Regulation of PTH1 receptor expression by uremic ultrafiltrate in UMR osteoblast-like cells

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1 Kidney International, Vol. 65 (24), pp Regulation of PTH1 receptor expression by uremic ultrafiltrate in UMR 16 1 osteoblast-like cells SINEE DISTHABANCHONG, HATIM HASSAN, CHARLES L. MCCONKEY, KEVIN J. MARTIN, and ESTHER A. GONZALEZ Division of Nephrology, Saint Louis University, St. Louis, Missouri Regulation of PTH1 receptor expression by uremic ultrafiltrate in UMR 16 1 osteoblast-like cells. Background. Homologous down-regulation/desensitization of the parathyroid hormone receptor (PTH1R)/adenylate cyclase system has been demonstrated in uremia, and may contribute to parathyroid hormone (PTH) resistance; however, additional studies have shown that parathyroidectomy fails to normalize the down-regulation of the PTH1R. The present studies were designed to test directly, in vitro, the hypothesis that factors circulating in the uremic environment, other than PTH, decrease the response of osteoblastic cells to PTH. Methods. Studies were conducted in confluent cultures of UMR 16 1 osteoblast-like cells. Uremic ultrafiltrate (UUF) was obtained from patients on hemodialysis. Cells were exposed to media containing 5% uremic ultrafiltrate for periods of up to 72 hours. Control cultures were exposed to a buffered salt solution containing a comparable ionic composition to that of the UUF. PTH-stimulated cyclic adenosine monophosphate (camp) generation was determined by radioimmunoassay (RIA), PTH binding and PTH1R mrna levels were determined by radioligand binding and Northern analysis, respectively. Results. PTH-stimulated camp generation from cultures treated with uremic ultrafiltrate for 48 hours was / pmol/culture/5 minutes, whereas control cultures generated / 271 pmol camp/culture/5 minutes (P <.5). PTH binding was decreased by 3% in cultures incubated with UUF as compared to controls. The decrease in binding induced by UUF was accompanied by a decrease in PTH1R mrna levels. Conclusion. These findings demonstrate that factors present in UUF decrease PTH-stimulated camp generation by a mechanism that involves a decrease in the levels of PTH1R mrna levels. Thus, the skeletal resistance to PTH in the setting of chronic kidney disease, may be explained, at least in part, by circulating factors other than PTH. Key words: osteoblast, parathyroid hormone, PTH receptor, chronic kidney disease. Received for publication May 1, 23 and in revised form August 2, 23 Accepted for publication October 2, 23 C 24 by the International Society of Nephrology Osteitis fibrosa cystica or high turnover renal osteodystrophy as a result of secondary hyperparathyroidism is the most common skeletal abnormality found in patients on hemodialysis. Several factors contribute to the pathogenesis of secondary hyperparathyroidism in patients with chronic kidney disease, including retention of phosphorus, low levels of calcitriol, hypocalcemia, abnormal parathyroid gland function, and skeletal resistance to the actions of parathyroid hormone (PTH) [1 4]. Homologous down-regulation of the PTH receptor (PTH1R)/adenylate cyclase system has been implicated as one of the factors contributing to PTH resistance, and evidence suggests that decreased expression of the PTH1R per se contributes to the decreased response of target cells to PTH. Previous studies in our laboratory have demonstrated that exposure of UMR 16 1 osteoblast-like cells to PTH results in decreased cyclic adenosine monophosphate (camp) response, decreased receptor binding and decreased expression of PTH1R [5]. Studies in animals with chronic kidney disease have also shown downregulation of PTH1R mrna in rat femoral bone and epiphyseal cartilage as well as in liver and kidney tissue [6 9]. Further studies in rats with chronic kidney disease demonstrating decreased PTH1 mrna levels in kidney have questioned the role of PTH in the down-regulation of PTH1R since the defect was not prevented or corrected by parathyroidectomy [8]. Studies in humans examining bone specimens from patients with high turnover renal osteodystrophy have shown a decrease in PTH1R mrna in osteoblasts when compared to patients with high bone turnover not related to chronic kidney disease [1]. These observations suggest that chronic kidney disease is associated with decreased expression of PTH1R; however, factors other than parathyroid hormone may contribute to this abnormality. The present studies were designed to test the direct effects of factors circulating in the uremic environment on PTH1R in UMR 16 1 osteoblast-like cells. 897

