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Keywords:

  • Bone turnover;
  • Hemodialysis;
  • Indoxyl sulfate;
  • Oxidative stress;
  • Parathyroid hormone resistance

Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. REFERENCES

Skeletal resistance to parathyroid hormone (PTH) in uremia is known, although the mechanism of resistance is not fully elucidated. To clarify the roles of indoxyl sulfate, which is a uremic toxin, in skeletal resistance, we examined the relationship between indoxyl sulfate and biochemical markers of bone turnover in hemodialysis patients. We obtained blood samples from 47 hemodialysis patients and measured serum indoxyl sulfate, intact PTH, oxidative stress marker 8-hydroxy-2′-deoxyguanosine (8-OHdG), and various biochemical markers. The serum concentrations of alkaline phosphatase (ALP) and bone-specific alkaline phosphatase (BAP) were used as bone formation markers, and the concentration of tartrate-resistant acid phosphatase 5b (TRACP-5b) was used as a bone resorption marker. Serum indoxyl sulfate levels were much higher in hemodialysis patients than healthy subjects. Multiple regression analysis shows that indoxyl sulfate correlated negatively with ALP (β = −1.897, P = 0.042) and BAP (β = −0.310, P = 0.029), independent of intact PTH; however, indoxyl sulfate did not correlate with TRACP-5b or 8-OHdG. These findings suggest that indoxyl sulfate may relate skeletal resistance to PTH in uremia.

The effect of parathyroid hormone (PTH) on bone is down-regulated in uremia; therefore, PTH levels two to three times higher than the upper limit of normal range are required to keep bone turnover within a normal range in dialysis patients (1). This impaired PTH function in uremia is termed “skeletal resistance to PTH”.

The mechanism of PTH resistance in uremia involves several factors: the reduction of PTH receptor in bones (2–5), 7–84 PTH (6,7), uremic toxin (8–10), osteoprotegerin (OPG) (11,12), and a decrease in bone morphogenetic protein-7 (BMP-7) (13–15). Indoxyl sulfate, a uremic toxin, is regarded as a factor that induces skeletal resistance to PTH. It is known to increase in patients with chronic kidney disease (16) and is considered to accelerate the progression of chronic kidney disease (17), atherosclerosis (18), and cardiac damage (19) through oxidative stress.

As a mechanism of skeletal resistance to PTH, we have previously demonstrated that uptake of indoxyl sulfate by osteoblasts occurs via the organic anion transporter (OAT) 3 and augments oxidative stress, thereby impairing osteoblast function and down-regulating PTH receptor expression (9). Down-regulation of PTH receptor mRNA in osteoblasts along with the development of renal dysfunction has been shown in a rat model (3,5). The administration of an oral charcoal adsorbent, which inhibits the accumulation of indoxyl sulfate, has been shown to reverse the down-regulation of PTH receptor gene in osteoblasts and improve low bone turnover disease (8). In humans, osteoblast PTH receptor mRNA in end-stage renal failure has been down-regulated in comparison to healthy individuals (4); however, no study has yet investigated the relationship between indoxyl sulfate and skeletal resistance to PTH in patients with renal dysfunction. In this study we examine the relationship between indoxyl sulfate and biochemical markers of bone turnover in hemodialysis patients to determine whether indoxyl sulfate is involved in skeletal resistance to PTH in human.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. REFERENCES

Patient selection

We enrolled patients from a single dialysis unit (Hara Genitourinary Hospital, Kobe, Japan). Medically stable patients undergoing hemodialysis for at least one year were selected as candidates for this study. Diabetic patients and patients taking vitamins C or E were excluded from this study. Written consent was obtained from all participants prior to their enrolment in the study, and the study was approved by the Local Ethics Committees. Forty-seven patients fulfilling the eligibility criteria participated in the study.

