Profile of chronic kidney disease related-mineral bone disorders in newly diagnosed advanced predialysis diabetic kidney disease patients: A hospital based cross-sectional study

Accepted Manuscript Title: Profile of chronic kidney disease related-mineral bone disorders in newly diagnosed advanced predialysis diabetic kidney disease patients: A hospital based cross-sectional study Authors: S. Ray, A.M. Beatrice, A. Ghosh, S. Pramanik, R. Bhatacharjee, S. Ghosh, A. Raychowdhury, S. Mukhopadhyay, S. Chowdhury PII: DOI: Reference:

S1871-4021(17)30199-6 http://dx.doi.org/doi:10.1016/j.dsx.2017.07.019 DSX 821

To appear in:

Diabetes & Metabolic Syndrome: Clinical Research & Reviews

Please cite this article as: Ray S, Beatrice AM, Ghosh A, Pramanik S, Bhatacharjee R, Ghosh S, Raychowdhury A, Mukhopadhyay S, Chowdhury S.Profile of chronic kidney disease related-mineral bone disorders in newly diagnosed advanced predialysis diabetic kidney disease patients: A hospital based cross-sectional study.Diabetes and Metabolic Syndrome: Clinical Research and Reviews http://dx.doi.org/10.1016/j.dsx.2017.07.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title: Profile of chronic kidney disease related-mineral bone disorders in newly diagnosed advanced predialysis diabetic kidney disease patients: A hospital based cross-sectional study Short title: Chronic kidney disease related-mineral bone disorders

Authors: S. Ray 1, A.M. Beatrice 1, A. Ghosh 1, S. Pramanik 1, R. Bhatacharjee1, S. Ghosh 1, A. Raychowdhury 2, S. Mukhopadhyay1, S. Chowdhury 1

1. Sayantan Ray. MD, DM Senior Resident E-mail- [email protected] 2. Subhodip Pramanik, MD Senior Resident E-mail- [email protected] 3. Beatrice Anne M, MD, DM Senior Resident E-mail- [email protected] 4. Amritava Ghosh, MD,DM Senior Resident E-mail- [email protected] 5. Rana Bhatacharjee,MD,DM.MRCP RMO E-mail- [email protected] 6. Sujoy Ghosh, MD,DM, FRCP Associate Professor E-mail- [email protected] 7. Arpita Raychowdhury, MD,DM Associate Professor E-mail- [email protected] 8. Satinath Mukhopadhyay, MD,DM 1

Professor E-mail: [email protected] 9. Subhankar Chowdhury, MD,DM,MRCP Professor E-mail: [email protected]

Affiliations 1

Department of Endocrinology, Institute of Post Graduate Medical Education & Research

(IPGMER) and SSKM Hospital, Kolkata 2

Department of Nephrology, Institute of Post Graduate Medical Education & Research

(IPGMER) and SSKM Hospital, Kolkata Corresponding Author: Dr. Sayantan Ray Department of Endocrinology IPGMER and SSKM Hospital 244, AJC Bose Road Kolkata 700020 West Bengal, India e-mail- [email protected] Tel- 91-9231674135 ABSTRACT Aim: Chronic kidney disease related-mineral bone disorder (CKD‑MBD) has also been poorly studied in pre-dialysis CKD patients. The present study was conducted to find out the profile of metabolic bone disorders in pre-dialysis CKD patients with type 2 diabetes mellitus (T2DM). Methods: In this cross-sectional design, diabetic patients with newly-diagnosed stage 4 and 5 CKD were evaluated. Serum levels of calcium, phosphorus, intact parathyroid hormone (iPTH), 25 hydroxy vitamin D and total alkaline phosphatase were measured in all patients. Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry (DXA).

