Ofloxacin: new applications for the prevention of urinary tract infections in renal graft recipients.

BACKGROUND: Urinary tract infections (UTIs), the most common form of bacterial infection in kidney transplant recipients, recently have been demonstrated to be detrimental for long-term graft outcome. Therefore, reinforcing antibiotic prophylaxis might be vital, in addition to basic hygiene recommendations, surgical care, and prophylaxis by trimethoprim-sulfamethoxazole. METHODS: In 2006, a Legionella pneumophila contamination of our department's water pipes meant that all the patients undergoing renal transplantation underwent a 1-month regimen of ofloxacin (OFLO) (200 mg every other day). We took this opportunity to measure the incidence of UTI, including acute pyelonephritis (APN), in 100 consecutive patients transplanted before (n = 50) and after (n = 50) this treatment decision was reached. We also studied the antimicrobial resistance profiles in our department and in the rest of the hospital. RESULTS: No patient developed Legionnaire's disease. A dramatic decrease in the incidence of UTI (-63%) was also seen in patients undergoing OFLO treatment. Logistic regression analysis demonstrated that the use of OFLO was independently associated with a reduction in UTI (odd ratio [OR] = 0.31%, 95% confidence interval [CI] 0.11-0.84, P = 0.02) and APN (OR = 0.21%, 95% CI 0.07-0.98, P = 0.045). This protection was sustained during the whole first year post transplantation. As for resistance rates, we observed a decrease in the susceptibility of Pseudomonas aeruginosa to ciprofloxacin in our nephrology department, compared with that observed in the rest of the hospital. The incidence of multi-resistant bacteria was stable. DISCUSSION: Our unintentional extension of prophylactic antibiotherapy with OFLO gave rise to a dramatic decrease in the 1-year incidence of UTI and APN in kidney recipients. Emergence of resistant strains is, however, a major concern.

Cortical cell populations from rabbit kidney isolated by free-flow electrophoresis: characterization by measurement of hormone-sensitive adenylate cyclase.

Free-flow electrophoresis allows the separation of different cell populations from a cell suspension isolated from rabbit kidney cortex after perfusion of the kidneys with a calcium-binder, followed by gentle mechanical treatment. After electrophoretic separation, analysis of the adenylate cyclase activities after stimulation by various hormones allows the precise determination of the origin of the cell populations with different electrophoretic mobilities. Adenylate cyclase from the slow-moving main cell population was only sensitive to parathyroid hormone. These cells had also high alkaline phosphatase content, further demonstrating their proximal origin. The various fast-moving cell populations had adenylate cyclase sensitive to isoproterenol and arginine vasopressin but were less sensitive to parathyroid hormone than the slow-moving cells. Their alkaline phosphatase content was also much lower. This indicates that these fast-moving cell populations originate from both the granulous segment of the distal tubule and from the collecting ducts. The adenylate cyclase activity and the cyclic AMP contents of isolated proximal cells maintained in culture medium were also investigated.

Estradiol-dependent uterine leiomyomas in transgenic mice.

Uterine leiomyomas are a major health problem for women of reproductive age. The molecular biology of these tumors is poorly understood partly because of the lack of relevant animal models. We have produced transgenic mice expressing the simian virus 40 T antigen driven by the promoter of the Calbindin-D9K (CaBP9K) gene and either -1,000 or -117 bp of regulatory sequences so as to establish in vivo, uterine smooth muscle tumor models. Six transgenic mouse lines were obtained. Leiomyomas developed in all of them, with an almost complete penetrance of the phenotype. The smooth muscle tumors arose in different parts of the female reproductive tract. Leiomyomas usually developed in the corpus of the uterus, but one mouse line developed leiomyomas in the horn of the uterus, and another in the vagina. The CaBP9K regulatory sequences directing the expression of the Tag gene possess an estradiol responsive element, and accordingly, development of the tumors was strictly under the control of estrogen. Expression of the Tag gene is not only necessary for the initiation of the tumor but also for its development and maintenance. These transgenic mouse models should be useful for studying the pathobiology of uterine leiomyomas and could be instrumental in designing new therapeutic approaches to this disease.

Cl- absorption across the thick ascending limb is not altered in cystic fibrosis mice. A role for a pseudo-CFTR Cl- channel.

