Mutant Tamm-Horsfall Glycoprotein Accumulation in Endoplasmic Reticulum Induces Apoptosis Reversed by Colchicine and Sodium 4-Phenylbutyrate
Sung Won Choi*,
Ok Hee Ryu*,
Sun Jin Choi*,
In Sun Song*,
Anthony J. Bleyer and
Thomas C. Hart*
* Human Craniofacial Genetics Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland; and Section on Nephrology, Wake Forest University School of Medicine, Winston-Salem, North Carolina
Address correspondence to: Dr. Thomas C. Hart, NIH-NIDCR, 10 Center Drive, Building 10, Room 5-2531, Bethesda, MD 20892-1432. Phone: 301-402-1706; Fax: 301-480-4455; E-mail: thart{at}mail.nih.gov
Received for publication May 4, 2005.
Accepted for publication July 18, 2005.
As a consequence of uromodulin gene mutations, individuals developprecocious hyperuricemia, gout, and progressive renal failure.In vitro studies suggest that pathologic accumulation of uromodulin/Tamm-Horsfallglycoprotein (THP) occurs in the endoplasmic reticulum (ER),but the pathophysiology of renal damage is unclear. It was hypothesizedthat programmed cell death triggered by accumulation of misfoldedTHP in the ER causes progressive renal disease. Stably transfectedhuman embryonic kidney 293 cells and immortalized thick ascendinglimb of Henle’s loop cells with wild-type and mutateduromodulin cDNA were evaluated to test this hypothesis. Immunocytochemistry,ELISA, and deglycosylation studies indicated that accumulationof mutant THP occurred in the ER. FACS analyses showed a significantincrease in early apoptosis signal in human embryonic kidney293 and thick ascending limb of Henle’s loop cells thatwere transfected with mutant uromodulin constructs. Colchicineand sodium 4-phenylbutyrate treatment increased secretion ofTHP from the ER to the cell membrane and into the culture mediaand significantly improved cell viability. These findings indicatethat intracellular accumulation of THP facilitates apoptosisand that this may provide the pathologic mechanism responsiblefor the progressive renal damage associated with uromodulingene mutations. Colchicine and sodium 4-phenylbutyrate reversethese processes and could potentially be beneficial in amelioratingthe progressive renal damage in uromodulin-associated kidneydiseases.
Mutations in the uromodulin (UMOD) gene result in an autosomaldominantly transmitted syndrome characterized by precocioushyperuricemia, gout, and progressive kidney failure frequentlyleading to dialysis (1–3). This condition has been termedfamilial juvenile hyperuricemic nephropathy (FJHN), medullarycystic kidney disease type 2 (MCKD-2), and glomerulocystic kidneydisease (2,4). Most UMOD mutations involve alteration of highlyconserved cysteine residues that compromise processing of uromodulin/TammHorsfall glycoprotein (THP) (3,5). Uromodulin and THP are twolarge glycoproteins with an identical amino acid sequence, andboth are a product of the UMOD gene. Uromodulin was isolatedfrom the urine of pregnant women and has been found to havea small increase in Man7GlcNac2 molar percentage compared withTHP, which is isolated by salt precipitation in nonpregnantindividuals (6). We refer to the uromodulin gene product asTHP in this article, as the antibody to uromodulin was createdby using THP obtained by salt precipitation. Synthesis of THPis modified in the endoplasmic reticulum (ER), before secretion.In addition to the assembly of N-glycans, THP biosynthesis ischaracterized by two peculiar posttranslational steps: (1) Theaddition of glycosyl phosphatidylinositol (GPI)-anchor by ERtranspeptidase (7), an event that ensures THP exposure at theluminal face of thick ascending limb of Henle’s loop (TALH)cells, and (2) the formation of a large number of intrachaindisulfide bridges by ER protein disulfide isomerase.
Transfection experiments of the UMOD gene into Hela cells havedemonstrated that intrachain disulfide bonding is importantin the maturation and secretion of THP (8). Because of theireffect on disulfide bonds, mutations that affect cysteine residuesare often associated with defective folding of the global proteinstructure. Misfolded and incompletely assembled proteins, particularlyas a result of cross-linking by nonnative interchain disulfidebonds (9), are prone to aggregation and retention in the ER(10). Such retention can lead to the unfolded protein response(11,12), which may activate a number of pathways, includinggrowth arrest and programmed cell death (13).