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Results: A total of 72 eligible patients participated (44 males, 28 females; age 54.2 ±11.7). Patients with CKD Stage 5 had a lower level of corrected serum calcium and significantly higher level of inorganic phosphorus, total alkaline phosphatase and iPTH as compared to stage 4 patients. Overall, 38.5% were hypocalcemic, 31.43 % were hyperphosphatemic. 24.2 % of CKD subjects were vitamin D deficient (< 10 ng/ml) and 41.4 % having vitamin D insufficiency (10-20 ng/ml). In stage 4, hyperparathyroidism (iPTH > 110 pg/ml) was detected in nearly 43 % of patients. However, in stage 5 only 32 % patients was found to have hyperparathyroidism (iPTH > 300 pg/ml). There was a good correlation between iPTH and total ALP (r=0.5, p= 0.0001) in this cohort. 25 (OH) vitamin D was inversely correlated with ALP (r= -0.39, P= 0.001) and showed negative correlation with urine ACR (r= -0.37, P= 0.002). As a group, the osteoporotic CKD subjects exhibited higher iPTH (220.1±153.8 vs. 119± 108 pg/ml, p< 0.05) as compared to those who were osteopenic or had normal bone density. There was significant correlation between BMD and iPTH (adjusted r= -0.436; P=0.001). In the multivariate regression model, we found intact PTH to predict BMD even after adjustment of all the confounders. Conclusion: The current study showed that adynamic bone disease is prevalent even in predialysis CKD population. High bone turnover disease may not be the most prevalent type in diabetic CKD. However, it could contribute to the development of osteoporosis in CKD subjects. Serum total ALP can serve as a biochemical marker to identify pattern of bone turnover where intact PTH is not available.

Key words: Chronic kidney disease, mineral and bone disorder, type 2 diabetes mellitus, osteoporosis

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1. Introduction Diabetes Mellitus (DM) is recognized as the most common cause of chronic kidney disease (CKD), and accounts for about 40% of end-stage renal disease (ESRD) cases in the United States [1,2]. In India, DM is an important cause of CKD in approximately 30–40% of the patients. In comparison to patients without diabetes, those with diabetes experience faster progression to ESRD and higher rates of cardiovascular disease (CVD) events and mortality [1]. Chronic Kidney Disease–Mineral and Bone Disorder (CKD-MBD) refers to the clinical syndrome of laboratory abnormalities, bone disease, and extraskeletal calcification, including the arterial system [3]. Disturbances of mineral metabolism, which occur in nearly all patients with CKD stages 3 to 5D, are associated with bone loss and fractures, cardiovascular disease, decreased quality of life, and increased mortality [4-6]. With the declining renal function, there is a progressive deterioration in mineral homeostasis leading to altered serum concentrations of calcium, phosphorus, parathyroid hormone (PTH) and vitamin D. Assessment of bone health is required not only for adjustment of therapy for abnormal mineral metabolism but also to evaluate fracture and cardiovascular risk. However, CKD‑MBD has been poorly studied, especially in predialysis population. Recent evidences suggest that the disordered mineral metabolism is more common and more severe in patients with diabetic nephropathy [7]. Nevertheless, there are limited data on the pattern of MBD induced by CKD in diabetic patients in Indian context. Furthermore, a considerable higher prevalence of adynamic bone disease (ABD) has been found in diabetic than in a non-diabetic pre-dialysis population [8-10]. Adynamic bone in patients with CKD is a clinical concern because of its potential increased risk for fracture and CVD. The pattern of abnormalities in diabetic patients with CKD has not adequately been addressed in previously. Recent studies in 4

Indian population [11, 12] have also shown that diabetic patients are more likely to have ABD but true prevalence of adynamic bone in Indian CKD population is unknown. In this study, we aimed to document the laboratory parameters and different forms of mineral disorders in treatment-naive stage 4 and 5 non-dialysed diabetic CKD patients.We evaluated the correlation between the biochemical parameters in identifying bone turnover pattern in this cohort using non-invasive methods for assessment of bone health in terms of bone turnover and mass. The bone mineral density (BMD) patterns in diabetic patients with stage 4 and 5 CKD and its relation with bone turnover pattern was also explored.

2. Materials and methods 2.1 Subjects This cross-sectional study was carried out on T2DM adults 30–75 years old (mean age: 54.2 years, N = 72). Study population was collected from outpatients attending Diabetes and Nephrology clinic of Institute of Post Graduate Medical Education and Research (IPGME&R) and SSKM Hospital, Kolkata (from March 2015 and September 2016). All participants were informed about the study procedure through a written consent form before participation. The study was complied with the Declaration of Helsinki and the research protocol was approved by the IPGME & R and SSKM Hospital Ethics Committee.

