Primary Rabbit Kidney Research Articles

Isolation and Partial Sequencing of Equid Herpesvirus 5 from a Horse in Iceland

Horses are hosts to 2 types of gammaherpesviruses, Equid herpesvirus 2 and 5 (EHV-2 and EHV-5, respectively). Both EHV-2 and EHV-5 are common in horses in Iceland. An Icelandic EHV-5 isolate was recovered by sequential culture in primary fetal horse kidney and rabbit kidney cells. Glycoprotein B, glycoprotein H, and DNA terminase genes of the isolate were fully sequenced, and the DNA polymerase gene was partly sequenced. To date, the glycoprotein B gene of EHV-5 was the only gene that has been reported to be completely sequenced in addition to small parts of the glycoprotein H, DNA polymerase, and DNA terminase genes. The present report, therefore, is a significant addition to previously reported EHV-5 sequences.

ETIOLOGICAL AND MOLECULAR BIOLOGICAL INVESTIGATION OF CAPRINE HERPESVIRUS 1 ISOLATED IN BULGARIA

From goat and bucks with genital disorders and abortions the attempts for isolation of caprine herpesvirus 1 (CHV 1) were carried out. For virus exaltation Dexametazone was used. For viruses isolation vaginal, nasal, rectal, preputial swabs and organ samples were used. Primary and continuous cell cultures rabbit kidney (RK), Madin Darby bovine kidney (MDBK), and embrional bovine trachea (EBTR) were used for cultivation. For determination of DNA type and lipid envelop 60 μg/ml iod desoxiuridine (JDUR) and the ether treat ment was used. Neutralization by specific hyperimmune serum obtained from Switzerland was performed. Five CHV 1 strains were isolated by cell cultures and identified as goat herpesviruses from dif ferent Bulgarian regions. After electron microscopy the viral agents with typical size and morphology for herpesviruses were established. For demonstration gC gene of CHV 1 the polymerase chain reaction (PCR) with primers designed from sequences deposited in gene bank were developed. Isolated on cell cultures herpesviruses were proved as caprine herpesvirus 1 by using applied PCR variant. The products after gC gene amplification from Bulgarian isolates were separated on the same place as the amplicons of ref er ence CHV 1 strains.

Comparative Evaluation of the Immune Responses and Protection Engendered by LC16m8 and Dryvax Smallpox Vaccines in a Mouse Model

The immune response elicited by LC16m8, a candidate smallpox vaccine that was developed in Japan by cold selection during serial passage of the Lister vaccine virus in primary rabbit kidney cells, was compared to Dryvax in a mouse model. LC16m8 carries a mutation resulting in the truncation of the B5 protein, an important neutralizing target of the extracellular envelope form of vaccinia virus (EV). LC16m8 elicited a broad-spectrum immunoglobulin G (IgG) response that neutralized both EV and the intracellular mature form of vaccinia virus and provoked cell-mediated immune responses, including the activation of CD4+ and CD8+ cells, similarly to Dryvax. Mice inoculated with LC16m8 had detectable but low levels of anti-B5 IgG compared to Dryvax, but both Dryvax and LC16m8 sera neutralized vaccinia virus EV in vitro. A truncated B5 protein (approximately 8 kDa) was expressed abundantly in LC16m8-infected cells, and both murine immune sera and human vaccinia virus immunoglobulin recognized the truncated recombinant B5 protein in antigen-specific enzyme-linked immunosorbent assays. At a high-dose intranasal challenge (100 or 250 50% lethal doses), LC16m8 and Dryvax conferred similar levels of protection against vaccinia virus strain WR postvaccination. Taken together, the results extend our current understanding of the protective immune responses elicited by LC16m8 and indicate that the relative efficacy in a mouse model rivals that of previously licensed smallpox vaccines.