2 898 Disthabanchong et al: Regulation of PTH1R by uremic ultrafiltrate METHODS Uremic ultrafiltrate The studies were approved by the Saint Louis University Institutional Review Board. Informed consent was obtained from each patient. Ultrafiltrate from uremic plasma (UUF) was collected from 12 patients who were stable on maintenance hemodialysis admitted to the hospital for hemodialysis access-related procedures. Patients were dialyzed with the same type of dialyzer (CA 21) (Baxter, Deerfield, IL USA). The ultrafiltrates were collected during the first 15 to 3 minutes after the initiation of hemodialysis from the effluent side of the membrane and were then submitted to ultrafiltration through.22 m Vacuum Filter Unit (Costar, Cambridge, MA, USA). The ultrafiltrates were then aliquoted and frozen at 8 C until the time of the study. Cell culture Studies were performed in cultures of UMR 16 1 osteoblast-like cells (Nicola Partridge, Robert Woods Johnson Medical School, Piscataway, NJ, USA) between passages 14 and 24. Cells were grown to confluence in minimum essential medium (MEM) supplemented with 5% fetal bovine serum (FBS), penicillin, and streptomycin. Confluent cultures were then incubated in medium containing 5% UUF for up to 72 hours. Control cultures were incubated in medium containing 5% of Hanks balanced salt solution (HBSS) with a comparable ionic composition. Electrolyte concentrations in UUF (experimental) and HBSS (control) media were sodium / 2.23 and 141 mmol/l, potassium 4.8 +/.97 and 5 meq/l, ionized magnesium.53 +/.7 and.61 mmol/l, and ionized calcium /.1 and 1.25 mmol/l, respectively. For the experiments evaluating the direct effect of chronic exposure to PTH on PTH-stimulated camp generation, synthetic rat PTH [1 34] was added at the concentrations indicated for 48 hours. PTH-stimulated camp production The medium was aspirated from confluent cultures of UMR 16 1 cells grown in 12-well plates. The cells were washed three times with Krebs-Henseleit solution. PTH was added at concentrations 1 8 mol/l for 5 minutes in the presence of 1 mmol/l 3-isobutyl-1-methylxanthine in.1% bovine serum albumin (BSA) in a total volume of.5 ml. The reactions were terminated with the addition of.1 ml of 1.8 mol/l perchloric acid. After 5 minutes, the cell extracts were neutralized with.1 ml of 3 mol/l KHCO 3, the volume was brought to 1 ml with 2 mmol/l MES (2-[n-morpholino] ethanesulfonic acid), ph 6.2, and camp was measured by radioimmunoassay (RIA) as previously described [11]. Iodination of PTH Five micrograms of Nle 8,21 Tyr 34 -rat PTH [1-34]NH 2 were iodinated with 25 g of chloramine T in the presence of 2 mci of 125 I. The reaction was terminated with 75 g of sodium metabisulfite and.5 ml of 2% acetonitrile (ACN) in.1% trifluoroacetic acid (TFA) was added. This was applied to a C 18 SepPak, which had been equilibrated sequentially with 3 ml of 1% ACN, 5 ml of 8% ACN,.1% TFA, and finally with 5 ml of 2% ACN,.1% TFA. Following application of the iodination reaction, the cartridge was washed with 2.5 ml of 2% ACN,.1% TFA, and the iodinated hormone was eluted with three successive.5 ml aliquots of 5% ACN,.1% TFA. The peak tube was diluted in 1% BSA and aliquots were stored at 8 C. PTH binding studies The medium was removed from confluent cultures of UMR16 1 cells, and the cells were washed twice with MEM containing 5% FBS at 1 C. Following addition of 1 6 mol/l PTH to duplicate wells for determination of nonspecific binding, cpm of 125 I Nle 8,21 Ty r34 - rat PTH [1-34]NH 2 were added to all wells. Incubations were continued for 3 hours at 1 C. The cells were then washed three times with cold MEM-5%FBS and phosphate-buffered saline (PBS). The cells were solubilized