Mutant Tamm-Horsfall Glycoprotein Accumulation in Endoplasmic Reticulum Induces Apoptosis Reversed by Colchicine and Sodium 4-Phenylbutyrate

Construction of Wild-Type and Mutated UMOD Expression Vector

Wild-type (WT) UMOD cDNA were generated from a human kidney cDNA library by PCR and subcloned into pcDNA3 mammalian expression vector (Invitrogen, Carlsbad, CA). Mutant constructs were based on previously reported UMOD mutations (2). Mutant F1 that contains a 27-bp deletion that results in the in-frame deletion of amino acids 177 to 185 (g.1966_1992del; c.529_555del; p.H177_185del) and mutant F2 that contains a missense mutation that changes a conserved cysteine to tyrosine (g.1880G>A; c.443G.A; p.C148Y) were constructed by a mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol using mutant primer sets: F1 SS, 5′-GCGCGGATCCGTGTCAGGCGAGCACCGAGTACGGGGAGGG-3′; F1 AS, 5′-CCCTCCC CGTACTCGGTGCTCGCCTGACACGGATCCGCGC-3′; F2 SS, 5′-GGGGGATGGATGGC ACTATGAGTGCTCCCCGGG-3′; F2 AS, 5′-CCCGGGGAGCACTCATAGTGCC ATCCATC CCCC-3′.

Selection of Stably Transfected Clones

Human embryonic kidney 293 (HEK293) cells (American Type Culture Collection, Rockville, MD) were cultured in DMEM supplemented with 10% FBS and standard antibiotics in a 5% CO₂ atmosphere at 37°C. An immortalized TALH cell line derived from kidneys of New Zealand white rabbits was used (17). The TALH cell line was maintained as a monolayer culture in DMEM supplemented with 10% FBS and standard antibiotics in a 5% CO₂ atmosphere at 37°C. Transfection was performed using lipofectamine reagents (Invitrogen) according to the manufacturer’s protocol. Stably transfected cells were selected by culturing in the presence of DMEM media supplemented with 10% FBS, standard antibiotics, and 0.75 mg/ml G418 in a 5% CO₂ atmosphere at 37°C for 2 wk. For determining the capacity of THP expression, cells were cultured in a 24-well plate with 80% confluence, washed, and incubated with serum-free DMEM media that contained antibiotics for 24 to 72 h. Expressed THP was determined with the conditioned media by Western blot.

Cell Culture, THP Expression, and Effect of Chemical Agents

Stably transfected cells were cultured for 24 h in DMEM media that contained 10% FBS, antibiotics, and 0.375 mg/ml G418. The cells were washed (×3) with serum-free DMEM media that contained antibiotics and further incubated with the same media for 24 to 72 h. Secreted THP in the conditioned media was determined by Western blot and ELISA. For evaluating chemical agents, the cells were incubated in a serum-free medium supplemented with colchicine, sodium 4-phenylbutyrate (4-PBA; Calbiochem, San Diego, CA), and allopurinol (Sigma, St. Louis, MO) at concentration ranges of 0.001 to 1 mM, 1 to 10 mM, and 0.001 to 5 mM, respectively.

Western Blot and ELISA for THP

The conditioned media were harvested and concentrated with cold ethanol. Incubated cells were lysed in lysis buffer (20 mM Tris-HCl [pH 8.0], 1 mM PMSF, 1 mM 4-[2-aminoethyl] benzenesulfonyl fluoride, 10 μM E-64, 100 μM bestatin, 100 μM leupeptin, 100 μM aprotinin, and 5 mM EDTA). Proteins were quantified by using the Bio-Rad Proteins Assay (Bio-Rad, Hercules, CA), and equal amounts of proteins were loaded. For Western blotting, samples were separated by SDS-PAGE in reducing conditions and transferred to nitrocellulose. After blocking with 5% skim milk, membranes were incubated overnight at 4°C with sheep polyclonal primary antibody against human THP (Chemicon International, Inc., Temecula, CA; 1:2000 dilution), followed by the incubation of AP-conjugated rabbit secondary antibody against sheep (Chemicon; 1:5000 dilution) for 1 h at room temperature and visualized with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad). β-Actin (Abcam, Cambridge, MA) was used as an internal control for equal loading.