Rapid Detection and Differentiation of Wild-Type and Three Attenuated Lapinized Vaccine Strains of Classical Swine Fever Virus by Reverse Transcription Polymerase Chain Reaction

A simple one-step reverse transcription polymerase chain reaction (RT-PCR) method was developed based on T-rich insertions in the viral genome for simultaneous detection and differentiation of wild type and vaccine strains of Classical swine fever virus (CSFV). The CSFV-specific primers were designed to contain the sequences of the T-rich insertion sites that exist uniquely in the 3' nontranslated regions (3' NTR) of the genome of lapinized CSFV vaccine strains. By using a one-step RT-PCR or a nested PCR followed by an agarose gel electrophoresis or a multicapillary electrophoresis, the wild-type and lapinized vaccine strains of CSFV in clinical samples could be detected and accurately distinguished. These assays can be applied to at least 3 attenuated lapinized vaccine strains, lapinized Philippines Coronel (LPC), hog cholera lapinized virus (HCLV), and Chinese strain (C strain). The detection limit of the wild-type virus was 6.3 TCID(50) (50% tissue culture infective dose)/ml for RT-PCR and 0.63 TCID(50)/ml for nested PCR. In previous studies, notable T-rich insertions of 12-13 nucleotides (nt) were found in the 3' NTR of the genome of lapinized vaccine strains of CSFV. However, this study discovered that 2 T-rich insertions, 42 and 36 nt in length, are present in the viral genome of lapinized vaccine strains LPC/PRK (primary rabbit kidney) and LPC/TS (Tam-Sui), respectively. These T-rich insertions of 12, 36, and 42 nt length increases the size of PCR fragments, which are favorable genetic markers for rapid detection of and differentiation between wild-type and different lapinized vaccine strains of CSFV.

Examination for a viral co-factor in postweaning multisystemic wasting syndrome (PMWS)

In order to test the hypothesis that a putative co-factor for the development of postweaning multisystemic wasting syndrome (PMWS) in pigs could be of viral origin, we performed extensive virological examinations on organ material from pigs diagnosed with PMWS originating from within a Danish PMWS-transmission study. Virus isolation attempts were carried out on a large panel of different cell types including primary pig kidney cells and lung macrophages, primary rabbit kidney cells and seven established cell lines (MARC-145, ST117, PK15, BHK21, HeLa, Vero, and MDCK). Although these represent cells with susceptibility to a wide range of known viruses, the results did not provide evidence for a specific virus other than PCV2 contributing to the development of PMWS. Furthermore, in order to test whether specific genotypes of PCV2 may trigger the switch from PCV2 infection to clinical disease, we compared complete DNA genome sequences of PCV2 derived from PMWS-positive as well as PMWS-negative pigs. On the basis of the DNA sequences, the PCV2 isolates were divided into two groups. Group 1 consisting of one isolate originating from a herd unaffected by PMWS, with group 2 consisting of nine isolates originating from four PMWS-affected herds, four PMWS-positive pigs plus one unaffected herd. The PCV2 genomes from the two groups showed 95.5% identity. Alignment analyses of the sequences encoding the replicase and capsid protein from group 1 and group 2 PCV2 isolates showed two amino acid differences encoded in the replicase protein, while 19 amino acid differences were predicted among the capsid protein sequences. The PCV2 DNA sequence analysis supports recent observations from studies in USA as well as Europe, which suggest that strain variations may influence the clinical outcome of PCV2 infection.

Isophosphoramide Mustard-Lysine (ZIO-201): Active Moiety of Ifosfamide (IFOS) without Kidney Toxicity.

