Effect of Hypothalamic-Hypophysary Inhibitory Factor on Mesangial Cell Activation

Collagenase type IA from Clostridium histolyticum, TMB-8, l-glutamine, sodium selenite, transferrin and insulin (human), and fura 2–AM were purchased from Sigma Chemical Co. Penicillin was obtained from Laboratorios Level SA. Streptomycin sulfate was obtained from Antibioticos SA. RPMI 1640, Hanks’ balanced salt solution, trypsin-EDTA solution, and FCS were obtained from Whittaker Laboratories. [³H]Thymidine and ⁸⁶Rb were purchased from New England Nuclear. Verapamil was a gift of Knoll AG. Wistar rats were purchased from Charles River (Rouan, France). All other reagents were of the highest grade commercially available.

HHIF was extracted from 1 kg frozen bovine hypothalamus and hypophysis, purified following chromatographic procedures, and physicochemically characterized as previously reported.¹¹ The purified material has chemical and chromatographic characteristics different from any known cardioglycoside.^{11 12} Each bath of purified material was tested for purity as previously described.^{11 12 13} In essence, the homogeneous material underwent chromatography in two different chromatographic systems,¹² and we again obtained a single, symmetrical peak with an identical spectrum and biological activity as the one of the purified material, suggesting the purity of the final product. This purified factor revealed by mass spectrometric analysis a single unique molecular ion with an accurate mass of 412 277 and mass spectra different from ouabain (unpublished data, 1995). Its ability to inhibit Na,K-ATPase from porcine renal outer medulla has been tested for each batch as previously reported,¹¹ and 1 U was defined as the amount of HHIF required to inhibit the activity of 8 μg purified Na,K-ATPase by 50%.

Glomeruli isolated by successive mechanical sieving (150 and 50 μm) from Wistar rats (150 to 200 g) previously anesthetized with ether were treated with 300 U/mL collagenase, plated in 25-mm² plastic tissue culture bottles (Nunc), and maintained under previously described conditions.^{17 18} The culture medium consisted of RPMI 1640 supplemented with 0.1 FCS, l-glutamine (1 mmol/L), penicillin (50 IU/mL), and streptomycin sulfate (0.034 mmol/L) and was buffered with bicarbonate. This culture medium was changed every 2 days. Studies were performed on day 21 or 22. Characterization of culture cells as mesangial cells was confirmed by morphological and functional criteria, strellate aspect, absence of factor VII, contraction with angiotensin II, and absence of growth inhibition with the substitution of l-valine for d-valine, described previously.^{17 18} Experiments were performed on cultures approaching confluence from the first passage in order to avoid cell dedifferentiation.

Cells were subcultured by treatment with 0.05% trypsin and 0.02% EDTA and plated in 6×4-well plates (Nunc). Cells were incubated with agonists at 37°C over 10 minutes and then pulsed with 1 μCi/mL ⁸⁶Rb (0.5 mmol/L) during 15 minutes. The cell layers were washed three times with ice-cold culture medium and fixed with 0.5 mL ice-cold 0.1 trichloroacetic acid; acid-insoluble material was solubilized with 0.5 mL of 0.1 mol/L NaOH for 30 minutes at 60°C. After NaOH exposure cells were scraped off and removed; radioactivity was measured on a gamma scintillation counter (Gammamatic 1, Kontron Instruments).

Direct observation of mesangial cells in plastic flasks was carried out at room temperature under phase contrast with an inverted Nikon photomicroscope with the use of a CCD videocamera (model KP 110, Hitachi Denshi Ltd) and monitor (model VM-921E, Hitachi). With the use of an on-line videoprinter (Sony UP-910) serial photographs of the cells were taken before and after the addition of test substances. Five to 10 cells were analyzed per photograph. In each experimental block all the cells were from a single culture. Experiments were done in triplicate. Planar surface was determined by computerized image analysis with an IBAS II image-analyzer system (Kontron Instruments). Actual areas were calculated after correction for microscope and photographic magnification.