in.1 mol/l sodium hydroxide and the radioactivity was then quantitated. PTH1R mrna levels Total RNA was isolated from cultured cells using the TRIzol method as described by the manufacturer (Life Technologies, Grand Island, NY, USA). PTH1R mrna levels were determined by Northern analysis using a probe consisting of nucleotides 439 to 768 of the rat PTH1R cdna prepared by reverse transcriptionpolymerase chain reaction (RT-PCR) from rat kidney RNA, cloned into pcrii and sequenced to confirm identity with the published sequence of the cloned rat PTH/PTHrP receptor [5]. Using 2 lg of total RNA, Northern analysis was performed using Northern Max- Gly TM Kit as described by the manufacturer (Ambion, Austin, TX, USA). All reagents mentioned in the following procedure were obtained from Ambion unless otherwise specified. Briefly, 2 lg of total RNA was mixed 1:1 with glyoxal load dye and the mixture was incubated for 3 minutes in a 5 C water bath. The samples were then applied to 1% agarose LE gel and electrophoresed at 1 volts for 1 hour. After transferring to nylon membranes, the RNA was bound by ultraviolet cross-linking (Stratalinker) (Strategene Cloning systems, La Jolla, CA, USA). The filters were prehybridized for 1 hour at 42 C in Ultrahyb

3 Disthabanchong et al: Regulation of PTH1R by uremic ultrafiltrate 899 solution. The filters were probed with 32 P-deoxycytidine 5 triphosphate radiolabeled PTHR1 cdna for 16 to 2 hours (Megaprime) (Amersham Life Science, Arlington Heights, IL, USA), washed with low stringency solution for 1 minutes at 42 C. The filters were analyzed by phosphorimager (Molecular Dynamics, Sunnyvale, CA, USA). After analysis, the filters were then stripped and reprobed with radiolabeled cdna for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The PTH1R bands were normalized relative to the GAPDH band using Image Quant software. Intact PTH assays Intact PTH levels in UUF and serum were measured by two-site immunoradiometric assay (IRMA) (Nichols, San Juan Capistrano, CA, USA). The procedures were performed according to the manufacturer s instructions. Calculations Statistical analyses were performed using Student t test. Analysis of variance (ANOVA) was used when multiple groups were being analyzed. Differences between groups were considered significant when P <.5. Data are expressed as mean +/ standard error of the mean. RESULTS Effect of UUF on PTH-stimulated camp generation We first determined the effect of individual UUFs obtained from each of 12 patients on both basal and PTH-stimulated camp response in confluent cultures of UMR16 1 osteoblast-like cells. As shown in Figure 1, following a 48-hour incubation with UUF, the ability of the cells to respond to acute stimulation with PTH was impaired without significant difference in basal camp levels. In cultures treated with UUF, the values for PTHstimulated camp generation were / 2.7% lower as compared to the values obtained in control cultures (P <.1). This degree of suppression was similar for all UUF samples and was not influenced by the concentration of intact PTH in serum, which ranged from 18 to 1465 pg/ml. Figure 2 shows the time course for the effect of UUF on PTH-stimulated camp generation. The suppression of PTH-stimulated camp generation was evident at 24 hours and reached statistical significance at 48 hours (P <.1 as compared to time ). No further effect was noted after 72 hours of incubation with UUF. Cell viability was not significantly different in control vs. UUF-treated cultures as determined by protein content (data not shown). Intact PTH levels in UUF and effect on PTH-stimulated camp generation In order to exclude the possibility that the impaired response to PTH as a result of exposure to UUF was due to camp generation, pmol/5 min/culture Basal P <.1 PTH Fig. 1. Effect of uremic ultrafiltrate (UUF) on basal and parathyroid hormone (PTH)-stimulated cyclic adenosine monophosphate (camp) generation. Confluent cultures of UMR 16 1 cells were incubated in the presence ( ) or absence ( ) of UUF for 48 hours prior to acute stimulation with PTH (1 8 mol/l). PTH-stimulated camp generation was measured by radioimmunoassay (RIA). The