In vitro studies of MCKD2/FJHN/glomerulocystic kidney disease–associatedUMOD mutations corroborate histologic evidence from kidney biopsiesof affected patients, demonstrating that intracellular accumulationof THP occurs in the ER (3–5,14). These findings are consistentwith the decrease of THP in the urine of individuals with diseaseassociated UMOD mutations (4,14,15). Whereas studies indicatea pathologic accumulation of THP in the ER, the pathophysiologyof renal damage is unclear.
We hypothesized that accumulation of misfolded THP in the ERleads to apoptosis. We further hypothesized that colchicineand chemical chaperones that are known to stabilize the conformationof proteins that are defective in patients with a number ofinherited diseases (16) would facilitate THP excretion, reduceabnormal THP deposition, and decrease programmed cell death.
Construction of Wild-Type and Mutated UMOD Expression Vector
Wild-type (WT) UMOD cDNA were generated from a human kidneycDNA library by PCR and subcloned into pcDNA3 mammalian expressionvector (Invitrogen, Carlsbad, CA). Mutant constructs were basedon previously reported UMOD mutations (2). Mutant F1 that containsa 27-bp deletion that results in the in-frame deletion of aminoacids 177 to 185 (g.1966_1992del; c.529_555del; p.H177_185del)and mutant F2 that contains a missense mutation that changesa 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 mutantprimer sets: F1 SS, 5'-GCGCGGATCCGTGTCAGGCGAGCACCGAGTACGGGGAGGG-3';F1 AS, 5'-CCCTCCC CGTACTCGGTGCTCGCCTGACACGGATCCGCGC-3'; F2 SS,5'-GGGGGATGGATGGC ACTATGAGTGCTCCCCGGG-3'; F2 AS, 5'-CCCGGGGAGCACTCATAGTGCCATCCATC CCCC-3'.
Selection of Stably Transfected Clones
Human embryonic kidney 293 (HEK293) cells (American Type CultureCollection, Rockville, MD) were cultured in DMEM supplementedwith 10% FBS and standard antibiotics in a 5% CO2 atmosphereat 37°C. An immortalized TALH cell line derived from kidneysof New Zealand white rabbits was used (17). The TALH cell linewas maintained as a monolayer culture in DMEM supplemented with10% 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 transfectedcells were selected by culturing in the presence of DMEM mediasupplemented with 10% FBS, standard antibiotics, and 0.75 mg/mlG418 in a 5% CO2 atmosphere at 37°C for 2 wk. For determiningthe capacity of THP expression, cells were cultured in a 24-wellplate with 80% confluence, washed, and incubated with serum-freeDMEM media that contained antibiotics for 24 to 72 h. ExpressedTHP 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 mediathat contained 10% FBS, antibiotics, and 0.375 mg/ml G418. Thecells were washed (x3) with serum-free DMEM media that containedantibiotics and further incubated with the same media for 24to 72 h. Secreted THP in the conditioned media was determinedby Western blot and ELISA. For evaluating chemical agents, thecells were incubated in a serum-free medium supplemented withcolchicine, sodium 4-phenylbutyrate (4-PBA; Calbiochem, SanDiego, CA), and allopurinol (Sigma, St. Louis, MO) at concentrationranges 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 coldethanol. 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 quantifiedby 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 andtransferred to nitrocellulose. After blocking with 5% skim milk,membranes were incubated overnight at 4°C with sheep polyclonalprimary antibody against human THP (Chemicon International,Inc., Temecula, CA; 1:2000 dilution), followed by the incubationof AP-conjugated rabbit secondary antibody against sheep (Chemicon;1:5000 dilution) for 1 h at room temperature and visualizedwith nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate(Bio-Rad). -Actin (Abcam, Cambridge, MA) was used as an internalcontrol for equal loading.
An ELISA was established with a precoated-antibody (rabbit anti-humanTHP antibody; Santa Cruz Biotechnology, Santa Cruz, CA; 0.5µg/ml biocarbonate buffer [pH 8.4]; 1:200 dilution) boundto the solid phase, a primary antibody (sheep anti-human THPpolyclonal antibody; Chemicon; 1:2000 dilution), a secondaryantibody (AP-conjugated rabbit anti-sheep antibody; Chemicon;1:5000 dilution), and an alkaline phosphatase liquid substratesystem (Sigma). Urinary THP was purified by the salt-precipitationmethod (18).
Deglycosylation of THP
For differentiating THP precursor and mature forms, oligosaccharidecleavage was evaluated with two different deglycosidases, N-glycosidaseF (PNGase F; New England Biolab, Beverly, MA) and endo--N-acetylglucosaminidaseH (Endo H; New England Biolab) according to the manufacturer’sprotocol.