2.2. Inclusion & exclusion criteria Patients with newly diagnosed Stage 4 and 5CKD (based on history, estimated glomerular filtration rate [eGFR] of <30 mL/min/1.73 m2by the abbreviated modification of diet in renal disease (MDRD) formula, biochemical and ultrasonographic evidence of CKD) who were not yet

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on dialysis at the time of enrolment in the study. We excluded patients who were on calcium supplements, phosphate binders, vitamin D analogues well as who received glucocorticoids, anticonvulsants, NSAIDS and bisphosphonates. Patients were excluded if they had liver disease and history of bone fracture in preceding 6 months. Figure 1 outlines the flow chart followed for patient selection. Thorough general survey with special emphasis on blood pressure and dilated fundoscopy. In our cohort hypertension was defined as systolic blood pressure ≥ 140mmHg or diastolic blood pressure ≥ 90 mmHg or on therapy for hypertension or had past medical records consistently showing hypertension. Dilated fundus examination was carried with direct ophthalmoscope (dilation of the pupil is accomplished with medicated eye drops before funduscopy)

2.3 Biochemical assays A fasting blood sample was drawn for measurement of biochemical parameters. Serum levels of creatinine, albumin, total calcium, inorganic phosphorus, total alkaline phophatase (ALP), 25 (OH) vitamin D and intact parathyroid hormone (iPTH) were measured. The test methodologies used were as follows: Intact PTH assay was done using the solid-phase, two-site chemiluminescent enzymelabeled immunometric assay (Immulite/Immulite 1000) with an intra and inter-assay coefficient of variation of 5.5% and 8.6% respectively. Serum 25(OH) vitamin D was estimated using the ELISA technique (EUROIMMUN AG, Lubeck, Germany) with an intra and inter-assay coefficient of variation of 4.9% and 7 % respectively. The detection limit of the assay was 1.6 ng/ml. Estimation of serum ALP was performed using p- NPP kinetic method.

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2.4 Categories definition The definitions for hypocalcemia (corrected Ca<8.5 mg/dL), hypercalcemia (corrected Ca>10.5 mg/dL), hyperphosphatemia (PO4 >4.5 mg/dL), hypophosphatemia (PO4 <2.5 mg/dL), vitamin D deficiency (<10 ng/mL), vitamin D insufficiency (10-20 ng/mL), and vitamin D sufficiency (>20 ng/mL) were same for CKD stages 4and 5. In CKD 4 and 5. Hyperparathyroidism was defined as iPTH levels of > 110 pg/ml and > 300 pg/ml, respectively. Adynamic bone disease in CKD 4 and 5 was defined as iPTH level of <70 pg/mL and <100 pg/mL, respectively [13, 14]. 2.5 Bone mineral density BMD was measured by dual-energy x-ray absorptiometry (DXA) scan (Lunar DPX DXA system (GE Healthcare), with software version: 9.3) at the distal 1/3 of the radius, consistent with the recommendations of International Society of Clinical Densitometry (Official position statement 2005) [15]. It was estimated in gm/cm2 and comparative T- and Z-scores were calculated.

2.6 Statistical Analyses The statistical software namely SAS 9.2 (for windows), SPSS Ver. 20 (for windows), was used for the analysis of the data and Microsoft word and Excel were used to generate graphs, tables etc. Descriptive statistical analysis was performed in the present study. Results on continuous measurements were presented as Mean ± Standard deviation or Median (with interquartile range) for data on continuous scale depending on the distribution of data. The normality of the study variables was tested through Anderson Darling test, Shapiro-Wilk and QQ

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plot. The results on categorical measurements are presented in number (%). Significance was assessed at 5 %. P<0.05 was considered as statistically significant. Wilcoxon Mann-Whitney test was used to find the significance of study parameters between two groups of patients (CKD Stage 4 and 5) depending on the non-parametric distribution of data. Correlation among numerical was assessed by Spearmen correlation test (Rho) depending on the non-parametric distribution of data. Adjustment for any confounding factors was done by partial correlation test. Simple linear regression was used to establish the antecedent-outcome relationship between two study variables (BMD and iPTH).

