation (0/50). A significant association was found between LINE-1 hypomethylation and clinical pathologic features including HBsAg positivity (p=0.009), tumor size (p=0.001) and AFP levels (p<0.001). Besides, significant correlation was detected between RASSF1A promoter hypermethylation and lymph nodes metastasis (p=0.045).The results of Kaplan-Meier estimates of survival suggested that LINE-1 hypomethylation was highly associated with poor survival of patients (disease-free survival p=0.002, overall survival p=0.0123). More importantly, co-evaluation of LINE-1 hypomethylation and RASSF1A promoter hypermethylation was found to be significantly correlated to early recurrence and poor prognosis (disease-free survival p=0.0001, overall survival p=0.05) in patients after curative resection.In conclusion, our study showed that the combined examination of LINE-1 hypomethylation and RASSF1A promoter hypermethylation was effective in predicting early recurrence of HCC after curative resection. Patients with dual positivity of LINE-1 hypomethylation and RASSF1A promoter hypermethylation should be supplied with more intensive care and close follow-up after they undergo tumor resection.

Prospective ECG-triggering cardiac CT for infants with complex congenital heart disease using low-dose contrast medium, low tube voltage, and adaptive statistical iterative reconstruction.

AIM: To demonstrate the clinical value of prospective electrocardiography (ECG)-triggered cardiac computed tomography (CT) with low concentration contrast medium, low tube voltage, and adaptive statistic iterative reconstruction (ASIR) to reduce both radiation and contrast dose in examining infants with complex congenital heart disease (CHD). MATERIALS AND METHODS: Forty-four consecutive infants (19 male, 25 female, age: 8.06±4.33 months, weight: 7.31±1.36 kg) with complex CHD underwent prospective ECG-triggered low-dose cardiac CT using 80 kVp and 120 mA. The contrast agent was iodixanol (270 mg iodine/ml, Visipaque, GE Healthcare, Co. Cork, Ireland). Cardiac CT images were reconstructed with 70% ASIR. The quantitative CT image quality was assessed by image noise in adipose tissue and contrast-to-noise ratio (CNR) in the aorta. The qualitative image analysis was performed on a five-point grading scale by two independent reviewers and interobserver variability was calculated. The results of 32 CT examinations were also compared with the available surgical results for diagnostic accuracy evaluation. RESULTS: The effective dose was 0.55±0.10 mSv for the patient population. The iodine load was 3.95±0.73 g iodine. Image noise in adipose tissue was 16.24±1.42 HU and CNR in aorta was 21.90±7.10. All images were acceptable for diagnosis with an average score of 4.52±0.38 and good agreement between reviewers (kappa=0.75). Compared to the surgery results in 32 cases, CT was 97% and 88% accurate diagnosing extracardiac and intracardiac defects, respectively. CONCLUSION: Prospective ECG-triggered cardiac CT using 80 kVp, low-concentration iodinated contrast agent (270 mg iodine/ml) and 70% ASIR reconstruction provides excellent image quality and accurate diagnosis for complex congenital heart disease in infants with reduced contrast medium dose and low radiation dose.

Hrd1 facilitates tau degradation and promotes neuron survival.

Intraneuronal accumulation of abnormal phosphorylated tau (p-tau) is a molecular pathology in many neurodegenerative tauopathies, including Alzheimer's disease (AD) and frontotemporal dementia with parkinsonism-linked to chromosome 17 (FTDP-17). However, the underlying mechanism remains unclear. Here, we showed an inverse relationship between endoplasmic reticulum membrane ubiquitin ligase (E3) Hrd1 expression and p-tau accumulation in the hippocampal neurons of AD, and proposed that Hrd1 may be a negative regulator of p-tau. This notion was further supported by in vitro study demonstrating that Hrd1 interacted with tau and promoted the degradation of total tau and p-tau as well. The degradation of tau depended on its Hrd1 E3 activity. Knockdown of endogenous Hrd1 with siRNA stabilized tau levels. In addition, inhibition of proteasome maintained tau level and increased Hrd1-mediated tau ubiquitination, suggesting the proteasome was involved in tau/p-tau degradation. Over-expression of Hrd1 significantly alleviated tau cytotoxicity and promoted cell survival. These results indicated that Hrd1 functions as an E3 targeting tau or abnormal p-tau for proteasome degradation. The study provides an important insight into the molecular mechanisms of human tauopathies.