Immunocytochemistry
Stably transfected WT and mutant THP-expressing HEK293 and TALHcells 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 blockedby incubating for 1 h with PBS that contained 5% normal goatserum and 1% BSA for 1 h. Cells then were incubated with sheepanti-human THP polyclonal antibody (Chemicon; 1:200 dilution)in PBS that contained 1% normal goat serum and 1% BSA and followedby incubation with FITC-conjugated donkey anti-sheep IgG antibody(Chemicon; 1:200 dilution) in PBS that contained 1% normal goatserum and 1% BSA. Immunocytochemical signals were visualizedand photographed with a x400 magnification using a fluorescencemicroscope (Olympus 1X71; Olympus, Melville, NY).
FACS Analysis for Apoptosis Assay
Apoptotic cells were measured using annexin V-FITC and propidiumiodide staining (BD Bioscience, San Jose, CA) with FACS analysis.Cell sample preparation, Annexin V, and propidium iodide stainingfor FACS analysis were performed according to the manufacturer’sinstructions. The percentage of apoptotic cells was determinedby the fluorescence of individual cells measured by FACS flowcytometry (Becton Dickinson, San Jose, CA).
Evaluation of Cell Viability
WT and two mutant cell clones (1 x 106) were inoculated in eachwell of a six-well plate and cultured in DMEM supplemented with10% FBS and standard antibiotics in a 5% CO2 atmosphere at 37°Cfor 24 h. The cells were incubated further in a serum-free DMEMmedium in the absence and presence of 0.5 mM colchicine, 10mM 4-PBA, and 5 mM allopurinol. After 24- to 72-h incubations,cells were detached from a plate using a cell scraper, suspendedin PBS, and stained with trypan blue solution. Viable cells,which were resistant to trypan blue staining, were counted usinga hemacytometer under light microscopy (Olympus 1X71). Viabilitywas calculated with 100% being the viable cell number at 0 hincubation. 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 withan apparent molecular weight (MW) of 86 kD, the same MW as urinaryTHP (Figure 1). HEK293 and TALH cells do not express endogenousTHP. Negative controls and vector or enhanced green fluorescentprotein (EGFP) transfected cells did not express THP. ELISAquantification of THP expressed in conditioned media indicatedthat mutant clones excreted significantly less THP. Comparedwith WT THP-expressing HEK293 cells (54.7 ± 1.2 ng/mgprotein), THP expression was decreased by approximately 70%for F1 (16.0 ± 2.0 ng/mg protein) and decreased by approximately77% for F2 (12.7 ± 1.2 ng/mg protein). Compared withWT 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 werereproducible in six independent clones for each WT, F1, andF2.
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 fromWT and mutant HEK293 cells (Figure 2A). Although the ratio of75 to 86 kD was similar in the WT cell, the amount of the 86-kDforms was reduced in both mutant cells. To characterize theoligosaccharide modifications of the two different intracellularTHP, they were treated with deglycosidases PNGase F and EndoH. PNGase F hydrolyzes nearly all types of N-linked oligosaccharidesof glycoproteins, from both the ER and the Golgi. In contrast,Endo H cleaves only high-mannose glycoproteins from the ER butdoes not cleave glycoproteins modified in the Golgi. Deglycosylationexperiments showed that both the 75- and the 86-kD forms werecleaved by PNGase (Figure 2B). Whereas Endo H cleaved the 75-kDTHP, the 86-kD form was resistant to Endo H digestion, indicatingthat the protein was a terminally glycosylated precursor inthe Golgi apparatus. These results suggest that the 75-kD THPexists as a precursor in the ER and is exported into the Golgiapparatus for further oligosaccharide processing and becomesthe mature 86-kD form that is excreted into the conditionedmedia. Similar results were obtained for TAL cells (data notshown).
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 -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--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 hasbeen reported to induce programmed cell death (11), we measuredapoptosis by labeling THP-expressing HEK293 and TALH cells withannexin V-FITC followed by FACS analysis (Figure 3). FACS datashowed 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 TALHcells (Figure 3B), respectively. The percentage of cells inlate apoptosis was also higher in both mutant cells than inthe WT cells. These data indicate that the accumulation of mutantTHP in the ER is associated with significantly greater apoptosis.