Measurement of [Ca²⁺]_i was performed as previously reported.¹⁹ Fluorescence was measured at 37°C with a fluorescence spectrophotometer provided with a thermostatically controlled cuvette holder (Perkin-Elmer LS50) at an emission wavelength of 510 nm. An excitation wavelength of 340 nm was chosen for monitoring of the Ca²⁺-induced shift in fura 2 fluorescence as determined by previous calibration determinations. Autofluorescence, as measured in cells under similar conditions except that they were not loaded with fura 2, was found to be less than 10% of the total fluorescence determined in fura 2–loaded cells in all the experiments. [Ca²⁺]_i was calculated as described by Olivera and López-Novoa.¹⁹

Cell proliferation was measured by [³H]thymidine uptake into DNA and by measurement of viable cell numbers. For this purpose cells were subcultured by treatment with 0.05% trypsin and 0.02% EDTA and plated in 6×4-well plates. Mesangial cells were rendered quiescent by removal of the serum-containing growth medium. Cells were incubated for 2 days with similar culture medium except that it contained a low amount (0.005) of FCS. During this time the cells were observed to incorporate a very low amount of [methyl-³H]thymidine, indicating the quiescent state. At this point the cells were reactivated by exposure to culture medium containing the substances to be tested. The conditions tested included cell exposure to quiescent medium alone (basal conditions); 0.2, 0.02, and 0.002 U/mL HHIF; 10⁻⁵ mol/L verapamil; 10⁻⁵ mol/L TMB-8; and medium containing 0.1 FCS as a positive control. After 18 hours of incubation the cells were pulsed for 6 hours with 1 μCi/mL [methyl-³H]thymidine. Cell layers were washed with ice-cold phosphate-buffered saline and fixed with 1 mL ice-cold 0.1 trichloroacetic acid for 15 minutes; acid-insoluble material was solubilized with 1 mL of 0.1 mol/L NaOH for 30 minutes at 60°C. After NaOH exposure radioactivity was determined on a scintillation counter (Betamatic IV, Kontron Instruments).

We also checked the number of viable cells in each well using a commercial modification (Cell Titter 96, Promega) of the method of Hansen et al.²⁰ In brief, cells subcultured to subconfluence in 24-well plates were rendered quiescent as described above. Cells were then incubated for 24 hours with culture medium and the substances described above. The medium was removed and 1 mL fresh incubation medium was added. Then, 150 μL of a solution of tetrazolium bromide salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL] was added to each well. The plate was returned to the incubator (Ultima; Revco Scientific, Inc) for 4 hours, after which 1 mL of the solubilization solution containing sodium dodecyl sulfate and N,N-dimethyl formamide, pH 4.7, was added. After 1 hour at room temperature the contents of the well were mixed thoroughly with a Pasteur pipette to obtain a uniform color. The contents of the wells were then transferred to a cuvette and read at 570 nm on a spectrophotometer (Shimadzu UV-120-02). Optical density at 570 nm was proportional to the number of viable cells in each well.⁵

Mesangial cells were rendered quiescent by removal of the serum-containing growth medium and incubation for 3 days with similar culture medium without FCS. Cells were treated with 0.2 U/mL HHIF, and cells treated with 0.1 FCS were used as positive controls. After 30 minutes of stimulation mRNA was extracted from 5×10⁵ cells by NP-40 lysis.²¹ The RNA (1 to 2 μg) was subjected to a reverse transcription reaction with the use of oligo(dT) primers (Promega). cDNA was amplified by PCR with oligonucleotides derived from two adjacent exons of the actin gene, c-fos gene, and c-jun gene with the use of a thermal cycler (Dual Cycler, Linus) and amplitaq DNA polymerase.²² Oligonucleotides were synthesized at the Department of Microbiology and Genetics, University of Salamanca, and their sequences were as follows: FOS 1: 5′-CAGCCGACTCCTTCTCCAG-3′; FOS 2: 5′-GCCACGGAGGAGACCAGAGT-3′; JUN 1: 5′-GACCTTCTACGACGATGC-3′; JUN 2: 5′-CAGCGCCAGCTACTGAGGC-3′; ACT 1: 5′-AGGCCAACCGCGAGAAGATGACC-3′; and ACT 2: 5′-GAAGTCCAGGGCGACGTAGCAC-3′.