An ELISA was established with a precoated-antibody (rabbit anti-human THP antibody; Santa Cruz Biotechnology, Santa Cruz, CA; 0.5 μg/ml biocarbonate buffer [pH 8.4]; 1:200 dilution) bound to the solid phase, a primary antibody (sheep anti-human THP polyclonal antibody; Chemicon; 1:2000 dilution), a secondary antibody (AP-conjugated rabbit anti-sheep antibody; Chemicon; 1:5000 dilution), and an alkaline phosphatase liquid substrate system (Sigma). Urinary THP was purified by the salt-precipitation method (18).

Deglycosylation of THP

For differentiating THP precursor and mature forms, oligosaccharide cleavage was evaluated with two different deglycosidases, N-glycosidase F (PNGase F; New England Biolab, Beverly, MA) and endo-β-N-acetylglucosaminidase H (Endo H; New England Biolab) according to the manufacturer’s protocol.

Immunocytochemistry

Stably transfected WT and mutant THP-expressing HEK293 and TALH cells were grown on eight-well chamber slides, fixed with 100% cold methanol (−20°C) for 10 min at room temperature, and washed with PBS. Nonspecific antibody binding was blocked by incubating for 1 h with PBS that contained 5% normal goat serum and 1% BSA for 1 h. Cells then were incubated with sheep anti-human THP polyclonal antibody (Chemicon; 1:200 dilution) in PBS that contained 1% normal goat serum and 1% BSA and followed by incubation with FITC-conjugated donkey anti-sheep IgG antibody (Chemicon; 1:200 dilution) in PBS that contained 1% normal goat serum and 1% BSA. Immunocytochemical signals were visualized and photographed with a ×400 magnification using a fluorescence microscope (Olympus 1X71; Olympus, Melville, NY).

FACS Analysis for Apoptosis Assay

Apoptotic cells were measured using annexin V-FITC and propidium iodide staining (BD Bioscience, San Jose, CA) with FACS analysis. Cell sample preparation, Annexin V, and propidium iodide staining for FACS analysis were performed according to the manufacturer’s instructions. The percentage of apoptotic cells was determined by the fluorescence of individual cells measured by FACS flow cytometry (Becton Dickinson, San Jose, CA).

Evaluation of Cell Viability

WT and two mutant cell clones (1 × 10⁶) were inoculated in each well of a six-well plate and cultured in DMEM supplemented with 10% FBS and standard antibiotics in a 5% CO₂ atmosphere at 37°C for 24 h. The cells were incubated further in a serum-free DMEM medium in the absence and presence of 0.5 mM colchicine, 10 mM 4-PBA, and 5 mM allopurinol. After 24- to 72-h incubations, cells were detached from a plate using a cell scraper, suspended in PBS, and stained with trypan blue solution. Viable cells, which were resistant to trypan blue staining, were counted using a hemacytometer under light microscopy (Olympus 1X71). Viability was calculated with 100% being the viable cell number at 0 h incubation. Data were obtained from three individual experiments.

THP Excretion from HEK 293 and TALH Cells Transfected with Mutant UMOD Genes Is Significantly Reduced