Ifosfamide (IFOS) and cyclophosphamide (CPA) are widely used anti-cancer drugs. Both are pro-drugs which must be metabolized before their metabolites cross-link DNA. Use of these drugs, especially high-dose IFOS, is complicated by substantial adverse effects of metabolites not directly involved in DNA-cross-linking including kidney, bladder and CNS toxicities. Recently, a lysine-stabilized form of isophosphoramide mustard (IPM-lysine; ZIO-201), an IFOS-metabolite, was developed. ZIO-201 directly cross-links DNA and is active against human cancer cell lines and in mice with human cancer xenografts. To evaluate nephrotoxicity of ZIO-201 we used an in vitro primary rabbit kidney proximal tubule (RPT) cell culture system, which retains many of the characteristics of renal proximal tubule cells including a polarized morphology, a Na+/glucose cotransport system and a p-aminohippurate transport system, glutathione status, and hormone responses (including a parathyroid hormone sensitive adenylate cyclase). We studied effects of ZIO-201 and other IFOS metabolites on primary RPT cells. IFOS is activated by the cytochrome P450 system producing a mixture of 2- and 3-dechloroethylifosfamide and resulting in formation of chloroacetaldehyde (CAA). Also, metabolism of IFOS by the cytochrome P450 system produces 4-hydroxyifosfamide, ultimately resulting in the production of acrolein (ACR). CAA and ACR are IFOS metabolites implicated in kidney toxicity in humans. Effects of ZIO-201 and CAA on the viability of confluent monolayers of primary rabbit kidney proximal tubule (RPT) cells were tested at 15–100 μM. Viability was determined by neutral red dye uptake by the primary cultures after 8 h incubation. ZIO-201 did not significantly reduce viability of primary RPT cells; the LD50 for CAA was about 40 μM with complete inhibition at 75 μM. Effects of 4-hydroperoxyifosfamide (HIFA), a synthetic prodrug of 4-hydroxyifosfamide and ACR were also studied. Although ACR had an LD50 of 80 μM HIFA did not significantly affect viability. The IFOS metabolite 4-hydroxyifosfamide, was prepared from 4-hydroperoxyifosfamide (1). At 200 μM, 4-hydroxyifosfamide resulted in extensive cell death after 4 h incubation. These data show that ZIO-201 avoids the kidney toxicity caused by metabolites of pro-drugs like IFOS. Whereas 2 IFOS metabolites, CAA and ACR were cytotoxic to kidney cells, ZIO-201 was not. An explanation for the toxic effects of 200 μM 4-hydroxyifosfamide t might be metabolism to ACR in vitro.

Primary kidney proximal tubule cells.

Primary rabbit kidney epithelial cell cultures can be obtained that express renal proximal tubule functions. Toward these ends, renal proximal tubules are purified from the rabbit kidney by the method of Brendel and Meezan. To summarize, each kidney is perfused with iron oxide, which becomes associated with glomeruli. The renal cortex is sliced and homogenized to liberate nephron segments. Renal proximal tubules and glomeruli are purified by sieving. The glomeruli, covered with iron oxide, are removed using a magnet. After a brief collagenase treatment (to disrupt basement membrane), the tubules are plated in hormonally defined serum-free medium supplemented with 5 micro g/mL bovine insulin, 5 microg/mL human transferrin, and 5 x 10(-8) M hydrocortisone. After 5-6 d of incubation, confluent monolayers are obtained that possess multicellular domes, indicative of their capacity for transepithelial solute transport.

Effects of TCDD and estradiol-17β on the proliferation and Na+/glucose cotransporter in renal proximal tubule cells

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is a highly toxic environmental toxicant that alters cell proliferation and function. Estrogens are noted for their ability to stimulate cell proliferation in various tissues. However, little is known about any interaction between TCDD and estradiol-17β (E2) that affects renal proximal tubule cell proliferation and Na+/glucose cotransporters' activity. Thus, the effects of TCDD and E2 on [3H]-thymidine incorporation and on α-methyl-d-glucopyranoside (α-MG) uptake were investigated in the primary rabbit kidney proximal tubule cells (PTCs). TCDD (>10−10 M >1 h) inhibited [3H]-thymidine incorporation and c-fos transcripts in real-time RT-PCR, whereas E2 (>10−9 M, 24 h) stimulated them. Aryl hydrocarbon receptor (AhR) agonists, β-naphthoflavone (β-NF) and polychlorinated biphenyls (PCBs) (10−6 M) synergistically increased the TCDD-induced inhibition of [3H]-thymidine incorporation. However, the AhR antagonist, α-naphthoflavone (α-NF) as well as E2 blocked TCDD-induced inhibition of [3H]-thymidine incorporation. TCDD (10−8 M, 48 h) specifically inhibited α-MG uptake and its effect was due to Vmax value but not Km value. Indeed, TCDD decreased Na+/glucose cotransporter 1, 2 (SGLT1, 2) protein level compared with control. In addition, TCDD-induced inhibition of α-MG uptake was blocked by α-NF or E2. In conclusion, TCDD inhibited [3H]-thymidine incorporation and α-MG uptake, and E2 blocked TCDDs effects in primary cultured renal proximal tubule cells.