The cortical thick ascending limb (CTAL) absorbs Cl- via a Na+-K+-Cl- cotransport at the apical membrane and several Cl- channels at the basolateral membrane, including a 9-pS channel having several properties of the cystic fibrosis transmembrane conductance regulator (CFTR). Having checked that CFTR mRNA is present in the mouse CTAL, we investigated whether this channel is a CFTR molecule by applying the patch-clamp technique to CTALs microdissected from CFTR knockout mice (cftrm1Unc). The 9-pS channel was active in cell-attached patches from tubules of mice homozygous for the disrupted cftr gene [CFTR (-/-)] at the same frequency and with the same activity (NPo) as in normal [CFTR (+/+)] or heterozygous [CFTR (+/-)] mice. The conductive properties of the channel, studied on inside-out patches, were identical in CFTR (-/-), CFTR (+/+), and CFTR (+/-) tubules, as were the sensitivities to internal pH and internal ATP, two typical features of this channel. In addition, the Cl- absorption in isolated, microperfused CTALs and the Na+-K+-Cl- cotransport activity were identical in CFTR (-/-), CFTR (+/+), and CFTR (+/-) mice. These results show that the 9-pS Cl- channel is distinct from CFTR, and that the CFTR protein has no influence on the Cl- absorption in this part of the renal tubule.

Overproduction of a truncated hepatocyte nuclear factor 3 protein inhibits expression of liver-specific genes in hepatoma cells.

Transcription of hepatocyte-specific genes requires the interaction of their regulatory regions with several nuclear factors. Among them is the hepatocyte nuclear factor 3 (HNF3) family, composed of the HNF3 alpha, HNF3 beta, and HNF3 gamma proteins, which are expressed in the liver and have very similar fork head DNA binding domains. The regulatory regions of numerous hepatocyte-specific genes contain HNF3 binding sites. We examined the role of HNF3 proteins in the liver-specific phenotype by turning off the HNF3 activity in well-differentiated mhAT3F hepatoma cells. Cells were stably transfected with a vector allowing the synthesis of an HNF3 beta fragment consisting of the fork head DNA binding domain without the transactivating amino- and carboxy-terminal domains. The truncated protein was located in the nuclei of cultured hepatoma cells and competed with endogenous HNF3 proteins for binding to cognate DNA sites. Overproduction of this truncated protein, lacking any transactivating activity, induced a dramatic decrease in the expression of liver-specific genes, including those for albumin, transthyretin, transferrin, phosphoenolpyruvate carboxykinase, and aldolase B, whereas the expression of the L-type pyruvate kinase gene, containing no HNF3 binding sites, was unaltered. Neither were the concentrations of various liver-specific transcription factors (HNF3, HNF1, HNF4, and C/EBP alpha) affected. In partial revertants, with a lower ratio of truncated to full-length endogenous HNF3 proteins, previously extinguished genes were re-expressed. Thus, the transactivating domains of HNF3 proteins are needed for the proper expression of a set of liver-specific genes but not for expression of the genes encoding transcription factors found in differentiated hepatocytes.

Cyclic AMP increases cell surface expression of functional Na,K-ATPase units in mammalian cortical collecting duct principal cells.

Cyclic AMP (cAMP) stimulates the transport of Na(+) and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCD(c14) collecting duct cells. db-cAMP (10(-3) M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of (86)Rb(+) uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20 degrees C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.

Intestinal dysplasia and adenoma in transgenic mice after overexpression of an activated beta-catenin.

Mutations in the adenomatous polyposis coli gene or activating mutations in the beta-catenin gene itself are thought to be responsible for the excessive beta-catenin signaling involved in intestinal carcinogenesis. We generated transgenic mice that expressed large amounts of a NH2-terminally truncated mutant beta-catenin (deltaN131beta-catenin) in the intestine. These mice had multifocal dysplastic lesions in the small intestine, reminiscent of the early lesions observed in the mouse models of familial adenomatous polyposis. The number of apoptotic cells in the villi of these transgenic mice was 3-4-fold higher than in nontransgenic mice. Expression of the truncated beta-catenin mutant in the kidney led to the development of severe polycystic kidney disease. Our findings support the concept that deregulation of the beta-catenin signaling pathway is the major oncogenic consequence of adenomatous polyposis coli mutations in intestinal neoplasia.

Early development of polycystic kidney disease in transgenic mice expressing an activated mutant of the beta-catenin gene.