The WT and the two mutant (F1 and F2) clones expressed THP with an apparent molecular weight (MW) of 86 kD, the same MW as urinary THP (Figure 1). HEK293 and TALH cells do not express endogenous THP. Negative controls and vector or enhanced green fluorescent protein (EGFP) transfected cells did not express THP. ELISA quantification of THP expressed in conditioned media indicated that mutant clones excreted significantly less THP. Compared with WT THP-expressing HEK293 cells (54.7 ± 1.2 ng/mg protein), THP expression was decreased by approximately 70% for F1 (16.0 ± 2.0 ng/mg protein) and decreased by approximately 77% for F2 (12.7 ± 1.2 ng/mg protein). Compared with WT THP-expressing TALH cells (14.0 ± 1.0 ng/mg protein), THP expression was decreased by approximately 73% for F1 (3.7 ± 1.2 ng/mg protein) and decreased by approximately 80% for F2 (2.7 ± 0.6 ng/mg protein). These results were reproducible in six independent clones for each WT, F1, and F2.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Expression of Tamm-Horsfall glycoprotein (THP) from human embryonic kidney 293 (HEK293; A) and thick ascending limb of Henle’s loop (TALH) cells (B) that were stably transfected with wild-type (WT) and mutant uromodulin (UMOD) genes. (a) Western blot of the conditioned media of HEK293 and TALH cell culture. After WT and two mutant UMOD constructs (F1 containing c.529_555 del and F2 containing c.443G>A) were stably transfected into HEK293 and TALH cells, the cultured cells were incubated with serum-free DMEM media for 24 h, and the conditioned media were tested for THP expression by Western blot. HEK293 and TALH cells that were transfected with expression vector pCDNA3 alone (EV) or enhanced green fluorescent protein (EGFP) were used as negative and transfection efficiency controls. (b) Secreted THP in the conditioned media was measured by ELISA. The amount of secreted THP was expressed as mean ± SD from triplicate experiments. The amount of THP in HEK293 cells was WT, 54.7 ± 1.2; F1, 16.0 ± 2.0; F2, 12.7 ± 1.2 ng/mg protein per 24 h (**P < 0.005). The amount of THP in TALH cells was WT, 14.0 ± 1.0; F1, 3.7 ± 1.2; F2, 2.7 ± 0.6 ng/mg protein per 24 h (**P < 0.005). These results were reproducible in six independent clones for each WT, F1, and F2.

Accumulation of Mutant THP within ER

Two MW intracellular THP, 75 kD and 86 kD, were detected from WT and mutant HEK293 cells (Figure 2A). Although the ratio of 75 to 86 kD was similar in the WT cell, the amount of the 86-kD forms was reduced in both mutant cells. To characterize the oligosaccharide modifications of the two different intracellular THP, they were treated with deglycosidases PNGase F and Endo H. PNGase F hydrolyzes nearly all types of N-linked oligosaccharides of glycoproteins, from both the ER and the Golgi. In contrast, Endo H cleaves only high-mannose glycoproteins from the ER but does not cleave glycoproteins modified in the Golgi. Deglycosylation experiments showed that both the 75- and the 86-kD forms were cleaved by PNGase (Figure 2B). Whereas Endo H cleaved the 75-kD THP, the 86-kD form was resistant to Endo H digestion, indicating that the protein was a terminally glycosylated precursor in the Golgi apparatus. These results suggest that the 75-kD THP exists as a precursor in the ER and is exported into the Golgi apparatus for further oligosaccharide processing and becomes the mature 86-kD form that is excreted into the conditioned media. Similar results were obtained for TAL cells (data not shown).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

Retention of mutant THP in endoplasmic reticulum (ER) of HEK293 cells. (A) Detection of intracellular and extracellular THP. WT and mutant (F1 and F2) THP-expressing cells were incubated with serum-free DMEM media for 24 h, and expressed THP isoforms were determined with cell extracts or conditioned media by Western blot. The expression of β-actin is shown as a control for protein loading. Intracellular THP consisted of two forms, 75 and 86 kD, in which the latter is the same size as extracellular THP. The amount of 86-kD THP was dramatically reduced in the two mutant cells. (B) Deglycosylation of THP isoforms. The samples from cell extract and culture media were treated with N-glycosidase F (PNGase F) or endo-β-N-acetylglucosaminidase H (Endo H) as described in the Materials and Methods sections, and changes of molecular weight were visualized by Western blot. Both intracellular 75- and 86-kD isoforms were cleaved by PNGase and migrated to 60 kD. The 86-kD form was resistant to the Endo H digestion. These data indicate the 75-kD form is a THP precursor in the ER and the 86-kD form is a mature form with oligosaccharides modified in the Golgi apparatus, consistent with delay of mutant THP transport to the Golgi.