Clonal Analysis of Immortalized Renal Proximal Tubule Cells: Na +/Glucose Cotransport System Levels Are Maintained Despite a Decline in Transport Function

Primary rabbit kidney proximal tubule (RPT) cells (S.D. Chung et al., 1982, J. Cell Biol. 95, 118–126) were transfected with the vector pRSV-T, which contains SV40 early region genes. After the third passage (when normal cells had stopped dividing), individual colonies formed in cultures transfected with pRSV-T. Clonal isolates (RPT-I cells) could be obtained in a simple and reproducible manner. Southern analysis of clone RPT-I8 indicated the presence of SV40 early region genes. Nuclear SV40 T was detected. After 23 passages, and subcloning, RPT-I8 (and subclones) was found to express renal proximal tubule markers to a similar extent, indicating that the phenotype was stable. Nevertheless, the activities of the Na +/glucose cotransport system, γ-glutamyl transpeptidase and alkaline phosphatase, were reduced as compared with primary cultures. Western analysis indicated that the level of Na +/glucose cotransporters was maintained in RPT-I8 cells, when compared with intact proximal tubules and primary cultures. Thus, the reduction in α-MG uptake in RPT-I8 cells may be attributed to other types of cellular alterations, including changes in energy metabolism. Indeed, growth in glucose-free medium was not observed in RPT-I8 cell cultures, suggesting that unlike primary RPT cells (J. C. Chung et al., 1992, J. Cell. Physiol. 150, 243–250), the gluconeogenic pathway was not intact.

Estradiol-17β Stimulates Phosphate Uptake and Is Mitogenic for Primary Rabbit Renal Proximal Tubule Cells

The direct effects of estradiol-17β (E₂) on phosphate (P_i) uptake and on DNA synthesis in the primary rabbit kidney proximal tubule cells (PTCs) have been investigated. In the present study, E₂ (>10^–9M, over 9 days) causes an increase both in [³H]thymidine incorporation and the number of PTCs. The anti-estrogen tamoxifen completely prevented the E₂-induced increase in [³H]thymidine incorporation, and ameliorated the stimulatory effect of E₂ on growth. E₂ (>10^–9M, over 5 days) also stimulated the P_i uptake and its effect was due to the V_max values but not to the K_m value for P_i uptake. Estriol and estrone also exerted significant stimulatory effects on P_i uptake. Progesterone, tamoxifen, actinomycin D and cycloheximide prevented the E₂-induced stimulation of P_i uptake. In conclusion, estrogens at physiological concentrations stimulate P_i uptake and DNA synthesis in the renal proximal tubule cells, and these effects are estrogen receptor mediated.

Estradiol-17beta-BSA stimulates Ca(2+) uptake through nongenomic pathways in primary rabbit kidney proximal tubule cells: involvement of cAMP and PKC.