Laboratory methods

Blood samples were obtained from the participants after an overnight fast before dialysis, two days after the last hemodialysis treatment. Serum was separated by centrifugation immediately after the blood samples were collected and aliquots stored at −80°C. Hemoglobin, albumin, creatinine, urea nitrogen, calcium, phosphate, alkaline phosphatase (ALP), and C-reactive protein (CRP) were measured by standard laboratory techniques using automatic analyzers. We measured the serum levels of intact PTH by an electro-chemiluminescence immunoassay (Roche-Diagnostics, Basel, Switzerland), serum levels of bone-specific alkaline phosphatase (BAP) by an enzyme immunoassay (Quidel Corporation, San Diego, CA, USA), serum levels of tartrate-resistant acid phosphatase 5b (TRACP 5b) by a novel fragment absorbed immunocapture enzyme assay (FAICEA) method using two monoclonal antibodies (Nitto Boseki, Fukushima, Japan), serum levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) using a commercially available competitive ELISA kit (Japan Institute for Control of Aging, Shizuoka, Japan) by diluting the samples, and serum levels of indoxyl sulfate by a high-performance liquid chromatography technique (Fushimi Pharmaceutical, Kagawa, Japan). The ELISA kit can measure 8-OHdG values ranging from 0.125 to 10 ng/mL using a monoclonal specific antibody, N45.1 (20).

Statistical analysis

All data are represented as mean ± SD or frequency (%). We conducted univariate and multivariate linear regression analyses to evaluate factors influencing biochemical markers of bone turnover (ALP, BAP, and TRACP 5b) and 8-OHdG. In multiple regression analysis, indoxyl sulfate, gender, age, duration of hemodialysis, and variables that achieved statistical significance (P < 0.05) in univariate analysis were included. Comparison of 8-OHdG between patients using a cellulose membrane and those using polymethylmethacrylate (PMMA) or polysulfone (PS) membranes was performed with the Student's t-test. The value P < 0.05 was considered to be statistically significant. All analyses were performed with SPSS II for Windows, version 11.01J (SPSS, Chicago, IL, USA).

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. REFERENCES

Patient characteristics

The clinical characteristics of the study population are shown in Table 1. Sixty-eight percent of the participants were male and the average age was 61.5 ± 11.7 years. All women were postmenopausal. The mean duration of hemodialysis was 18.1 ± 5.4 years. Average serum levels of indoxyl sulfate in the participants were 37.6 ± 16.5 µg/mL, which were much higher than those in healthy subjects previously reported (21). Eighteen patients used a cellulose membrane and twenty-nine patients used synthetic (PMMA or PS) membranes.

Table 1. Clinical characteristics of the study population
 All patients (N = 47)
  1. ALP, alkaline phosphatase; BAP, bone-specific alkaline phosphatase; CRP, C-reactive protein; HD, hemodialysis; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; P, phosphate; PMMA, polymethylmethacrylate; PTH, parathyroid hormone; TRACP 5b, tartrate-resistant acid phosphatase 5b.

Age (years)61.5 ± 11.7 (range 30–83)
Male, N (%)32 (68%)
Duration of HD (years)18.1 ± 5.4 (range 11.0–31.4)
HD time (hours per week)12.2 ± 0.5
Hemoglobin (g/dL)10.5 ± 1.1
Albumin (g/dL)3.8 ± 0.4
Creatinine (mg/dL)11.9 ± 3.0
Urea nitrogen (mg/dL)71.1 ± 14.0
Ca (mg/dL)9.3 ± 0.6
P (mg/dL)5.8 ± 1.3
Intact PTH (pg/mL)253 ± 270
ALP (IU/L)291 ± 115
BAP (U/L)36.8 ± 15.9
TRACP 5b (mU/dL)193 ± 147
CRP (mg/dL)0.17 ± 0.16
8-OHdG (ng/mL)0.82 ± 0.25
Indoxyl sulfate (µg/mL)37.6 ± 16.5
Type of dialysis membrane 
 Cellulose, N (%)18 (38%)
 PMMA, N (%)12 (26%)
 Polysulfone, N (%)17 (36%)

Effect of indoxyl sulfate and other clinical variables on biochemical markers of bone turnover

The correlation between biochemical markers of bone turnover (ALP, BAP, and TRACP 5b) and clinical variables in univariate analysis is shown in Table 2. A significant negative correlation was observed between indoxyl sulfate and BAP. Indoxyl sulfate also correlated negatively with ALP, although the correlation was not statistically significant. There was no correlation between indoxyl sulfate and TRACP 5b. ALP and BAP significantly correlated with the duration of hemodialysis and intact PTH. TRACP 5b significantly correlated with phosphate and intact PTH.