Autosomal dominant polycystic kidney disease (ADPKD) is common and is a major cause of renal failure. Although the genetics of ADPKD are well known and have led to the discovery of polycystins, a new protein family, the pathogenesis of the disease remains largely unknown. Recent studies have indicated that the beta-catenin signaling pathway is one of the targets of the transduction pathway controlled by the polycystins. We have generated transgenic mice that overproduce an oncogenic form of beta-catenin in the epithelial cells of the kidney. These mice developed severe polycystic lesions soon after birth that affected the glomeruli, proximal, distal tubules and collecting ducts. The phenotype of these mice mimicked the human ADPKD phenotype. Cyst formation was associated with an increase in cell proliferation and apoptosis. The cell proliferation and apoptotic indexes was increased 4-5-fold and 3-4-fold, respectively, in cystic tubules of the transgenic mice compared to that of littermate controls. Our findings provide experimental genetic evidence that activation of the Wnt/beta-catenin signaling pathway causes polycystic kidney disease and support the view that dysregulation of the Wnt/beta-catenin signaling is involved in its pathogenesis.

Analysis of the mouse Scnn1a promoter in cortical collecting duct cells and in transgenic mice.

We have isolated and characterised the promoter of the mouse Scnn1a (alpha ENaC) gene. Using transient transfections of serial deletion mutants into Scnn1a-expressing cells, we demonstrate that 1.56 kb of 5' upstream sequence is required for cell-specific expression and corticosteroid-mediated regulation. These 5' sequences are not sufficient to drive expression of a lacZ reporter gene or a rat Scnn1a cDNA in transgenic mice, where they failed to rescue Scnn1a deficiency.

Functional and growth properties of a myometrial cell line derived from transgenic mice: effects of estradiol and antiestrogens.

This study describes the properties of a myometrial cell line, m-M116, that was derived from a leiomyoma developed in an adult female transgenic mouse harboring the simian virus 40 large T antigen (Tag) under the control of the 5'-regulatory sequence of the calbindin D9k (CaBP9k) gene. As the expression of this transgene is governed by the CaBP9k estrogen-responsive element, m-M116 cells were grown in medium supplemented with 17 beta-estradiol. The cells were long lived, had Tag-positive nuclei, and were nontumorigenic when injected into nude mice. They formed irregular layers of elongated cells with typical features of uterine, smooth muscle cells, as assessed by the presence of alpha-smooth muscle actin and desmin filaments, estradiol and progesterone receptors, and expression of the CaBP9k gene. The rate of cell doublings and the expression of the Tag gene in early passaged cells depended on the presence of 17 beta-estradiol. Tamoxifen, a mixed estrogen agonist-antagonist, also stimulated the growth of m-M116 cells, whereas ICI 182 780, a pure antiestrogen, blocked cell growth. Later passages of m-M116 cells still had a smooth muscle phenotype, but proliferated even in the absence of 17 beta-estradiol. These mouse uterine smooth muscle cells obtained by targeted oncogenesis provide a useful model for studies of the progression of steroid-independent carcinomas.

Deep brain stimulation of the globus pallidus pars interna in advanced Parkinson's disease.

Pallidotomy is now widely performed for the treatment of advanced Parkinson's disease (PD). Preliminary reports of the effect of globus pallidus pars interna deep brain stimulation (GPi DBS) have also been promising. We have analyzed a cohort of 22 consecutive patients enrolled in a multicenter study. Surgery was bilateral in 17 and unilateral in five patients. At 6-month follow-up, the bilaterally GPi-implanted patients demonstrated a marked improvement when examined after drug withdrawal ("off") and under optimal medication ("on") using the Unified Parkinson's Disease Rating Scale (UPDRS). The benefit induced by the stimulation in the "off" medication condition in the total motor score was 31% and in the activities of daily living (ADL) scores was 39%. During the "on" medication period, the reduction in the total "on" dyskinesias score was 66% and in the ADL score was 32%. A similar pattern of improvement was seen in the group of patients with unilateral GPi stimulation, although a second cohort of 12 patients not included in the multicenter study showed greater improvements in "on" motor functioning. Although the effect of DBS is predominantly reversible, electrode insertion alone resulted in measurable clinical effects in the absence of stimulation. Thus, at 6-month follow-up, the benefit observed without stimulation was up to 44% in the "on" dyskinesias score and 29% in timed tapping scores undertaken in the "off" medication state. Complications among 34 patients from all centers included perioperative infection (n=3), hardware fracture (n=2), and premature battery failure (n=3). These results show a positive antiparkinsonian effect of pallidal DBS. No specific complications were observed with bilateral procedures.

Protection of autotransplanted pig kidneys from ischemia-reperfusion injury by polyethylene glycol.