values represent the mean ± SEM from 12 different samples. camp generation, pmol/5 min/culture Time, hours Fig. 2. Time course of the effect of uremic ultrafiltrate (UUF) on parathyroid hormone (PTH)-stimulated cyclic adenosine monophosphate (camp) generation. Confluent cultures of UMR 16 1 cells were exposed to UUF for the times indicated prior to acute stimulation with PTH (1 8 mol/l). PTH-stimulated camp generation was measured by radioimmunoassay (RIA). The values represent the mean ± SEM from four different samples. P <.1 as compared to time. homologous down-regulation of the PTH1R due to the presence of PTH in the UUF, we measured the concentration of intact PTH in the UUF. The values ranged from 6 to 79 pg/ml; therefore, the concentration of intact PTH in the media containing 5% UUF used for the present studies ranged from 3 to 4 pg/ml. Thus, we exposed UMR 16 1 cells to equimolar amounts of the hormone (1 12 to 1 11 mol/l) for 48 hours. As shown in Figure 3, chronic exposure of the cultures to PTH at concentrations similar to those of intact PTH present in UUF did not have an effect on camp accumulation following acute stimulation with PTH. These findings indicate that factors present in UUF, other than PTH, are responsible for the impaired ability of the cells to respond to PTH stimulation. Effect of UUF on PTH binding In order to determine if the inhibitory effect of UUF on PTH-stimulated camp generation was due to a defect

4 9 Disthabanchong et al: Regulation of PTH1R by uremic ultrafiltrate camp generation, pmol/5 min/culture Control PTH, mol/l Fig. 3. Effect of chronic exposure to parathyroid hormone (PTH) concentrations present in uremic ultrafiltrate UUF on PTH-stimulated cyclic adenosine monophosphate (camp) generation. Confluent cultures of UMR 16 1 cells were exposed to PTH 1 12, 1 11, and 1 1 mol/l for 48 hours prior to acute stimulation with PTH (1 8 mol/l). The values represent the mean ± SEM from four different experiments. in PTH binding to its receptor, we examined the effect of UUF on 125 I-PTH binding in confluent cultures of UMR 16 1 cells. As shown in Figure 4, exposure to UUF for 48 hours resulted in a 3% decrease in PTH binding as compared to control cultures (P <.5). Competitive inhibition binding experiments revealed that binding was decreased at all concentrations of unlabeled PTH tested. Scatchard analysis indicated that the decrease in binding was due to a decrease in receptor number with no change in receptor affinity. Effect of UUF on PTH1R mrna We next examined whether the decrease in PTH binding in response to exposure to UUF was related to a decrease in PTH1R mrna levels. The effect of UUF on the levels of PTH1R mrna levels was determined by Northern analysis and corrected for GAPDH mrna. As shown in Figure 5, exposure to UUF for 12 hours results in a / 5.9% decrease in PTH1R mrna levels (P <.5 as compared to time ). The effect of UUF on PTH1R mrna peaked at 48 hours when the levels had decreased by / 5.7% (P <.5 as compared to time ), and no further decrease was noted after 72 hours of exposure. DISCUSSION Previous studies have demonstrated that high circulating levels of PTH may lead to homologous downregulation/desensitization of the PTH1R/adenylate cyclase system thus leading to PTH resistance. Studies in our laboratory have shown that PTH directly decreases the expression of PTH1R in osteoblastic cells [5]. Consistent with our findings in vitro, studies in uremic animals have also shown decreased expression of PTH1R in target tissues [7 9, 12]. Interestingly, however, Ureña et al [8] reported that parathyroidectomy failed to prevent the downregulation of PTH1R in rats with chronic kidney disease. These findings suggest that in % Specific binding Bound/ free Bound, nmol/l PTH, nmol/l Fig. 4. Competitive inhibition of equilibrium binding of 125 I Nle 8,21 Ty r34 -rat PTH [1-34]NH 2. Confluent cultures of UMR 16 1 cells were incubated in the presence ( ) or absence ( ) of uremic ultrafiltrate (UUF) for 48 hours. Binding of 125 I Nle 8,21 Ty r34 -rat PTH [1-34]NH 2 was examined in the presence of increasing concentrations of unlabeled parathyroid hormone (PTH). The inset shows