Comparison of Apoptosis between WT and Mutant THP-Expressing HEK293 and TALH Cells

Because the accumulation of misfolded proteins in the ER has been reported to induce programmed cell death (11), we measured apoptosis by labeling THP-expressing HEK293 and TALH cells with annexin V-FITC followed by FACS analysis (Figure 3). FACS data showed more early apoptotic signal in mutant cells (Figure 3, b and c) than in WT cells (Figure 3a), 3.7 or 3.5 versus 0.8% in HEK293 cells (Figure 3A), and 3.6 or 5.1 versus 1.4% in TALH cells (Figure 3B), respectively. The percentage of cells in late apoptosis was also higher in both mutant cells than in the WT cells. These data indicate that the accumulation of mutant THP in the ER is associated with significantly greater apoptosis.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3.

Determination of apoptosis of WT (a), mutant F1 (b), and F2 (c) THP-expressing HEK293 (A) and TALH cells (B) by annexin V labeling followed by FACS analysis. WT and mutant cells (1 × 10⁶) were stained with annexin V–FITC and propidium iodide. The percentage of cells that underwent apoptosis was measured quantitatively using FACS flow cytometry. Mutant THP-expressing HEK293 cells show significantly greater early apoptotic signal (right bottom) than WT cells (3.7% [F1] versus 3.5% [F2] versus 0.8% [WT]). The percentage of end-stage apoptotic cells (right top) was also higher in the two mutant cells than in WT cells (5.3% [F1] versus 5.2% [F2] versus 2.7% [WT]). Mutant THP-expressing TALH cells also show significantly greater early apoptotic signal (right bottom) than WT cells (3.6% [F1] versus 5.1% [F2] versus 1.4% [WT]). The percentage of end-stage apoptotic cells (right top) was also higher in the mutant cells than in WT cells (17.6% [F1] versus 15.3% [F2] versus 10.5% [WT]). A similar pattern of results was seen in three independent clones for each WT, F1, and F2, respectively.

Effects of Chemical Agents on Mutant THP Release from Cells

Chemical chaperones have been shown to rescue misfolded proteins from the ER. We examined the effect of the chemical chaperone sodium 4-PBA as well as colchicine and allopurinol on THP excretion using ELISA (Figure 4). THP secretion in both WT and mutant cells was increased by treatment of 0.001 to 1.0 mM colchicine and by 5 to 10 mM 4-PBA. At the optimal colchicine concentration of 0.5 mM, THP excretion was increased by 6.4-, 11.2-, and 9.0-fold for WT, F1, and F2 mutant THP-expressing HEK293 cells, respectively (Figure 4Aa). Treatment with 10 mM 4-PBA increased THP excretion by 4.1-, 3.9-, and 4.6-fold for WT, F1, and F2 mutant THP-expressing HEK293 cells, respectively (Figure 4Ab). At the optimal colchicine concentration of 0.5 mM, THP excretion was increased by 6.1-, 12.2-, and 10.6-fold for WT, F1, and F2 mutant THP-expressing TALH cells, respectively (Figure 4Ba). Treatment with 10 mM 4-PBA increased THP excretion by 3.4-, 3.8-, and 4.0-fold for WT, F1, and F2 mutant THP-expressing TALH cells, respectively (Figure 4Bb). In contrast, allopurinol tested at concentrations ranging from 0.001 to 5 mM did not affect THP secretion (Figure 4c).

• Download figure
• Open in new tab
• Download powerpoint

Figure 4.

Effect on THP secretion by colchicine (a), sodium 4-phenylbutyrate (4-PBA; b), and allopurinol (c) in THP-expressing HEK293 (A) and TALH cells (B). WT and mutant THP-expressing HEK293 and TALH cells were cultured for 24 h and incubated further for 24 h in the serum-free DMEM media in the absence or presence of various concentrations of colchicine, 4-PBA, and allopurinol. The secreted THP in the conditioned media was measured by ELISA. THP secretion was increased by treatment of 0.001 to 1 mM colchicine or 5 to 10 mM 4-PBA for both WT and the two mutant expressing cells.

Chemical Agents Facilitate Movement of Mutant THP from Cytoplasm to Plasma Membrane

As colchicine and 4-PBA increased the excretion of mutant THP into conditioned media, we performed additional immunologic assays to study their effect on intracellular localization of THP in HEK293 and TALH cells. In WT transfected cells, THP localized predominantly to the cell membrane (Figure 5a), whereas in HEK293 and TALH cells that were stably transfected with mutant F1 and F2, THP localization was mainly cytoplasmic (Figure 5, d and g), consistent with accumulation of mutant protein in the ER. When mutant cells were incubated with either 0.5 mM colchicine or 10 mM 4-PBA, THP signal was reduced in cytoplasm and increased at plasma membrane.