3. Results

3.1 Demographic profile Seventy-two patients were included in this study with a mean age of 54.2 ±11.7 (range 1285) years. Majority of the subjects were in age group between 40 to 60 years (62.8 %). Males outnumbered females (44 males, 28 females). Age distribution of the population is represented in Figure S1 (supplementary file). Patients were divided into two groups: Group I included patients with CKD stage 4 and group II composed of patients with CKD stage 5. No exposure to dialysis was present in patients of CKD stage 5. Group I had 42 and group II had 30 patients. Duration of diabetes in CKD stage 4 group was 12.11±4.76 years and in stage 5 group was 13.53± 4.18 years. Hypertension was present in 73.6 % of patients. Retinopathy (both NPDR and PDR) was detected in 64 % of patients. There was no difference between stage 4 and 5 CKD patients in the prevalence of CKD‑MBD related symptoms.

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3.2 Biochemical parameters Comparison of biochemical parameters in patients with CKD stages 4 and 5 are given in Table 1. Patients with CKD Stage 5 had a lower level of corrected serum calcium and significantly higher level of inorganic phosphorus, total alkaline phosphatase and iPTH as compared to stage 4 patients. Serum 25(OH) D level was significantly lower in CKD stage 5 patients. Overall, 38.5% were hypocalcemic, 31.43 % were hyperphosphatemic. 24.2 % of CKD subjects were vitamin D deficient (< 10 ng/ml) and 41.4 % having vitamin D insufficiency (10-20 ng/ml). 27.1% patients had ALP in the lower third of reference range (70-100 U/L). PTH level <100 pg/mL was present in 52.8% in CKD Stage 4/5 patients. Distribution of different biochemical parameters in study subjects are shown in Fig. S2-S6 (supplementary file). In stage 4, hyperparathyroidism (iPTH > 110 pg/ml) was detected in nearly 43 % of patients. However, in stage 5 only 32 % patients was found to have hyperparathyroidism (iPTH > 300 pg/ml). Nearly 40 % patient in each CKD group showed iPTH levels consistent with low turnover bone disease (iPTH < 70 pg/ml in stage 4 and iPTH 100 pg/ml in stage 5 CKD). Stage specific categorization of patients according to IPTH levels is shown in Fig. S7 and S8. We examined the correlation between the various biochemical markers. There was a good correlation between iPTH and total ALP (r=0.5, p= 0.0001) in this cohort (Fig. 2). Significant negative correlation was noted between 25 (OH) vitamin D and iPTH levels (r= -0.32, P= 0.007) (Fig. 3). 25 (OH) vitamin D was inversely correlated with ALP (r= -0.39, P= 0.001). The correlation between corrected serum calcium and iPTH was weaker (r = -0.26, P=0.028). No significant correlation was found between corrected serum calcium/phosphorus and 25 (OH)

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vitamin D. 25 (OH) vitamin D showed negative correlation with urine ACR (r= -0.37, P= 0.002) (Fig. 4).

3.3 Dual-energy X-ray absorptiometry scan DXA scan showed that the prevalence of osteopenia (T-score -1 to -2.5) and osteoporosis (T-score <-2.5/Z-score < -2) in the CKD subjects was 41.6 % and 26.6 %, respectively. As a group, the osteoporotic CKD subjects exhibited higher iPTH (220.1±153.8 vs. 119± 108 pg/ml, p< 0.05) as compared to those who were osteopenic or had normal bone density. There was significant correlation between BMD and iPTH (adjusted r= -0.436; P=0.001) (Fig.5). Regression analysis was performed to see prediction of BMD with iPTH adjusting the effect of variables (age, gender, eGFR, serum creatinine and vitamin D status). In the multivariate regression model, we found intact PTH to predict BMD even after adjustment of all the confounders.

4. Discussion Despite the efforts of the CKD Registry of India, which collates data from an estimated 199 affiliated centers, [16] data regarding the characteristics of untreated CKD‑MBD in predialysis patients in India is scarce. More so, the pattern of abnormalities in diabetic patients with CKD has not adequately been addressed in previous studies. The study population comprised predominantly young and middle aged patients belonging to the low and middle income group. Our study involved newly diagnosed, treatment naive patients with CKD and all were diabetic. The mean age of our study population was slightly higher than other studies [11, 13, 17-18]. The main reason for these differences in the age may be due to the fact that we have included only diabetic patients. Males outnumbered females in our cohort (M:F 10