a Scatchard analysis of the data. addition to PTH, there are other factors circulating in the uremic environment that may account for the down-regulation of PTH1R. The present studies show that exposure of UMR 16 1 osteoblast-like cells to UUF obtained from patients on hemodialysis results in impaired PTH-stimulated camp generation as well as decreased PTH binding. The decrease in PTH binding was due to decreased PTH1R number by a mechanism involving a decrease in steady-state levels of PTH1R mrna. This effect is due to factors present in the uremic environment other than PTH. Skeletal resistance to PTH in the setting of chronic kidney disease was initially described by Evanson [13]. Subsequently, Llach et al [14] found a delayed recovery from ethylenediaminetetraacetic acid (EDTA)-induced hypocalcemia in patients with renal failure despite high levels of PTH. Massry et al [15] demonstrated a reduced calcemic response to infusion of parathyroid extract to 15 subjects with varying degrees of renal failure. A diminished calcemic response to PTH in chronic kidney disease has also been demonstrated in animal studies [16 24], and using dogs with chronic kidney disease, Galceran et al [24] found that the calcemic response to PTH was restored following parathyroidectomy. Furthermore, a blunted camp response to PTH extract was observed by Olgaard et al [25] in isolated perfused bones obtained from uremic dogs. The mechanisms responsible for PTH resistance in uremia remain unclear. It is well accepted that exposure to PTH results in homologous desensitization and downregulation of the PTH1R/adenylate cyclase system [5, 26 36], and recovery from this phenomenon has been

5 Disthabanchong et al: Regulation of PTH1R by uremic ultrafiltrate 91 A PTH1R mrna/gapdh mrna, % change from baseline PTH1R mrna GAPDH mrna Time, hours B Time, hours Fig. 5. Effect of uremic ultrafiltrate (UUF) on parathyroid hormone receptor (PTH1R) mrna levels. Confluent cultures of UMR 16 1 cells were exposed to UUF for the times indicated. Total RNA was isolated and PTH1R mrna levels were determined by Northern analysis. The membranes were stripped and reprobed using a cdna for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (A). Densitometric analysis of the levels of PTH1R mrna corrected for GAPDH mrna (B). The values represent the mean ± SEM from five different samples. P <.1. observed following withdrawal of the hormone [28, 29]. We have previously shown that PTH directly downregulates its own receptor in UMR 16 1 osteoblast like-cells by a mechanism that involves decreased PTH1R mrna levels [5]. These findings are in agreement with studies in experimental animals with secondary hyperparathyroidism related to chronic kidney disease and suggest that decreased expression of PTH1R in this setting is a result of increased circulating levels of PTH [6, 7, 9]. However, several lines of evidence indicate that there may be additional circulating factors in the uremic environment that contribute to decreased PTH1R expression. In this regard, Ureña et al [8] found a decrease in PTH1R mrna in liver and kidney of uremic rats; however, parathyroidectomy did not prevent the downregulation of the receptor. Tian et al [9] showed partial normalization of the PTH1R mrna levels in tissues of uremic rats after parathyroidectomy. Thus, although chronic exposure to PTH in the setting of chronic kidney disease can certainly decrease the expression of PTH1R, these data, as well as the present studies, suggest that other factors present in the uremic milieu may also be involved in this process. In skeletal tissue, PTH1R mrna levels have been found to be decreased in bone and epiphyseal cartilage growth plate of uremic rats. Recent studies by Picton et al [1] have provided additional insight into the cellular mechanism of skeletal resistance to PTH in the setting of chronic kidney disease. The investigators used in situ hybridization in order to examine PTH1R mrna expression in bone biopsy specimens obtained from patients with abnormalities of bone turnover. The groups included patients with high and low turnover renal bone disease, high turnover bone disease in patients with