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 x 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 proteinsfrom the ER. We examined the effect of the chemical chaperonesodium 4-PBA as well as colchicine and allopurinol on THP excretionusing ELISA (Figure 4). THP secretion in both WT and mutantcells was increased by treatment of 0.001 to 1.0 mM colchicineand by 5 to 10 mM 4-PBA. At the optimal colchicine concentrationof 0.5 mM, THP excretion was increased by 6.4-, 11.2-, and 9.0-foldfor WT, F1, and F2 mutant THP-expressing HEK293 cells, respectively(Figure 4Aa). Treatment with 10 mM 4-PBA increased THP excretionby 4.1-, 3.9-, and 4.6-fold for WT, F1, and F2 mutant THP-expressingHEK293 cells, respectively (Figure 4Ab). At the optimal colchicineconcentration of 0.5 mM, THP excretion was increased by 6.1-,12.2-, and 10.6-fold for WT, F1, and F2 mutant THP-expressingTALH cells, respectively (Figure 4Ba). Treatment with 10 mM4-PBA increased THP excretion by 3.4-, 3.8-, and 4.0-fold forWT, F1, and F2 mutant THP-expressing TALH cells, respectively(Figure 4Bb). In contrast, allopurinol tested at concentrationsranging from 0.001 to 5 mM did not affect THP secretion (Figure 4c).
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 THPinto conditioned media, we performed additional immunologicassays to study their effect on intracellular localization ofTHP in HEK293 and TALH cells. In WT transfected cells, THP localizedpredominantly to the cell membrane (Figure 5a), whereas in HEK293and TALH cells that were stably transfected with mutant F1 andF2, THP localization was mainly cytoplasmic (Figure 5, d andg), consistent with accumulation of mutant protein in the ER.When mutant cells were incubated with either 0.5 mM colchicineor 10 mM 4-PBA, THP signal was reduced in cytoplasm and increasedat plasma membrane.
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 mutantTHP in the ER induces apoptosis. Treatment of these cells withcolchicine and 4-PBA facilitated release of mutant THP fromthe ER to the plasma membrane and out of the cell. To evaluatewhether treatment with chemical agents affected the rate ofapoptosis in cells, we evaluated cell viability (Figure 6).Compared with WT cells, viability of the two mutant cells rapidlydecreased, with the extent more severe in F2 than F1. After72 h of incubation without chemical agent treatment, viabilitywas reduced to 79.1, 51.2, and 25.6% for WT, F1, and F2 THP-expressingHEK293 cells, respectively (Figure 6A). After 72 h of incubationwithout 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 colchicineor 10 mM 4-PBA, cell viability was improved for WT cells andgreatly improved for mutant cells. With treatment, viabilityin the mutant cells recovered to the level of WT cells withouttreatment. In contrast, 5 mM allopurinol did not change thecell viability for WT and mutant cells (Figure 6).
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 cellapoptosis were evaluated with FACS analysis (Table 1). FACSdata demonstrated that more early apoptotic signal was shownin cells without treatment than in cells with chemical treatment.The percentage of cells in late apoptosis was also higher incells without treatment than in cells with treatment.
Uromodulin (THP) is a GPI anchor glycoprotein produced exclusivelyby tubule cells of the thick ascending limb (TAL) of Henle (19,20).THP exposed at the luminal face of TAL is released in urineby proteolytic cleavage of the large ectodomain of GPI-anchoreddomain (21). THP is the most common protein in healthy urine.A characteristic of THP biosynthesis is the very slow conversionfrom precursor to mature form. Because THP has a very high numberof disulfide bridges (48 cysteine residues/mol), formation ofa correct set of intrachain disulfide bonds seems to be therate-limiting step for precursor export out of the ER (8,20).The majority of the >30 different disease-associated UMODmutations identified to date affect cysteine residues, and itis likely that all lead to protein misfolding and consequentlyto the retention of the mutant THP in the ER (2,3,5).
An increasing number of diseases seem to be due to improperprotein folding, including the ER storage diseases, resultingfrom the accumulation of proteins secondary to gene mutations(22–25). Identification of an apoptotic pathogenesis inducedby accumulation of THP in the ER provides a therapeutic target.Treatment that disrupts intracellular THP aggregates and facilitatesmovement of THP from the ER to the Golgi and out of the cellmay slow or prevent the characteristic tubular cell destruction.Low MW compounds can stabilize misfolded proteins, inhibitingaggregation and/or enabling the movement of these proteins tothe Golgi, where they are processed for cellular secretion (26,27).The recent utility of chemical chaperones to stabilize misfoldedproteins in a number of degenerative, lysosomal, and ER storagediseases spurred our interest (28–33). The utility of4-PBA in rescuing nephrin missense mutants encouraged us totest it (30), and we observed a beneficial effect in facilitatingTHP transport in our transfection model (Figures 4 and 5). Theincreased transport of mutant THP out of the cell was accompaniedby an increase in cell viability, suggesting possible clinicalutility (Figure 6).