Members of the Kv1 and Kv2 voltage-dependent K(+) channel families regulate insulin secretion.

In pancreatic beta-cells, voltage-dependent K(+) (Kv) channels are potential mediators of repolarization, closure of Ca(2+) channels, and limitation of insulin secretion. The specific Kv channels expressed in beta-cells and their contribution to the delayed rectifier current and regulation of insulin secretion in these cells are unclear. High-level protein expression and mRNA transcripts for Kv1.4, 1.6, and 2.1 were detected in rat islets and insulinoma cells. Inhibition of these channels with tetraethylammonium decreased I(DR) by approximately 85% and enhanced glucose-stimulated insulin secretion by 2- to 4-fold. Adenovirus-mediated expression of a C-terminal truncated Kv2.1 subunit, specifically eliminating Kv2 family currents, reduced delayed rectifier currents in these cells by 60-70% and enhanced glucose-stimulated insulin secretion from rat islets by 60%. Expression of a C-terminal truncated Kv1.4 subunit, abolishing Kv1 channel family currents, reduced delayed rectifier currents by approximately 25% and enhanced glucose-stimulated insulin secretion from rat islets by 40%. This study establishes that Kv2 and 1 channel homologs mediate the majority of repolarizing delayed rectifier current in rat beta-cells and that antagonism of Kv2.1 may prove to be a novel glucose-dependent therapeutic treatment for type 2 diabetes.

Microencapsulated bovine chromaffin cell xenografts into hemiparkinsonian rats: a drug-induced rotational behavior and histological changes analysis.

Bovine chromaffin cells were microencapsulated within alginate-polylysine-alginate (APA) membranes. Microencapsulated bovine chromaffin cells as well as unencapsulated cells and empty microcapsules were grafted into the brain of hemiparkinsonian rats with 6-hydroxydopamine (6-OHDA) lesions. Apomorphine-induced rotational behavior of the host animals and the survival of the grafted chromaffin cells were examined after transplantation. The animals receiving microencapsulated bovine chromaffin cells showed a significant decrease (17.6--35.6%) in apomorphine-induced rotation 1 week postimplantation that remained stable for the 10 month test period. Fluorescent histochemistry further revealed that microencapsulation increased the chromaffin cell survival with only a minimum host reaction for up to 10 months posttransplantation while the survival of free, unencapsulated chromaffin cells was only modest and was accompanied by a large inflammatory response. The reduction of apomorphine-induced rotations was correlated with the survival of bovine chromaffin cells in the host brain. The data indicate that encapsulation of bovine chromaffin cells in APA membranes reduces the host immune response to the xenograft and prolongs the viability of the grafted cells.

Xenotransplantation of microencapsulated bovine chromaffin cells into hemiparkinsonian monkeys.

This study examines the effects of xenografts of microencapsulated bovine chromaffin cells (BCCs) on the rotational behavior of hemiparkinsonian monkey recipients. In addition, it determines the content of monoamine neurotransmitters and their major metabolites in the neostriatum in hemiparkinsonian monkeys. The hemiparkinsonian model in monkeys was induced by a unilateral intracarotid injection of methyl-phenyl-tetrahydropyridine (MPTP). Unencapsulated BCCs, BCCs microencapsulated in alginate-polylysine-alginate (ALA) membranes as well as empty microencapsules were grafted into the neostriatum of the hemiparkinsonian monkeys. Following the transplantation the hemiparkinsonian symptoms subsided and the number of rotations induced by apomorphine decreased for up to nine months in the group of recipients grafted with microencapsulated BCCs, while only a temporary improvement (one month) was detected in the recipients of the unencapsulated BCCs. No change was observed in the recipients of empty microencapsules. Dopamine and its metabolites were found considerably depleted in the MPTP-lesioned side versus the unlesioned side of the neostriatum in the hemiparkinsonian monkeys(P<0.05).

In pursuit of perfection: a primary care physician's guide to body dysmorphic disorder.