Table 2. Correlation between bone turnover markers and clinical variables
VariablesALPBAPTRACP 5b
rP-valuerP-valuerP-value
  1. ALP, alkaline phosphatase; BAP, bone-specific alkaline phosphatase; BMI, body mass index; HD, hemodialysis; P, phosphate; PTH, parathyroid hormone; TRACP 5b, tartrate-resistant acid phosphatase 5b.

Age (years)0.1830.2180.1240.477−0.0630.717
Duration of HD0.3920.0060.4310.0100.2840.098
Ca−0.0510.732−0.0950.5870.2350.174
P−0.1160.4360.0780.6560.3500.039
Intact PTH0.3520.0150.656<0.0010.771<0.001
Albumin−0.1720.248−0.0500.7760.0910.603
BMI0.0190.901−0.0450.799−0.2390.167
Indoxyl sulfate−0.2870.050−0.3370.048−0.1200.494

In multiple regression analysis, indoxyl sulfate correlated negatively with ALP (Table 3) and BAP (Table 4), independent of PTH. There was no correlation between indoxyl sulfate and TRACP 5b (Table 5). ALP correlated with the duration of hemodialysis and intact PTH. BAP also correlated with intact PTH. Furthermore, TRACP 5b correlated with intact PTH.

Table 3. Independent effect of clinical variables on alkaline phosphatase by multiple regression analysis (adjusted R2 = 0.255)
Significant variablesβSEP-value
  1. This model included gender, age, duration of hemodialysis, intact parathyroid hormone, and indoxyl sulfate. HD, hemodialysis; PTH, parathyroid hormone.

Male gender15.94934.0030.642
Age (years)2.2981.3340.093
Duration of HD (years)6.0042.9080.045
Intact PTH (pg/mL)0.1280.0570.031
Indoxyl sulfate (µg/mL)−1.8970.9040.042
Table 4. Independent effect of clinical variables on bone-specific alkaline phosphatase by multiple regression analysis (adjusted R2 = 0.502)
Significant variablesβSEP-value
  1. This model included gender, age, duration of hemodialysis, intact parathyroid hormone, and indoxyl sulfate. HD, hemodialysis; PTH, parathyroid hormone.

Male gender−6.7664.4020.135
Age (years)0.1020.1580.524
Duration of HD (years)0.4760.4490.298
Intact PTH (pg/mL)0.0300.008<0.001
Indoxyl sulfate (µg/mL)−0.3100.1350.029
Table 5. Independent effect of clinical variables on tartrate-resistant acid phosphatase 5b by multiple regression analysis (adjusted R2 = 0.560)
Significant variablesβSEP-value
  1. This model included gender, age, duration of hemodialysis, phosphate, intact parathyroid hormone, and indoxyl sulfate. HD, hemodialysis; PTH, parathyroid hormone.

Male gender−19.56638.4480.615
Age (years)0.4411.6260.788
Duration of HD (years)−3.6093.9150.365
Phosphate (mg/dL)25.38517.8610.166
Intact PTH (pg/mL)0.3910.068<0.001
Indoxyl sulfate (µg/mL)−0.6151.1760.605

Effect of indoxyl sulfate and other clinical variables on 8-OHdG

The correlation between oxidative stress marker 8-OHdG and clinical variables in univariate analysis is shown in Table 6. There was no correlation between indoxyl sulfate and 8-OHdG; however, 8-OHdG correlated significantly with CRP.