The effect of estradiol-17beta-BSA (E(2)-BSA) on Ca(2+) uptake and its related signal pathways were examined in the primary cultured rabbit kidney proximal tubule cells. E(2)-BSA (10(-9) M) significantly stimulated Ca(2+) uptake from 2 h by 13% and at 8 h by 35% as compared to control, respectively. This stimulatory effect of E(2)-BSA was not inhibited by tamoxifen (10(-8) M, an intracellular estrogen receptor antagonist), actinomycin D (10(-7) M, a transcription inhibitor), and cycloheximide (4 x 10(-5) M, a protein synthesis inhibitor). However, E(2)-BSA-induced stimulation of Ca(2+) uptake was blocked by methoxyverapamil (10(-6) M, an L-type calcium channel blocker) and 5-(N-ethyl-N-isopropyl)-amiloride (10(-5) M, a Na(+)/H(+) antiporter blocker). These results suggest that E(2)-BSA stimulates Ca(2+) uptake through nongenomic pathways. Thus, we investigated which signal pathways were related to E(2)-BSA-induced stimulation of Ca(2+) uptake. 8-Br-cAMP (10(-6) M) alone increased Ca(2+) uptake by 22% compared to control. When E(2)-BSA combined with 8-Br-cAMP, Ca(2+) uptake was not significantly stimulated compared to E(2)-BSA. SQ 22536 (10(-6) M, an adenylate cyclase inhibitor) and myristoylated protein kinase A inhibitor amide 14-22 (10(-6) M, a protein kinase A inhibitor) blocked E(2)-BSA-induced stimulation of Ca(2+) uptake and E(2)-BSA also increased cAMP generation by 26% of that of control. In addition, TPA (0.02 ng/ml, an artificial PKC promoter) stimulated the Ca(2+) uptake by 14%, and the cotreatment of TPA and E(2)-BSA did not significantly stimulate Ca(2+) uptake compared to E(2)-BSA. E(2)-BSA-induced stimulation of Ca(2+) uptake was blocked by U 73122 (10(-6) M, a phospholipase C inhibitor) or bisindolylmaleimide I (10(-6) M, a protein kinase C inhibitor). Indeed, E(2)-BSA stimulated PKC activity by 26%. In conclusion, E(2)-BSA (10(-9) M) stimulated Ca(2+) uptake by nongenomic action, which is mediated by cAMP and PKC pathways.

Evaluation of ELVIS HSV ID/Typing System for the detection and typing of herpes simplex virus from clinical specimens.

Herpes simplex virus (HSV) is a common pathogen with two serotypes: HSV-1 and HSV-2. HSV infection does not pose much of a threat to an immunocompetent host but to an immunocompromised host or a neonate the infection can be fatal. The Enzyme-Linked Virus Inducible System (ELVIS) employs a genetically altered baby hamster kidney (BHK) cell line that allows for the rapid overnight detection of HSV but also includes an immunofluorescent stain for the simultaneous detection and typing of HSV-1 and HSV-2. To evaluate the ELVIS HSV ID/Typing System in comparison with HSV identification and typing in primary rabbit kidney (PRK) cells grown in shell vials. Over a period of 6 weeks, 130 specimens were submitted to the diagnostic virology laboratory and cultured for the presence of HSV. Two PRK shell vials and one ELVIS BHK shell vial were inoculated with patient specimen. PRK shell vials were observed for cytopathic effect (CPE) for up to 4 days. When CPE was observed the PRK shell vials were fixed and one shell vial was stained with HSV-1 monoclonal antibody (Mab) and the other was stained with HSV-2 Mab. The coverslips were observed under the fluorescent microscope for specific apple-green fluorescence. The BHK shell vials were incubated overnight, fixed, and stained with galactopyranoside (X-Gal). If blue cells were present, the specimen was positive for HSV. The coverslip was then observed under the fluorescent microscope for the presence of specific apple-green fluorescence, indicating HSV-2. If no specific apple-green stain was observed, the coverslip was stained with a fluorescent conjugated goat anti-mouse IgG to determine the presence of HSV-1. Of the 130 specimens, PRK shell vials detected 43 positive HSV; 30 were HSV-2 and 13 were HSV-1. The ELVIS BHK shell vials detected 42 positive HSV; 30 were HSV-2 and 12 were HSV-1. One low titer specimen was not identified as being HSV positive. Two specimens were not directly typed by the ELVIS system. One specimen had only one blue cell present and did not show specific staining for either HSV-1 or HSV-2. The other specimen had only five blue cells present and only one fluorescent cell present that was difficult to type. As suggested by the manufacturer's instructions, both specimens that were not directly typed were re-grown overnight from their supernatants and were correctly identified and typed. The ELVIS HSV ID/Typing System is a rapid, highly specific and sensitive method of overnight HSV detection and typing.

Response of primary rabbit kidney proximal tubule cells to estrogens

Response of primary rabbit kidney proximal tubule cells to estrogens

Response of primary rabbit kidney proximal tubule cells to estrogens.