BACKGROUND: Ischemia-reperfusion injury (IRI) is often responsible for graft rejection and leads to delayed graft function of cadaveric kidneys. We have shown that adding polyethylene glycol (PEG 20M) to the preservation solutions helps protect isolated perfused pig kidneys against cold ischemia and reperfusion injury. METHODS: We compared the effects of adding PEG to a simplified high-K+ perfusion solution of cold-stored kidneys to Euro-Collins or University of Wisconsin solutions on the function of reperfused autotransplanted pig kidneys. The left kidney was cold-flushed with the preservation solutions and stored for 48 hr at 4 degrees C before reimplantation. Creatinine clearance and fractional excretion of sodium were analyzed 2 days before surgery and over 7 days after transplantation. Histological sections were obtained 40 min after reperfusion and on day 7 after surgery. RESULTS: Adding PEG to the perfusate significantly reduced IRI from autotransplanted pig kidneys. Creatinine clearance was significantly higher and fractional excretion of sodium was significantly lower in pigs transplanted with kidneys cold-flushed with PEG-supplemented perfusate than in those flushed with Euro-Collins or University of Wisconsin solutions. PEG supplementation also better preserved the integrity of kidney cells and markedly reduced interstitial cell infiltrates. CONCLUSION: PEG protects against IRI and reduces early cellular inflammation. PEG may impair the recruitment and migration of leukocytes into retransplanted pig kidneys. Cold preservation of donor organs with PEG-supplemented solutions may therefore help limit IRI in human renal transplantation.

Characterization of monoclonal antibodies specific for rabbit renal brush-border hydrolases: application to immunohistological localization.

By use of immunodepletion studies, we characterized four monoclonal antibodies reactive with rabbit brush-border (BB) as specific for aminopeptidase N (AP), dipeptidylpeptidase IV (DPPIV), neutral endopeptidase (EP), and angiotensin-converting enzyme (ACE), and we used these antibodies for immunohistochemical detection of these four hydrolases. Expression within the kidney was studied by light and electron microscopy. All four hydrolases are expressed on the various segments of the proximal tubule. In addition, EP and DPPIV are detectable on visceral epithelial cells of the glomerulus and AP on the cells of Bowman's capsule. Outside the kidney, the four hydrolases are expressed within the digestive and genital tracts, where AP, EP, and DPPIV predominate on epithelial structures, whereas ACE is essentially located in vascular structures. The latter localization is also characteristic of ACE in the other organs studied, where clear-cut systematic distribution of the other hydrolases was often difficult to demonstrate. In addition, AP, DPPIV, and EP were detected on lymphoid cells. As compared to reports of data obtained essentially by enzymatic or immunoradiometric assays, these observations suggest considerable interspecies variations of extrarenal expression of the major BB hydrolases. This should be taken into account in attempting to define a general physiological role for a given enzyme.

Tissue and cell distribution of the multidrug resistance-associated protein (MRP) in mouse intestine and kidney.

The multidrug resistance-associated protein (MRP) that is involved in drug resistance and the export of glutathione-conjugated substrates may not have the same epithelial cell membrane distribution as the P-glycoprotein encoded by the MDR gene. Because intestinal and kidney epithelial cells are polarized cells endowed distinct secreting and absorptive ion and protein transport capacities, we investigated the tissue and cell distribution of MRP in adult mouse small intestine, colon, and kidney by immunohistochemistry. Western blot analyses revealed the 190-kD MRP protein in these tissues. MRP was found in the basolateral membranes of intestinal crypt cells, mainly Paneth cells, but not in differentiated enterocytes. All the cells lining the crypt-villous axis of the colon wall contained MRP. MRP was found in the glomeruli, ascending limb cells, and basolateral membranes of the distal and collecting tubule cells of the kidney but not in proximal tubule cells. Cultured mouse intestinal m-ICcl2 cells and renal distal mpkDCT cells that have retained the features typical of intestinal crypt and renal distal epithelial cells, respectively, also possess MRP in their basolateral membranes. The patterns of subcellular and cellular distribution indicate that MRP may have a specific role in the basolateral transport of endogenous compounds in Paneth, renal distal, and collecting tubule cells.

Dibutyryl cyclic adenosine monophosphate and forskolin alter the paracellular pathway in cultured corneal endothelial cells.