• Download figure
• Open in new tab
• Download powerpoint

Figure 5.

Cellular localization of THP before and after treatment with chemical agents. WT and mutant THP-expressing HEK293 (A) and TALH cells (B) were grown and incubated for 24 h in serum-free DMEM media in the absence or presence of 0.5 mM colchicine or 10 mM 4-PBA. THP was labeled with sheep anti-THP antibody and FITC-conjugated anti-sheep IgG antibody, and cellular localization was observed under a fluorescence microscope. Without treatment with chemical agents, THP signal was most intense in the plasma membrane for WT cells (a) and in the cytoplasm for F1 (d) and F2 (g) cells. When mutant cells were incubated with 0.5 mM colchicine (F1 + colchicine [b]; F2 + colchicine [h]) or 10 mM 4-PBA (F1 + 4-PBA [c]; F2 + 4-PBA [f]), THP signal was reduced in the cytoplasm and increased at the plasma membrane. Negative control mock-HEK293 and TALH cells are shown in j.

Effects of Chemical Agents on Cell Viability

Previous results in HEK293 and TALH cells indicated that mutant THP in the ER induces apoptosis. Treatment of these cells with colchicine and 4-PBA facilitated release of mutant THP from the ER to the plasma membrane and out of the cell. To evaluate whether treatment with chemical agents affected the rate of apoptosis in cells, we evaluated cell viability (Figure 6). Compared with WT cells, viability of the two mutant cells rapidly decreased, with the extent more severe in F2 than F1. After 72 h of incubation without chemical agent treatment, viability was reduced to 79.1, 51.2, and 25.6% for WT, F1, and F2 THP-expressing HEK293 cells, respectively (Figure 6A). After 72 h of incubation without chemical agent treatment, viability was reduced to 74.5, 48.8, and 25.6% for WT, F1, and F2 THP-expressing TALH cells, respectively (Figure 6B). In the presence of 0.5 mM colchicine or 10 mM 4-PBA, cell viability was improved for WT cells and greatly improved for mutant cells. With treatment, viability in the mutant cells recovered to the level of WT cells without treatment. In contrast, 5 mM allopurinol did not change the cell viability for WT and mutant cells (Figure 6).

• Download figure
• Open in new tab
• Download powerpoint

Figure 6.

Effect of chemical agents on cell viability. WT and mutant THP-expressing HEK293 (A) and TALH cells (B) were cultured for 24 h and incubated further for up to 72 h in the serum-free DMEM in the absence or presence of 0.5 mM colchicine, 10 mM sodium 4-PBA, or 5 mM allopurinol. Viable cells were determined by trypan blue staining at 24, 48, and 72 h of incubation. Viability was calculated with 100% as the viable cell number of 0 h incubation. Data were obtained from three individual experiments. At 72 h of incubation without chemical treatment, viability was reduced to 79.1, 51.2, and 25.6% for WT, F1, and F2 THP-expressing HEK293 cells, respectively (a). In the presence of 0.5 mM colchicine or 10 mM 4-PBA, the viability was improved to 90.9, 90.9, and 74.5% for WT (b), F1 (c), and F2 (d) mutant THP-expressing HEK293 cells. At 72 h of incubation without chemical treatment, viability was reduced to 74.5, 48.8, and 25.6% for WT, F1, and F2 THP-expressing TALH cells, respectively (a). In the presence of 0.5 mM colchicine or 10 mM 4-PBA, the viability was improved to 90.9, 83.4, and 67.5% for WT (b), F1 (c), and F2 (d) mutant THP-expressing TALH cells. In contrast, 5 mM allopurinol did not significantly influence in cell viability.

Effects of the chemical agents (colchicine and 4-PBA) on cell apoptosis were evaluated with FACS analysis (Table 1). FACS data demonstrated that more early apoptotic signal was shown in cells without treatment than in cells with chemical treatment. The percentage of cells in late apoptosis was also higher in cells without treatment than in cells with treatment.