Table 6. Correlation between 8-hydroxy-2′-deoxyguanosine and clinical variables
VariablesrP-value
  1. CRP, C-reactive protein; HD, hemodialysis; P, phosphate; PTH, parathyroid hormone.

Age0.3170.064
Duration of HD0.1280.463
Ca−0.2280.187
P−0.2240.197
Intact PTH0.1510.387
Hemoglobin−0.1280.464
Albumin−0.2630.126
CRP0.4150.015
Indoxyl sulfate−0.0900.534

All the study participants were divided into two groups based on membrane types (cellulose, N = 18; synthetic, N = 29). There was no significant difference in 8-OHdG between the two groups (cellulose vs. synthetic; 0.81 ± 0.28 vs. 0.82 ± 0.22 ng/mL, P = 0.91).

In multiple regression analysis, indoxyl sulfate did not correlate with 8-OHdG (Table 7). Only gender correlated significantly with 8-OHdG.

Table 7. Independent effect of clinical variables on 8-hydroxy-2′-deoxyguanosine by multiple regression analysis (adjusted R2 = 0.432)
Significant variablesβSEP-value
  1. This model included gender, age, duration of hemodialysis, C-reactive protein, and indoxyl sulfate. CRP, C-reactive protein; HD, hemodialysis.

Male gender−0.3060.074<0.001
Age (year)0.00050.0030.852
Duration of HD (year)0.0030.0070.621
CRP (mg/dL)0.4480.2380.070
Indoxyl sulfate (µg/mL)−0.0020.0020.354

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. REFERENCES

Several mechanisms have been suggested for skeletal resistance in uremia; however, the pathophysiology of skeletal resistance to PTH has not been fully elucidated. This study demonstrated a negative correlation between indoxyl sulfate and bone formation markers in hemodialysis patients, independent of PTH. Thus, this result indicates that patients who accumulate indoxyl sulfate show suppressed bone formation. This result is in agreement with our previous findings that have demonstrated improvement in rat bone formation by preventing the accumulation of indoxyl sulfate in blood (8). The PTH receptor gene in osteoblasts is down-regulated depending on the concentration of indoxyl sulfate in vitro (9), and inhibiting the accumulation of indoxyl sulfate reverses the down-regulation of the PTH receptor gene in osteoblasts (8). Human osteoblast PTH receptor is known to be down-regulated in end-stage renal failure (4); therefore, we hypothesize that the accumulation of indoxyl sulfate in blood is a cause of the down-regulation of PTH receptor gene in human osteoblasts.

The effect of PTH on both osteoclasts and osteoblasts similarly decreased in uremia (22). The mechanism of osteoclast differentiation and activation has been elucidated at molecular levels recently (23). Receptor activator of nuclear factor κB ligand (RANKL), which is produced by osteoblasts, stromal cells, T cells, and other sources, binds and activates receptor activator of nuclear factor κB (RANK) on the surface of osteoclasts. RANK–RANKL interactions lead to osteoclast differentiation and activation. These interactions are prevented by OPG that binds to and thereby inactivates RANKL. Since serum OPG levels are increased in chronic kidney disease patients (11,12), high serum OPG levels are considered to result in osteoclast inactivation. Mozar et al. demonstrated that indoxyl sulfate inhibits the differentiation of osteoclast-like cells (24). We could not show the relationship between indoxyl sulfate and the bone resorption marker in this study. We previously reported that bone resorption is suppressed less than bone formation in a uremic rat model (3). Reduced suppression of bone resorption in uremia could explain why we could not observe any correlation between indoxyl sulfate and the bone resorption marker in this study. Further studies in vitro and in vivo will be necessary to clarify whether indoxyl sulfate influences osteoclast function.