The effects of estrogens on the growth and function of primary rabbit kidney proximal tubule (RPT) cells have been examined in hormonally defined phenol red-free medium. 17beta-estradiol was observed to stimulate growth at dosages as low as 10(-10) M. The growth stimulatory effects of 17beta-estradiol were mitigated in the presence of hydrocortisone, suggesting that these two steroid hormones acted at least in part by common mechanisms. The effects of other steroids known to interact with the estrogen receptor were examined. Alpha estradiol was found to be growth stimulatory over a concentration range of 10(-9) to 10(-8) M, albeit to a lower extent than beta estradiol. In addition, the anti-estrogen tamoxifen was also growth stimulatory (unlike the case with the human mammary tumor cell line MCF-7). The effects of several metabolic precursors of 17beta-estradiol were examined, including testosterone, which was growth stimulatory, and progesterone, which was growth inhibitory. The growth stimulatory effects of 17beta-estradiol, alpha estradiol, and tamoxifen could possibly be explained by their interaction with an estrogen receptor. Indeed, metabolic labelling and immunoprecipitation studies indicated the presence of such an estrogen receptor in the primary cultures. The rate of biosynthesis of the estrogen receptor was found to be affected by the presence of exogenously added 17beta-estradiol. 17beta-estradiol was also observed to increase the activity of two brush border enzymes, alkaline phosphatase and gamma glutamyl transpeptidase, during the growth phase of the primary cultures.

Effect of Complexes of Zinc, Cobalt and Copper With D‐Aminosugars on the Replication of Herpes Simplex Virus Type 1 (HSV‐1)

Our previous results show that Zn(pic)2 and Zn(asp)2 inhibit key steps of the replication of HSV-1. Anti-HSV effect of complexes of Co(II) with aminoacids Lys and Ser was also found. In the present study we describe the effect of complexes of Zn(II), Co(II) and Cu(II) with D-aminosugars on the replication of HSV-1 and on the infectivity of free virions. The experiments were done using primary rabbit kidney cells (r.k.), diploid human embryonal fibroblasts (F) and Vero cells. No differences in the toxicity of metal complexes on diploid cells- r.k. and F, were found. Neither metal complexes, nor ligands-galactosoxime and glucosoxime, influenced the viral replication. During 1-4h prolonged contact only Cu(Gl.NOH)2 inactivated HSV-1 virions up to 90%. The results show that D-aminosugars are not suitable ligands for Zn(II), Cu(II) and Co(II) in respect of the inhibition of viral replication. However, only Cu(Gl.NOH)2 was able to inhibit the infectivity of free virions.

A New Clerodane Diterpenoid Isolated from Propolis

Abstract A physiologically active substance has been isolated from Brazilian propolis and charac­terized as a new clerodane diterpenoid, as indicated by human hepatocellular carcinoma HuH 13 cell cytotoxicity assays. This compound inhibited growth of the hepatoma cells at a concentration around 10 μg/ml and arrested the tumor cells at S phase as revealed by flow cytometry. At higher concentrations it exerted lethal damage. The compound showed cyto­toxicity on human lung carcinoma HLC-2, HeLa, KB and rat W3Y cells, whereas human diploid foreskin and primary rabbit kidney cells were less affected.

Growth and function of primary rabbit kidney proximal tubule cells in glucose‐free serum‐free medium

The properties of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium have been examined. Primary rabbit kidney proximal tubule cells were observed to grow at the same rate, 1.0 doublings/day, both in glucose-free and in glucose-supplemented medium. Growth in glucose-free medium was dependent upon the presence of an additional nutritional supplement, such as glutamine, pyruvate, palmitate, lactate, or beta hydroxybutyrate. Lactate, pyruvate, and glutamate are utilized for renal gluconeogenesis in vivo. The growth of the primary rabbit kidney proximal tubule cells in glucose-free medium was also dependent upon the presence of the three growth supplements insulin, transferrin, and hydrocortisone. Insulin was growth stimulatory to the primary proximal tubule cells in glucose-free medium, although insulin causes a reduction in the phosphoenolpyruvate carboxykinase (PEPCK) activity in these cells. PEPCK is a key regulatory enzyme in the gluconeogenic pathway. In order to evaluate whether or not the primary cells have gluconeogenic capacity, their glucose content was determined. The cells contained 5 pmoles D-glucose/mg protein. However, no significant glucose was detected in the medium. Presumably, the primary cells were either utilizing or storing the glucose made by the gluconeogenic pathway. Consistent with this latter possibility, cellular glycogen levels were observed to increase with time in culture. The effect of glucose on the expression of the alpha I(IV) collagen and laminin B1 chain genes was examined. Northern analysis indicated that the level of alpha I(IV) collagen mRNA was significantly elevated in glucose containing, as compared with glucose deficient, medium. In contrast, laminin B1 chain mRNA levels were not significantly affected by the glucose content of the medium.