PURPOSE: This study describes the effects of the cyclic adenosine monophosphate (cAMP) pathway on the tight junctional barrier of the corneal endothelium, which plays a critical role in maintaining the corneal stroma in an underhydrated, transparent state. METHODS: Subcultured bovine corneal endothelial cells grown on filters were used to study the effects of dibutyryl-cAMP and forskolin on transendothelial electrical resistance and [3H]inulin flux. The tight junction-associated protein ZO-1 (zonula occludens protein-1) and F-actin were visualized by indirect immunofluorescence, and the ultrastructural organization of junctional complexes was studied by freeze-fracture electron microscopy. RESULTS: Cells formed a continuous monolayer of closely apposed hexagonal-type cells separated by a discontinuous belt of tight junctions with a transendothelial electrical resistance of 20.8 +/- 0.6 omega.cm2. Dibutyryl-cAMP (10(-4) M) and forskolin (10(-5) M) increased cell cAMP, significantly decreased the transendothelial resistance by 54% and 43%, respectively, and increased the flux of [3H]inulin from the apical to the basal side of the cells by 56% and 40%, respectively. Both agents also induced condensation of F-actin at the cell borders without any marked changes in the immunostaining of ZO-1 that delineated cell peripheries. However, freeze-fracture studies showed that dibutyryl-cAMP and forskolin induced dispersion of the tight junction network. CONCLUSIONS: These data suggest that activation of the cAMP-dependent pathway, leading to structural changes of the tight junctional network, may modulate the passive fluxes mediated by the paracellular pathway of the corneal endothelial barrier.

Mineralocorticoid receptors in SV40-transformed tubule cell lines derived from rabbit kidney.

The presence of mineralocorticoid (MR) and glucocorticoid (GR) receptors was investigated in two renal tubular cell lines, derived from primary cultures of isolated rabbit kidney cortical cells infected with the wild-type SV40 virus, which exhibit thick ascending limb (RC.SV2) and collecting tubule (RC.SV3) phenotypes (Vandewalle et al. J. Cell. Physiol. 141, 1989, 203-221). MR and GR were quantified, in cell monolayers and cell cytosolic fractions, with [3H]aldosterone, [3H]dexamethasone and [3H]RU486, an antiglucocorticoid with no affinity for MR. Cytosolic receptors from RC.SV2 and RC.SV3 cells labeled with [3H]aldosterone, [3H]dexamethasone or [3H]RU486 sedimented at approximately 8 S in a 15-40% glycerol gradient. All steroids displaced bound [3H]dexamethasone to the same extent, suggesting that dexamethasone bound to both MR and GR: under the conditions of assay, [3H]aldosterone binds exclusively to MR, and [3H]RU486 to GR. In both RC.SV2 and RC.SV3 cells, [3H]aldosterone bound to one class of high affinity sites (Kd 0.14-0.8 nM; Nmax 8 to 22 fmol/mg protein). In both cell lines, the number of high affinity binding sites for [3H]dexamethasone ranged from 9 to 18 fmol/mg protein with an affinity of 0.5-1.3 nM. Compared to renal cortex, the most striking observation was a marked decrease in [3H]dexamethasone binding in primary cultures and SV40-transformed cells. These results indicate that MR and GR are expressed in two established mammalian kidney tubular cell lines providing new models of cultured renal cells for studies on the physiological effects of corticosteroid hormones.

Tissue specificity of L-pyruvate kinase transgenes results from the combinatorial effect of proximal promoter and distal activator regions.

The L-type pyruvate kinase (L-PK) gene is regulated by diet and hormones and expressed at high levels in the hepatocytes, enterocytes, and proximal tubular cells of the kidney and at low levels in the endocrine pancreatic cells. Two regulatory regions have been shown to be important in transgenic mice to confer on a reporter gene a similar tissue-specific and diet-responsive expression: a proximal promoter fragment, with binding sites for the tissue-specific hepatocyte nuclear factors 1 and 4, and presence of the glucose-response element (GIRE) and a distal activator corresponding to a liver-specific hypersensitive site at -3000 bp with respect to the cap site. Although the proximal promoter is able to confer by itself tissue-specific expression on a reporter gene, its activity in vivo is strongly stimulated by the distal activator. To determine the possible role of the distal region on diet responsiveness and tissue specificity of the L-PK gene expression, we have created lines of transgenic mice in which the gene for SV40 T antigen (Tag) was directed by composite regulatory sequences consisting of the L-PK promoter and different enhancers: either the SV40 early enhancer (SV) or the H enhancer of the aldolase A gene (H). The induction of the composite H-PK/Tag and SV-PK/Tag transgenes by a carbohydrate-rich diet in the liver was similar to that of the endogenous L-PK gene. This suggests that in fasted mice the L-PK promoter, and especially the GIRE, is able to silence the activating influence of a strong viral enhancer such as the SV40 enhancer. The H-PK/Tag mice expressed the transgene similarly to the endogenous gene, except in the pancreas, where expression was practically undetectable. Consistently, whereas L-PK/Tag mice develop insulinomas, H-PK/Tag mice develop only hepatomas. In contrast, the transgene expression was partly aberrant in SV-PK/Tag mice. In addition to a normal activation of the transgene in the liver, a strong expression was also detected in the kidney medulla, whereas the transgene was practically silent in enterocytes. Finally, the effect of the distal region (-2070 to -3200) on an ubiquitous promoter was tested by ligating the distal L-PK gene fragment in front of a thymidine kinase/CAT transgene. Such a transgene was constantly expressed in the pancreas and, strikingly, in the brain. It appears, therefore, that the L-PK distal activator exhibits, by itself, a certain neuropancreatic specificity required in combination with the proximal promoter for L-PK gene expression in pancreas endocrine cells.