1.5:1). Among Indian studies, Agarwal et al. [19] (community based) showed a male prevalence of 48% among patients with serum creatinine more than 1.8 mg/dL, while other hospital-based studies found males constituting 60-78% of CKD population. Although, there is male predominance among CKD population in most studies, in our study the Male:female ratio is somewhat lower, signifying greater female representation. In our study, serum level of phosphorus, iPTH, and total ALP was significantly higher in patients of CKD stage 5 when compared with CKD stage 4. Corrected serum calcium and vitamin D were lower in patients of CKD stage 5 as compared to CKD stage 4. Agarwal showed that the mean corrected serum calcium in stage 4 and 5 CKD was 8.8 and 8.1 mg/dL, and the values for phosphate, total ALP, and iPTH were 4.6 and 6.0 mg/dL, 256.8 and 245.3 IU/dL and 184.0 and 339.5 pg/mL, respectively [13]. Our study corroborated with the study of Agarwal, although the median ALP and iPTH in stage 4 and 5 CKD were lower in our patients. Ghosh et al. [11] found similar biochemical parameters, although the median iPTH in stage 4 and 5D CKD were higher than our patients. However, this study included patients who were on dialysis. Jabbar, et al. found a mean iPTH of 308.8±177.5 pg/mL in patients with stage 4 and 5 CKD [20]. The median iPTH levels of patients with CKD stage 4 and 5 in our study were 82 pg/mL and 163 pg/ml, respectively. The reasons for these differences can only be speculated upon, and include difference in dietary habits, PTH assay methodology, and presence of other confounders. Moscovici et al. [21] described a close correlatio between PTH level < 150 pg/mL and low turnover bone disease. Malluche et al. [22] found PTH level as a predictor of high bone turnover in both black and white patients and of low bone turnover in white but not black patients. In a recent study by Sprague et al., [23] serum PTH levels correlated with bone turnover. Nevertheless, PTH measurements have not proved to be specific or sensitive enough to accurately

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diagnose bone turnover in all patients. The reasons for this weak correlation are not clear, but may include differences in PTH assays, therapies, and the racial composition of study populations [24]. Despite its weaknesses as a biomarker, PTH represents perhaps the best current option for noninvasive assessment of bone turnover. Due to decreased GFR and altered endocrine function of kidneys, the parathyroid-vitamin D-renal axis gets deranged which results in phosphate retention, hypocalcemia, decreased active vitamin D, and secondary hyperparathyroidism in majority of patients [14]. 25-OH vitamin D deficiency is common in CKD stage 3-5. LaClair et al. found 25-OH vitamin D level <75 nmol/L in 83% of patients with CKD stage 4 [25]. In a study of Jabbar, et al., more than 90% of patients with CKD stage 4 and 5 had vitamin D level less than 30 ng/mL [26]. In our study, 65.72 % of patients with CKD stage 4 and 5D had vitamin D level <20 ng/mL.In fact, vitamin D level showed an inverse relationship to iPTH level in this cohort. In a study by Wahl et al. serum 25hydroxyvitamin D was significantly lower among those with diabetes compared with those without diabetes (P, <0.001) [7]. Patients with diabetes also had greater degrees of proteinuria, which has been linked to lower vitamin D levels [27]. We have also found significant negative correlation between and urine ACR and 25 (OH) vitamin D. Although supplementation of cholecalciferol may improve secondary hyperparathyroidism without the need for the active compound (calcitriol or other analogues), this issue has not been studied adequately till now [28]. MBDs are well described in patients with CKD. Agarwal described hypocalcemia in 29.9% and 49.6% in CKD stage 4 and 5, respectively, and hyperphosphatemia in 45% and 41.8%, respectively [13]. Ghosh et al. found much higher prevalence of hypocalcemia and hyperphosphatemia even in comparison to the findings by Agarwal. [11] In our study, hypocalcemia (Ca < 8.5 mg/dL) was found in 38.5 % and hyperphosphatemia (PO4 > 4.5 mg/dL)