The amplification conditions consisted of denaturing at 94°C for 2 minutes, annealing at 60°C for 2 minutes, and extension at 72°C for 2 minutes. The number of cycles was 25. PCR products were size-fractionated by agarose gel electrophoresis. After staining with ethidium bromide, DNA bands were visualized with a UV transilluminator (Vilber Lourmat TF-35M).

In proliferation studies each value is the mean of three wells. Data were obtained as the average of 4 to 12 values. Comparisons between means of multiple groups were analyzed by one-way ANOVA and Scheffé’s multiple comparisons test. Time course studies were analyzed by two-way ANOVA.

HHIF decreased ⁸⁶Rb uptake by mesangial cells in a concentration-dependent manner. Inhibition with the highest concentration of HHIF used (0.2 U/mL) was stronger than that with 10⁻⁴ mol/L ouabain, a submaximal concentration used to compare the effect of HHIF with a well-known Na⁺-pump inhibitor (Table).

After 60 minutes of incubation HHIF decreased mesangial planar cell surface area. This effect was concentration- and time-dependent (Figs 1 and 2). By contrast, no significant changes were observed at that time in cells incubated under control conditions.

Preincubation of mesangial cells with the calcium antagonist verapamil (10⁻⁵ mol/L), a voltage-dependent calcium channel inhibitor, significantly blunted the contractile response to HHIF (0.2 U/mL). In addition, TMB-8 (10⁻⁵ mol/L), an inhibitor of Ca²⁺ release from the endoplasmic reticulum, also blunted the response to HHIF (Fig 2).

HHIF induced a slow and progressive increase in [Ca²⁺]_i from resting levels in a concentration-dependent manner (Fig 3). The lowest concentration used in our experiments (0.002 U/mL) did not produce any change in [Ca²⁺]_i, but higher concentrations (0.02 and 0.2 U/mL) induced a statistically significant increase in [Ca²⁺]_i. This increase was time dependent, with a maximal increase in [Ca²⁺]_i of approximately twofold the basal values at 10 minutes (Fig 3). Verapamil (10⁻⁵ mol/L) and TMB-8 (10⁻⁵ mol/L) blunted the response to HHIF (0.2 U/mL) (Fig 4).

HHIF also stimulated [³H]thymidine incorporation into DNA. This effect was significant at concentrations of 0.2 and 0.02 U/mL (Fig 5A). Verapamil (10⁻⁵ mol/L) abolished HHIF (0.02 and 0.2 U/mL)–stimulated thymidine incorporation into DNA (Fig 6). TMB-8 (10⁻⁵ mol/L) also blocked the HHIF-induced effect on thymidine incorporation into DNA (Fig 6).

A dose-dependent increase in the number of viable cells after 24 hours of incubation in the presence of HHIF was observed (Fig 5B). This effect was partially inhibited by verapamil (10⁻⁵ mol/L) and TMB-8 (10⁻⁵ mol/L) (Fig 7).

Fig 8 shows representative agarose gel electrophoresis of amplification products by reverse transcriptase–PCR of c-fos, c-jun, and actin genes. In cells treated for 30 minutes with 0.1 FCS, cDNA amplification gave a band of approximately 195 bp, which is consistent with the calculated size of the c-fos gene (Fig 8A, lane 2). Treatment with 0.2 U/mL HHIF induced the expression of c-fos mRNA (Fig 8A, lane 3); this expression was not observed in quiescent cells (Fig 8A, lane 1). A band of approximately 310 bp, corresponding to the calculated size of the actin gene, appeared with all treatments (Fig 8).

Expression of the c-jun gene (440 bp) (Fig 8B) was observed in cells incubated with 0.2 U/mL HHIF (Fig 8B, lane 3) and in cells treated with 0.1 FCS (Fig 8B, lane 2) but not in quiescent cells.