N-acetyl-beta-D-glucosaminidase (NAG) isoenzymes in primary cultures of rabbit kidney proximal tubule cells: a cellular model for studies on nephrotoxicity?

N-Acetyl-beta-D-glucosaminidase (NAG) isoenzyme profile in primary cultures of rabbit kidney proximal tubule cells was studied. Confluent cells had high levels of NAG activity, but ion exchange chromatography showed that the NAG isoenzyme profile in cultured cells was different from that of rabbit renal cortex homogenates and freshly isolated cells. Confluent cultured cells contained an atypical acidic isoform, absent in homogenates and freshly isolated cells in which the predominant isoform is NAG-A (a heterodimer alpha beta). The fact that this atypical isoform was able to hydrolyse the synthetic substrate 4-methylumbelliferyl-beta-N-acetylglucosaminide-6-sulphate indicated that it probably was an alpha-subunit homodimer. These results suggest subunit rearrangement within NAG polypeptide chains linked to down-regulation of beta-subunit production in cultured rabbit proximal cells. The change in isoenzyme profile in cultured cells may make it difficult to use primary cultures of rabbit proximal tubule cells to establish correlations between in vitro and in vivo studies using NAG isoenzymes as a nephrotoxicity index, as illustrated by the effects of gentamicin.

Persistent intravascular C3 activation after bilateral nephrectomy in patients with thrombotic microangiopathy.

Four patients with thrombotic microangiopathy had evidence of intravascular C3 activation on presentation which persisted after bilateral nephrectomy. In several serum samples, C3-splitting activity was not associated with the presence of circulating immune complexes, which were detected in all four patients before nephrectomy and in three after nephrectomy.

Heat shock protein gp96 and NAD(P)H oxidase 4 play key roles in Toll-like receptor 4-activated apoptosis during renal ischemia/reperfusion injury.

Ischemia/reperfusion injury (IRI) causes inflammation and cell injury as a result of activating innate immune signaling. Toll-like receptor 4 (TLR4) has a key role in mediating kidney damages during IRI, but the downstream signaling pathway(s) stimulating apoptosis remains debated. In this study we show that TLR4 mediates MyD88-dependent activation of TNF receptor-associated factor 2, apoptosis signal-regulating kinase 1 (ASK1), and Jun N-terminal kinase (JNK) and p38 MAP kinases in ischemic-reperfused kidneys and posthypoxic renal tubule epithelial cells (RTECs). Hypoxia stimulated the expression of the endoplasmic-resident gp96, which co-immunoprecipitated TLR4, whereas silencing gp96 mRNA expression impaired hypoxia-induced apoptosis in TLR4-expressing RTECs. NAD(P)H oxidase 4 (NOX4) was shown to interact with TLR4 and to be required in lipopolysaccharide-induced production of reactive oxygen species (ROS). IRI stimulated the expression of a 28-kDa NOX4 spliced isoform abundantly expressed in wild-type RTECs, which co-immunoprecipitated with TLR4, but not with gp96 in TLR4-deficient RTECs. Silencing NOX4 mRNA expression impaired hypoxia-induced activation of ASK1 and both JNK and p38, leading to the inhibition of ROS production and apoptosis in posthypoxic TLR4-expressing RTECs. These findings show that, concomitantly to the activation of p38, the gp96/TLR4 interaction is required for activation of ASK1/JNK signaling in posthypoxic mouse RTECs, and that the 28-kDa NOX4 has a key role in TLR4-mediated apoptosis during renal IRI.