Body dysmorphic disorder is an under-recognized chronic problem that is defined as an excessive preoccupation with an imagined or a minor defect of a localized facial feature or body part, resulting in decreased social, academic and occupational functioning. Patients who have body dysmorphic disorder are preoccupied with an ideal body image and view themselves as ugly or misshapen. Comorbid psychiatric disorders may also be present in these patients. Body dysmorphic disorder is distinguished from eating disorders such as anorexia nervosa that encompass a preoccupation with overall body shape and weight. Psychosocial and neurochemical factors, specifically serotonin dysfunction, are postulated etiologies. Treatment approaches include cognitive-behavioral psychotherapy and psychotropic medication. To relieve the symptoms of body dysmorphic disorder, selective serotonin reuptake inhibitors, in higher dosages than those typically recommended for other psychiatric disorders, may be necessary. A trusting relationship between the patient and the family physician may encourage compliance with medical treatment and bridge the transition to psychiatric intervention.

Bioartificial pancreas: alternative supply of insulin-secreting cells.

In this study, insulin secretion function of INS-1 cells immunoisolated in microcapsules was evaluated. Following encapsulation, the immunoisolated INS-1 cells continued to propagate and flourish within the microcapsules during the entire two-month in vitro incubation period. The insulin secretion from encapsulated INS-1 cells following seven days of in vitro culture increased from 1.6 +/- 0.2 ng/2h/10(6) cells in a glucose-free medium to 11.5 +/- 2.1 ng/2h/10(6) cells at 16.7 mM glucose. In vivo, transplants of 1.2 x 10(7) cells into each of six diabetic C57BL/6 mice resulted in the restoration of normoglycemia in all graft recipients for up to 60 days post transplantation. Most capsules recovered from two animals 30 days post transplantation were free of cell overgrowth and physically intact. Immunostaining for insulin of the cells within the recovered capsules clearly indicated the presence of insulin. The presented data demonstrate the potential use of an immunoisolated beta-cell line for the treatment of diabetes.

Up-regulation of hepatocyte growth factor receptor: an amplification and targeting mechanism for hepatocyte growth factor action in acute renal failure.

BACKGROUND: Hepatocyte growth factor (HGF) and its c-met receptor comprise a signaling system that has been implicated in tissue repair and regeneration. HGF action is specifically targeted to the damaged organ following injury; however, the mechanism underlying this important targeting process remains to be elucidated. We reasoned that induction of c-met expression might be a critical factor in determining the site specificity of this receptor-ligand system. To test this hypothesis, we examined changes in activity of the HGF/c-met system in the folic acid model of acute tubular injury and repair. METHODS: Tissue HGF and c-met mRNA levels were detected by RNase protection assay and Northern blot analysis following acute renal injury induced by a single injection of folic acid. HGF and c-met proteins were examined by a specific enzyme immunoassay and Western blotting, respectively. C-met expression and trans-activation were investigated by exposing renal epithelial mIMCD-3 cells to various cytokines in vitro. RESULTS: Extremely rapid induction of renal HGF and c-met mRNA was observed beginning one hour following injection of folic acid. Circulating plasma HGF protein level rose dramatically (approximately 16-fold), peaking first at two hours and again at 24 hours following injection. Despite elevated HGF mRNA in the kidney, total kidney HGF protein actually decreased significantly at 24 hours following injury. On the other hand, both c-met mRNA and c-met protein were markedly increased in the kidney, where active renal tubule repair and regeneration take place. In vitro studies suggested that increased levels of HGF, as well as other cytokines, might account for enhanced c-met expression in renal tubular epithelial cells. Pretreatment of the cells with actinomycin D totally blocked c-met induction, suggesting that induced c-met expression occurs primarily at the transcriptional level. Using a cloned region of the c-met promoter coupled to a reporter gene, we demonstrated that HGF directly stimulated c-met promoter transactivation in renal epithelial cells. CONCLUSION: These results suggest that local up-regulation of c-met transcription in the kidney is crucial to renal tubule repair and regeneration, not only because it increases overall activity of this receptor-ligand system, but also as a mechanism targeting HGF action specifically to renal epithelia.