normal renal function such as Pagetic bone, and normal controls. The studies showed that the expression of PTH1R mrna in osteoblasts from patients with high turnover renal bone disease was only 36% of that found in Paget s disease and 51% of that found in normal bone. Also, the osteoblastic PTH1R mrna signal from adynamic bone was only 28% of that of normal bone. In addition, there was no correlation between the expression of PTH1R mrna and serum PTH levels. Therefore, studies in experimental animals as well as in humans indicate that PTH levels are not the sole factor involved in down-regulation of PTH1R expression in the setting of chronic kidney disease. The present studies further support such concept and point toward dialyzable uremic factors as playing a role in this process. The precise nature of the uremic factors involved in the down-regulation of PTH receptors is not clear at the present time. A variety of cytokines and growth factors are known to down-regulate the PTH1R/adenylate cyclase system. Evidence suggests that uremia is an inflammatory state with up-regulation of multiple cytokines, especially tumor necrosis factor (TNF) and interleukin-1 (IL-1) [37 39], which have both been shown to affect the PTH1R/adenylate cyclase system. Katz et al [4] demonstrated that exposure of UMR 16 1 osteoblast-like cells to TNF-a and IL-1 inhibited PTH-sensitive adenylate cyclase secondary to a decline in PTH receptor density. Schneider et al [41] showed that incubation of UMR 16 1 osteoblast-like cells with TNF-a lead to a decrease in PTH binding to its receptor which was due to a decrease in receptor number [41]. The effect of TNF-a to inhibit the PTHR1/adenylate cyclase system has also been demonstrated by other investigators [42 44]. In addition to TNF-a and IL-1, interferon-a, and TGF-b have also been shown to inhibit the expression of PTH1R in osteoblastic cells [45, 46]. Thus, it is possible that the presence of these or a variety of other cytokines in UUF may contribute to the down-regulation of the PTH1 receptor observed in our studies. Factors present in the uremic environment have been described to affect other cellular processes, which are likely to have an impact on skeletal biology. For example,

6 92 Disthabanchong et al: Regulation of PTH1R by uremic ultrafiltrate it is well accepted that calcitriol resistance is present in uremia, and evidence suggests that uremic toxins are also involved in this process [47 51]. In this regard, studies by Patel et al [51] have shown that UUF obtained from patients on hemodialysis decreases the binding of the vitamin D receptor to the vitamin D responsive elements in target genes, thus leading to impaired calcitriol-mediated gene transcription. These findings offer an explanation for the calcitriol resistance observed in the setting of uremia. More recently, Canalejo et al [52] reported that UUF obtained from patients on hemodialysis prevented the effect of calcitriol to inhibit parathyroid cell proliferation. CONCLUSION We have shown that UUF obtained from patients on hemodialysis contains factors that decrease PTHstimulated camp generation in UMR 16 1 osteoblastlike cells. The impaired response to PTH was not related to homologous down-regulation/desensitization of the PTH1R/adenylate cyclase system since preincubation of the cells with concentrations of PTH similar to those present in UUF did not have an effect on camp accumulation following acute stimulation with PTH. In addition, we have shown that UUF decreases PTH receptor number by a mechanism involving decreased PTH1R mrna. These findings suggest that circulating uremic factors may contribute to skeletal resistance to PTH in the setting of chronic kidney disease. ACKNOWLEDGMENTS Portions of this manuscript were presented in abstract form at the 33rd Annual American Society of Nephrology meeting. This work was supported in part by grant DK-5199 from the National Institutes of Health. Reprint requests to Esther A. Gonzalez, M.D., Saint Louis University, 3635 Vista Ave., St. Louis, MO gonzalea@slu.edu REFERENCES 1. HAMDY NA: The spectrum of renal bone disease. 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