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in 31.4 % patients with stage 4/5 CKD. Thus, our data showed a lower prevalence of hypocalcemia and hyperphosphatemia in comparison to the data from other Indian studies. The main reason for these differences may be due to presence of low turnover state in diabetic CKD patients. Agarwal found hyperparathyroidism in 57.8% of patients with CKD stage 4 and in 39.4% of patients with CKD stage 5 [13]. He defined hyperparathyroidism (iPTH>110 pg/mL in stage 4 and iPTH > 300 pg/mL in stage 5 CKD) using K/DOQI guidelines. Jabbar, et al. observed prevalence of hyperparathyroidism in 60% of their patients of CKD stage 4 and 5 taking a cutoff of iPTH>300 pg/mL for both stages [20]. In the study by Ghosh et al., the prevalence of hyperparathyroidsm was much higher in both CKD stage 4 and 5D in comparison to previous studies [11]. This may be due to the use of different definitions and cutoff values for target range of iPTH. We have used similar stage specific cut-offs of iPTH as used by Agarwal. In our study, hyperparathyroidism was found in 42.8 % of patients with CKD stage 4 and in 32.1 % of patients with CKD stage 5. This lower prevalence possibly signifies low turnover bone in CKD patients with diabetes. We have found elevated total ALP (>130 IU/L) in nearly 40 % of stage 4/5 CKD patients. However, elevated total ALP level (>112 IU/L) was present in 43.59% and 76.66% of patients of CKD stage 4 and 5D, respectively in Ghost et al. study [11]. Jabbar, et al. found raised bone alkaline phosphatase (>45 IU/L) in 60% of their stage 4 and 5 CKD patients [20]. Spasovski et al. [8] found relatively low prevalence of hyperparathyroidism and adynamic bone disease (ABD) was the most prevalent type of renal bone disease in their pre-dialysis patients. Patient characteristics associated with ABD included male gender and diabetes. Only one Indian study addressed the issue of possible difference in MBD pattern between CKD patients with and without diabetes. They found that patients with DM had significantly lower serum phosphate (P=0.038), total ALP (P>0.001), and iPTH (P=0.042) levels when compared

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with patients without DM. They also had higher corrected calcium level [11]. This could mean that diabetic patients are more likely to have ABD. We found the marker combinations to be consistent with bone turnover pattern. In our study a good correlation of iPTH with TAP was observed. As a low cost measure we have looked at the TAP level and not the bone specific alkaline phosphatase (bALP). This is consistent with previous studies and supported the biological significance of these results [29, 30]. Jabbar, et al. found good correlation between (bALP) and iPTH. [20] In another study, [29] bALP < 25 U/L was able to differentiate ABD or normal bone from other types of bone disease with 89% sensitivity and 82% specificity in pre-dialysis patients. However, in a recent cross-sectional retrospective study the combination of PTH and bALP levels offered minimal additional discrimination [23]. In our study, the prevalence of osteopenia and osteoporosis in the CKD subjects was 41.6 % and 26.6 %, respectively. This figure is corroborative with that reported from the West. However, Jabbar, et al. found lower prevalence of osteopenia and osteoporosis (37% and 12%), likely due to the younger age and the shorter duration of CKD. Rix et al. [31] demonstrated osteoporosis in 30% of 113 pre-dialysis CKD population (GFR 37 ml/min). Osteoporosis was noted in 80% of the subjects by Urena et al., but those patients were on hemodialysis for a mean duration of 6.4 years [32]. The higher levels of iPTH as well as the markers of bone formation and resorption among osteoporotic subjects suggest a contribution of high turnover in the reduction of bone mass. In Jabbar, et al. study, the osteoporotic CKD subjects exhibited higher iPTH (549.2 ± 289.7 vs. 318.9 ± 180.2 pg/ml, P = 0.04), compared to those who were osteopenic or had normal bone density. We have found significant negative correlation between BMD and iPTH. Previous studies have shown an inverse relationship between BMD and PTH and some

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other markers [31-34]. We observed similar findings. Although guidelines recommend correction of vitamin D deficiency, it needs to be studied through randomized trials whether this approach can lead to amelioration of hyperparathyroidism. In one study, [35] patients who received weekly cholecalciferol supplementation exhibited a decrease in PTH levels which fell short of statistical significance, probably because of small number of participants. Certain limitations of this study must be noted: First, the inherent limitation of a hospital based survey involving a referred patient population is that it cannot describe the pattern of CKD‑MBD in the community. Secondly, there was no comparator group to find out any possible difference or unique pattern of MBD in diabetic CKD population. Thirdly, bone histomorphometry for histological assessment of CKD‑MBD was not available at our center due to resource limitations. Lastly, circulating PTH alone may not clearly distinguish adynamic or normal bone from hyperparathyroid bone disease and we did not evaluate bone turnover markers in this study. More so, we have looked at a single value, not multiple value based trend.