Methods for the immunoisolation and transplantation of pancreatic cells.

Although the administration of insulin by injection is clearly a life-saving intervention for patients devoid of ß-cells, this approach falls short of the remarkable titration of insulin delivery and consequent control of glucose levels achieved by normal, healthy individuals. In the absence of the physiological control of the plasma glucose concentrations, the daily injection of insulin has not been able to prevent the common complications of the disease, namely nephropathy, retinopathy, and neuropathy, as well as vascular complications. This has been confirmed in the recent diabetes control and complications trial which has demonstrated that intensive treatment of patients with insulin-dependent diabetes mellitus (IDDM), with tight glycemic control close to the control range, effectively delays the onset, and slows the progression, of the various diabetic complications (1). Therefore, it becomes mandatory to develop methods, applicable early in the course of the disease, and in any type 1 diabetic patient, for obtaining perfect metabolic control without increasing the risk of severe hypoglycemia. Consequently, the transplantation of islet tissue, either as whole pancreas or as isolated islets, has been pursued, because these techniques can provide near-normal blood glucose control, and thus have the potential to prevent diabetic complications.

Effect of hypoxic exposure on Na+/H+ antiport activity, isoform expression, and localization in endothelial cells.

Little is known about the effects of prolonged hypoxic exposure on membrane ion transport activity. The Na+/H+ antiport is an ion transport site that regulates intracellular pH in mammalian cells. We determined the effect of prolonged hypoxic exposure on human pulmonary arterial endothelial cell antiport activity, gene expression, and localization. Monolayers were incubated under hypoxic or normoxic conditions for 72 h. Antiport activity was determined as the rate of recovery from intracellular acidosis. Antiport isoform identification and gene expression were determined with RT-PCR and Northern and Western blots. Antiport localization and F-actin cytoskeleton organization were defined with immunofluorescent staining. Prolonged hypoxic exposure decreased antiport activity, with no change in cell viability compared with normoxic control cells. One antiport isoform [Na+/H+ exchanger isoform (NHE) 1] that was localized to the basolateral cell surface was present in human pulmonary arterial endothelial cells. Hypoxic exposure had no effect on NHE1 mRNA transcript expression, but NHE1 protein expression was upregulated. Immunofluorescent staining demonstrated a significant alteration of the F-actin cytoskeleton after hypoxic exposure but no change in NHE1 localization. These results demonstrate that the decrease in NHE1 activity after prolonged hypoxic exposure is not related to altered gene expression. The change in NHE1 activity may have important consequences for vascular function.

Expression of Na+/H+ exchanger isoforms in inner segment of inner medullary collecting duct.

Na+/H+ exchangers (NHE) play a critical role in many cellular and transport processes in the inner medullary collecting duct (IMCD). Morphologically, the IMCD is divided into the outer (IMCD1), middle (IMCD2), and inner (IMCD3) segments. The inner, IMCD3 segment contains only one cell type, the IMCD cell, which is distinct in ultrastructure and in function from the principal and intercalated cells that are present in other portions of the IMCD. NHEs constitute a gene family containing several isoforms (NHE1, NHE2, NHE3, NHE4 and NHE5) which possess distinct characteristics and serve specialized functions. To understand the molecular basis of NHE-related processes in the IMCD, it is critical to know the molecular identity of the NHEs in this tubule segment. The purpose of the present study was to identify the NHE isoforms present and their polar distribution in IMCD3. Applying the reverse transcription-polymerase chain reaction (RT-PCR) technique to IMCD3 (obtained from distal 50% of inner medulla) of mouse and rat kidneys, we found that NHE1, NHE2 and NHE4, but not NHE3 were expressed in both species. The polar localization of NHE in IMCD3 was examined in tubules isolated from rats and perfused in vitro with HEPES-buffered solutions under isotonic conditions. pHi was measured by BCECF fluorescence. Na+-dependent, amiloride-inhibitable pHi recovery from cell acidification (consistent with NHE) was detected in the basolateral, but not the apical, membrane of IMCD3. We conclude that NHE1, NHE2 and NHE4, but not NHE3, are present in both the mouse and rat IMCD3. Functionally, NHE is limited to the basolateral membrane. Additional studies are needed to determine the physiological roles and regulation of basolateral NHE isoforms in this tubule segment.