We also evaluated the effect of two medications that are commonlyused to treat gout in individuals with UMOD mutations. Allopurinolis effective in controlling hyperuricemia and preventing goutin individuals with uromodulin-associated kidney diseases, butits ability to prevent renal damage is controversial (34–36).Colchicine, also used to treat gout, can affect microtubuleformation, and recent reports suggest that it may delay aggresomeformation by the myopathy-causing R120G B-crystallin mutant(37). We found that allopurinol did not facilitate transportof THP in either of the mutant HEK393 and TALH transfectionconstructs (Figure 4), and allopurinol did not seem to improvecell viability (Figure 6). Colchicine did facilitate movementof mutant THP from the ER to the cell membrane (Figure 5) andout of the cell (Figure 4). In vitro studies also show thatcolchicine improved cell viability in HEK293 and TALH cellsexpressing mutant THP (Figure 6). Improved cell viability wasassociated with a decrease in apoptotic cells (Table 1). Theeffect of chemical treatment on posttranslational modificationof THP in both lysates and conditioned media was evaluated byWestern blot analyses. Treatment increased the ratio of 86-/75-kDTHP, consistent with an increase in matured forms of THP. Thesedata suggest that chemical treatment reduces THP accumulationin the ER of cells, decreasing cellular apoptosis and increasingcell viability. This is the first study to demonstrate the useof colchicine as a chemical agent to prevent accumulation ofER deposits. This agent may be beneficial not only in uromodulin-associatedkidney disease but also in other ER storage diseases as well.
Variability in clinical findings associated with UMOD mutationshas been reported, but clear genotype–phenotype correlationshave not emerged (3). Longitudinal assessment of families thatsegregate for UMOD mutations indicates that the disease is moresevere in some families (2,6). In human families that segregatethe mutations studied in this report, we have observed thatthe onset of renal damage occurs earlier in individuals withthe F2 mutation, compared with the F1 mutation. It thereforeis interesting to see that the F2 mutation is associated witha decreased rate of cell viability in the HEK293 and TALH transfectionassays. The generality of this observation is unknown, but thismay explain the clinical variability in renal function seenin these conditions.
Taken together, our data suggest that UMOD mutations associatedwith MCKD-2 and FJHN result in the failure to process THP fromthe ER to the Golgi and secretion out of the cell. THP retentionin the ER seems to be associated with an increase in programmedcell death. Apoptosis of the TALH may account for the pathophysiologyof progressive renal function seen in these diseases. Treatmentof stably transfected HEK293 and TALH cells that express mutantTHP with either colchicine or 4-PBA reduced the accumulationof abnormal THP in the ER, facilitating its transport to theplasma membrane and out of the cell. This effect was associatedwith an increase in cell viability. These findings suggest thattreatment with colchicine or sodium 4-PBA may enhance the transportationof THP from the ER to the plasma membrane and secretion intothe tubular lumen, reducing programmed cell death of the TALH.These chemical agents may provide therapeutic alternatives toprevent kidney damage in THP-associated kidney diseases.
Acknowledgments
This work was supported in part by the intramural program ofthe National Institute of Dental and Craniofacial Research/NationalInstitutes of Health and by the National Institute of Diabetesand Digestive and Kidney Diseases (DK62252), Department of Healthand Human Services.
We thank Dr. Rolf Kinne for providing the TALH cell line usedin these experiments.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication May 4, 2005.
Accepted for publication July 18, 2005.
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Top
Abstract
Introduction
-Actin (Abcam, Cambridge, MA) was used as an internal control for equal loading. 
B-crystallin mutant (37). We found that allopurinol did not facilitate transport of THP in either of the mutant HEK393 and TALH transfection constructs (Figure 4), and allopurinol did not seem to improve cell viability (Figure 6). Colchicine did facilitate movement of mutant THP from the ER to the cell membrane (Figure 5) and out of the cell (Figure 4). In vitro studies also show that colchicine improved cell viability in HEK293 and TALH cells expressing mutant THP (Figure 6). Improved cell viability was associated with a decrease in apoptotic cells (Table 1). The effect of chemical treatment on posttranslational modification of THP in both lysates and conditioned media was evaluated by Western blot analyses. Treatment increased the ratio of 86-/75-kD THP, consistent with an increase in matured forms of THP. These data suggest that chemical treatment reduces THP accumulation in the ER of cells, decreasing cellular apoptosis and increasing cell viability. This is the first study to demonstrate the use of colchicine as a chemical agent to prevent accumulation of ER deposits. This agent may be beneficial not only in uromodulin-associated kidney disease but also in other ER storage diseases as well. 