5. Conclusions Nearly 40 % of patients in each group (CKD stage 4 and 5) showed biochemical parameters consistent with low turnover bone disease, highlighting that adynamic bone disease is prevalent even in pre-dialysis CKD population. Relatively low prevalence of hyperparathyroidism in our cohort could mean that high bone turnover disease may not be the most prevalent type in diabetic CKD. A good correlation was observed between serum iPTH and total ALP in this cohort. Serum ALP can serve as a biochemical marker to identify pattern of bone turnover where intact PTH is not available. We have found inverse relationship between BMD and intact PTH which remained after adjusting for other variables, suggesting a contribution of high turnover in the in the genesis of osteoporosis in this cohort. A significant negative correlation was observed 15

between and urine ACR and 25 (OH) vitamin D. Degree of proteinuria in diabetic patients may have a link with low vitamin D levels. Conflict of interest The authors have no conflict of interest. Disclosure summary The authors have nothing to disclose. Acknowledgments This study received financial support in the form of research grant from Research Society for the Study of Diabetes in India (RSSDI)

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21. Gal-Moscovici A, Popovtzer MM. New worldwide trends in presentation of renal osteodystrophy and its relationship to parathyroid hormone levels. Clin Nephrol 2005;63:28489. 22. Malluche HH, Mawad HW, Monier-Faugere MC. Renal osteodystrophy in the first decade of the new millennium: analysis of 630 bone biopsies in black and white patients. J Bone Miner Res 2011;26:1368-76. 23. Sprague SM, Bellorin-Font E, Jorgetti V, Carvalho AB, Malluche HH, Ferreira A, et al. Diagnostic accuracy of bone turnover markers and bone histology in patients with CKD treated by dialysis. Am J Kidney Dis 2016;67:559-66. 24. Covic A, Voroneanu L, Apetrii M. PTH and/or Bone Histology: Are We Still Waiting for a Verdict From the CKD-MBD Grand Jury? Am J Kidney Dis 2016;67:535-8. 25. LaClair RE, Hellman RN, Karp SL, Kraus M, Ofner S, Li Q, et al. Prevalence of calcidiol deficiency in CKD: A cross-sectional study across latitudes in the United States. Am J Kidney Dis 2005;45:1026-33. 26. Jabbar Z, Aggarwal PK, Chandel N, Sakhuja V, Jha V. Vitamin D deficiency in Indian CKD patients. Indian J Nephrol 2007;17:95. 27. Thrailkill KM, Jo CH, Cockrell GE, Moreau CS, Fowlkes JL. Enhanced excretion of vitamin D binding protein in type 1 diabetes: a role in vitamin D deficiency? J Clin Endocrinol Metab 2011; 96:142–9. 28. Zisman AL, Hristova M, Ho LT, Sprague SM. Impact of ergocalciferol treatment of vitamin D deficiency on serum parathyroid hormone concentrations in chronic kidney disease. Am J Nephrol 2007;27:36-43.

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Figure legends:

Figure1. Protocol followed for patient selection

Figure 2: The scatterplot showing continuous positive linear relationships between iPTH and ALP

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Figure 3: The scatterplot showing continuous negative linear relationships between iPTH and 25 (OH) vitamin D

Figure 4: The scatter plot showing continuous negative linear relationships between vitamin D and ACR

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Figure 5: The correlation between BMD and iPTH by Spearman Correlation analysis

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Table 1.Comparison of biochemical parameters between CKD stage 4 & 5

Laboratory parameters

CKD 4

CKD 5

Median (Q1,Q3)

Median (Q1, Q3)

Serum creatinine

2.8 (2.4, 3.1)

4.4 (3.9, 5.6)

<0.001

Serum calcium (corrected)

8.9 (8.4, 9.5)

8.2 (8, 8.6)

0.0004

Serum phosphate

3.9 (3.7, 4.4)

4.7 (4.2, 5.15)

0.0002

Serum total ALP

105 (87, 134)

142 (120, 201)

0.0008

Serum iPTH

82 (34, 134.5)

163 (94.4, 288.7)

0.0002

Serum 25 (OH) Vitamin D

19.15 (13.6, 23.4)

10.95 (9.3, 16.4)

0.006

p<0.05 considered as statistically significant, p value determined by Mann-whitney test.

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p value