Genetic engineering of glucose-stimulated insulin secretion in Chinese hamster ovary cells.

To engineer an a non-islet cell capable of glucose-stimulated insulin secretion, a chinese hamster ovary cell line (CHO) was transfected with a mammalian expression vector carrying the human insulin cDNA (pCB/hINS). More proinsulin than insulin was released daily by the stably transformed cell line (CHO-INS). Examination of acid-ethanol extracts confirmed that both insulin and proinsulin were stored. Immunohistochemical analysis of the cells also showed that (pro)insulin was stored. Unlike beta cells, CHO-INS cells did not secrete insulin in response to glucose. To investigate this lack of effect, we examined whether transfection of GLUT2 cDNA, which is ordinarily not expressed in CHO-INS cells, would confer glucose-stimulated insulin secretion. Consequently, we have demonstrated that glucose regulated insulin release occurs in the CHO-INS-GLUT2 cell line and that glucose potentiates the insulin secretory response to non-glucose secretagogues.

Constitutive expression of HGF modulates renal epithelial cell phenotype and induces c-met and fibronectin expression.

Hepatocyte growth factor (HGF) is a potent renotropic factor that has been shown to play important roles in kidney development and recovery from acute renal injury. To examine the effects of HGF on renal tubular epithelium, we generated HGF-producing renal epithelial cells by stably transfecting mIMCD-3 and OK cells with an expression plasmid containing human HGF cDNA. Expression of HGF in the transfected cells was confirmed by detection of HGF mRNA by Northern blot analysis and detection of HGF secretion into the conditioned medium by ELISA. HGF-transfected cells exhibited fibroblast-like scattered morphology and increased cell motility. They formed branching tubules when grown in 3-D collagen gel. In addition, HGF-producing cells grew faster than their parental cells, but failed to form colonies in soft agar. These phenotypic changes were inhibited by a specific, neutralizing anti-HGF antibody. Interestingly, both c-met transcript and c-met protein were increased in HGF-transfected cells, suggesting that HGF amplifies its own action via stimulation of c-met expression. Autocrine expression of HGF and c-met in renal epithelial cells also stimulated fibronectin gene expression, which was totally blocked by incubation with a neutralizing anti-HGF but not a pan-specific anti-TGF-beta antibody, suggesting that it is independent of TGF-beta production. Our data demonstrate that HGF as a single factor stimulates renal epithelial cell proliferation, migration, differentiation, and extracellular matrix remodeling, making it uniquely suited to promote renal tubulogenesis during development, as well as to reconstitute tubular integrity following acute injury.

In vitro and in vivo evaluation of insulin-producing beta TC6-F7 cells in microcapsules.

In the present study, the insulin secretory capacity of beta TC6-F7 cells in microcapsules was evaluated. The cell mass within capsules was found to expand in a three-dimensional fashion, in contrast to cells seeded on plates that grew as a monolayer. In in vitro studies, both free and encapsulated cells were found to secrete insulin in the absence of glucose, at 13.6 +/- 1.1 and 14.5 +/- 0.9 ng.10(6) cells-1.60 min-1, respectively, with the response rising to a maximum of 26.0 +/- 0.8 and 31 +/- 2.3 ng.10(6) cells-1.60 min-1 in the presence of 16.8 mM glucose. Encapsulated cells were able to produce Ca2+ responses in the presence of KCl (50 mM) and BAY K 8644 (100 microM). In in vivo studies, intraperitoneal transplantation of 3.0 x 10(6) microencapsulated cells into mice (n = 5) with streptozotocin-induced diabetes resulted in the restoration of normoglycemia up to 57 days. Insulin concentrations rose from 0.4 +/- 0.1 ng/ml before the graft administration to 2.2 +/- 0.8 ng/ml after the transplantation in the normoglycemic recipients. An oral glucose challenge in transplant recipients demonstrated a flat glucose response, suggesting extremely high glucose clearance rates. These data demonstrate the potential use of the immunoisolated beta-cell lines for the treatment of diabetes.