Growth characteristics and measurement of confluency of rabbit primary proximal tubule kidney cells grown on millicell-HA membrane filters

We have characterized primary rabbit kidney proximal tubule cells grown on Millipore cellulose filters (Millicell-HA) in which the cells are exposed to culture medium on both the apical and basolateral sides. Cells reach confluency 12 to 14 d after plating. Methods are described for removal of the cells from the filters and measurement of the integrity of the monolayer at confluency by exclusion of [14C]inulin.

Insulin and other regulatory factors modulate the growth and the phosphoenolpyruvate carboxykinase (PEPCK) activity of primary rabbit kidney proximal tubule cells in serum free medium

Insulin was observed to modulate the growth and the phosphoenolpyruvate carboxykinase (PEPCK) activity of primary cultures of rabbit renal proximal tubule cells in serum free medium. Insulin was stimulatory to primary proximal tubule cell growth at a concentration of 10(-8) M. In contrast, insulin was inhibitory to a proximal tubule function, PEPCK activity, following a 5-minute incubation period. An insulin dosage as low as 10(-10) M was inhibitory to PEPCK activity, suggesting the involvement of insulin receptors. Although insulin was required at a significantly higher dosage to stimulate the growth of the primary renal proximal tubule cells than to inhibit PEPCK activity, the elevated dosage required in order to observe a growth effect may be explained by the degradation of insulin by the primary renal proximal tubule cells. However the possible involvement of receptors for Insulin-like Growth Factor I (IGF-I) and Insulin-like Growth Factor II (IGF-II) in mediating the effects of insulin cannot be excluded. Other effector molecules were also examined with respect to their effects on PEPCK activity. The possible involvement of cyclic AMP in the control of the PEPCK activity of the primary renal cells was indicated by the stimulatory effects of 8 bromocyclic AMP, isobutyl methylxanthine (a cyclic AMP phosphodiesterase inhibitor), and forskolin (an activator of adenylate cyclase). Phorbol 12-myristate 13-acetate (TPA), which activates protein kinase C, was inhibitory. The actions of these effector molecules and insulin on the PEPCK activity of the primary renal cultures are remarkably similar to their effects on hepatic PEPCK. Several growth factors, fibroblast growth factor (FGF), and transforming growth factor beta (TGF beta) were also examined. FGF was observed to be stimulatory, whereas TGF beta was inhibitory to the PEPCK activity of the primary renal proximal tubule cells.

Herpes simplex virus antigen direct detection in standard virus transport medium by Du Pont Herpchek enzyme-linked immunosorbent assay

A commercial 5-h direct herpes simplex virus (HSV) antigen detection enzyme immunoassay kit (Du Pont Herpchek) was compared with a cell culture isolation system by using primary rabbit kidney and MRC-5 cells with 779 clinical specimens received in virus transport medium and with stock tissue culture preparations of HSV types 1 and 2. In the first study of 422 specimens from symptomatic patients, Herpchek detected 110 of 111 HSV-positive specimens (26.3% of all specimens), with a sensitivity of 99% and a specificity of 100%. In the second study of 357 specimens primarily from asymptomatic pregnant women, however, Herpchek detected 70 of 119 HSV-positive specimens (33% of all specimens), with a sensitivity of 58.8% and a specificity of 99.5%. Stock virus dilution experiments showed that Herpchek was 10 to 100 times less sensitive than culture. Herpchek was found to be an acceptable test for symptomatic patients, but for asymptomatic patients shedding a low titer of HSV it was not as sensitive and cell culture of Herpchek-negative specimens is recommended for such cases.