Uptake of indoxyl sulfate via OAT1 and OAT3 induces oxidative stress in proximal tubular cells (17). Oxidative stress induced by indoxyl sulfate activates plasminogen activator inhibitor-1 (PAI-1) and nuclear factor-κB (NF-κB), which is related to the progression of renal disease. Furthermore, indoxyl sulfate has shown a significant positive correlation with the oxidative stress marker pentosidine in hemodialysis patients (18). We demonstrated that indoxyl sulfate increases oxidative stress in osteoblasts via OAT3 (9); however, we could not show a positive correlation between indoxyl sulfate and oxidative stress in this study. Oxidative stress correlates positively with many factors, including age, diabetes, anemia, erythropoietin (25), intravenous iron administration, ferritin (26), CRP (27), cellulose membranes (28), and malnutrition (29). Although we analyzed some factors that are related to 8-OHdG, we did not identify a relationship between 8-OHdG and all the aforementioned factors; therefore, the factors that we did not analyze may influence the relationship between indoxyl sulfate and 8-OHdG.

Gender and menopausal status correlate with bone metabolism (30–34). Serum ALP, BAP, and TRACP5b levels in women were higher than those in men, but were not statistically significant in our study (data not shown). Since the number of female participants was only 15, the statistical power was possibly not sufficient to detect the correlation between gender and ALP, BAP, or TRACP5b. Indoxyl sulfate negatively correlated with BAP and ALP in multiple regression analysis including, gender as independent variables; therefore, we believe that indoxyl sulfate negatively correlated to BAP and ALP, independent of gender.

Nutritional status possibly influences bone metabolism in hemodialysis patients (35,36). In our study, the body mass index and serum albumin levels did not correlate with biochemical markers of bone turnover. In addition, indoxyl sulfate negatively correlated with BAP and ALP when we included the nutritional status and performed multiple regression analysis (data not shown); therefore, we believe that indoxyl sulfate negatively correlates with BAP and ALP, independent of the nutritional status.

When toxins or biomolecules in hemodialysis patients are evaluated, removal by hemodialysis sometimes influences the results. Indoxyl sulfate (37), ALP, BAP (38), and TRACP5b (39) are poorly removed by dialysis. Since PTH is adsorbed by some membranes, changes in PTH during hemodialysis are influenced by dialysis membranes (40); however, since PTH has a very short half life, serum PTH levels before hemodialysis are possibly less influenced by dialysis membrane. Serum levels of 8-OHdG after hemodialysis are different according to each dialysis modality (41–44); however, there was no statistically significant correlation between the serum levels of 8-OHdG and the type of membrane employed in this study. Therefore, we believe that the type of membrane used had little effect on the serum levels of these toxins and biomolecules in this study.

Our study has several limitations. First, the sample size is relatively small; therefore, we could not analyze many factors that influence the relationship between indoxyl sulfate and skeletal resistance to PTH. However, we could demonstrate that indoxyl sulfate is negatively correlated with bone formation independent of major factors such as PTH. Second, as this was a cross-sectional study, we could not determine whether indoxyl sulfate suppresses bone formation, although we showed that there is a negative correlation between indoxyl sulfate and bone formation. To confirm that accumulation of indoxyl sulfate in blood suppresses bone formation, it is necessary to investigate whether decreasing serum levels of indoxyl sulfate improves low bone turnover in humans. Third, we did not assess additional oxidative markers and antioxidants. Fourth, we did not assess other uremic toxins—phenylacetic acid may be one of candidate toxins that can interfere with the bone metabolism (10). Further studies will be necessary to investigate the relation between other uremic toxins and skeletal resistance to PTH in uremia. Finally, we did not assess bone histomorphometric parameters obtained by bone biopsy, which is the gold standard for the assessment of bone turnover. Although BAP and TRACP 5b have been useful markers and correlated well with bone histomorphometric parameters in patients with end-stage renal failure (38,39,45–48), biochemical markers of bone turnover still have some limitations for the assessment of bone turnover (49–51). Further studies assessed by bone histomorphology would be necessary to support our results.

CONCLUSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. REFERENCES

We demonstrated that indoxyl sulfate correlated negatively with bone formation markers independent of PTH. In contrast, indoxyl sulfate did not correlate with bone resorption and oxidative stress markers. These findings suggest that the accumulation of indoxyl sulfate in blood may induce, at least in part, low bone turnover in hemodialysis patients.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. REFERENCES
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