Hepatocyte growth factor protects renal epithelial cells from apoptotic cell death.

Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an essential role in renal tubular repair and regeneration following injury. Studies indicate that administration of exogenous HGF to animals stimulates renal epithelial cell DNA synthesis and accelerates recovery from acute renal failure (ARF). However, whether increased cell proliferation accounts for all of the beneficial effects of HGF in ARF is unknown. In this study, we demonstrate that HGF protects renal epithelial cells from undergoing apoptotic cell death. Treatment of renal epithelial mIMCD-3 cells with 25 microM cisplatin in the serum-free medium induced significant apoptosis, as assessed by fluorescent Dye H-33342 staining, TUNEL staining, light and electron microscopy, and DNA laddering analysis. However, constitutive expression of HGF by transfection in mIMCD-3 cells resulted in resistance to cisplatin-induced apoptotic death. The survival rate of HGF-producing C1 cells was more than 2-fold greater as compared to control, mIMCD-3 cells following treatment with 25 microM cisplatin for 2 days. These results suggest that HGF may not only activate tubular repair processes but also ameliorate the initial injury by protecting renal epithelial cells from undergoing apoptosis.

Expression of Cl-/HCO3- exchanger in the basolateral membrane of mouse medullary thick ascending limb.

Although a basolateral Cl-/HCO3- exchanger (AE) has been implicated in the arginine vasopressin (AVP)-dependent hypertonic regulatory increase in the medullary thick ascending limb (MTAL), there are conflicting data regarding whether this exchanger is indeed present in this tubule segment. In this study, mouse MTAL was examined whether Cl-/HCO3- exchange activity was present in the basolateral membrane and whether mRNAs from the known AE genes are expressed. Cl-/HCO3- exchange activity was examined in isolated perfused MTAL tubules under isotonic conditions and in the absence of arginine vasopressin. 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein was used to monitor intracellular pH. Removal of basolateral Cl- induced reversible cell alkalization that was independent of external Na+ and completely inhibited by peritubular 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (200 microM). The rate and extent of cell alkalinization were significantly greater in the presence than absence of external CO2/HCO3-. A voltage clamp did not inhibit cell alkalinization induced by basolateral Cl- removal. Consistently, addition of basolateral Cl- induced reversible cell acidification in MTAL depleted of intracellular Cl-. Furthermore, mRNA encoding two members (AE2 and AE3) of the AE gene family were demonstrated in microdissected mouse MTAL tubules by reverse transcription-polymerase chain reaction. It is concluded that AE is present in the basolateral membrane of mouse MTAL.

Na+/H+ exchanger isoform 2 (NHE2) is expressed in the apical membrane of the medullary thick ascending limb.

Apical Na+/H+ exchangers (NHE) in the proximal tubule and medullary thick ascending limb (MTAL) display similar functions and regulation, suggesting that similar NHE isoforms are present. In the rat proximal tubule, NHE2 and NHE3 are present in the apical membrane, however, in the MTAL, NHE3, but not NHE2, mRNA has been found. In this study, the expression and subcellular localization of NHE2 in both rat and mouse MTAL were studied. To detect NHE2 mRNA, reverse transcription-polymerase chain reaction (RT-PCR) was performed in microdissected MTAL tubules using primers specific for NHE2. Analysis of PCR products with and without digestion by restriction enzymes chosen from the published NHE2 sequence gave predicted sizes. Subcloning and sequencing of the PCR product from mouse MTAL revealed 91% and 75% identity to the published NHE2 nucleotide sequence of comparable regions in rat and rabbit, respectively. Thus, NHE2 mRNA is expressed in the MTAL of mouse and rat. The subcellular localization of NHE2 was determined by immunochemistry using a specific NHE2 antibody. Immunofluorescence staining was observed in the apical, but not basolateral, membrane of MTAL of both species. In addition, anti-NHE2 antibody recognized an 85 kD protein in plasma membranes prepared from mouse and rat renal outer medulla and a MTAL cell line by Western analysis, which further support that NHE2 protein is expressed in the MTAL of both species. We conclude that NHE2 is expressed in the apical membrane of MTAL in both mouse and rat.