Prevalence of CKD-MBD in pre-dialysis patients using biochemical markers in Enugu, South-East Nigeria.

BACKGROUND
As kidney function declines, there is a progressive deterioration in mineral homeostasis with disruption of normal serum and tissue concentration of phosphorus and calcium, and changes in circulating levels of hormones-parathyroid hormone (PTH), calcitriol (1,25(OH)2 D), and Fibroblast growth factor-23 (FGF-23).


OBJECTIVE
This study was aimed at determining the prevalence of markers of CKD-MBD in pre-dialysis patients.


METHODS
We evaluated consecutively 168 subjects made up of 85 CKD patients and 83 healthy controls, who were attending the renal clinics and medical outpatient of University of Nigeria Teaching Hospital, Enugu. GFR was estimated and serum calcium, phosphorus, alkaline phosphatase, PTH, and 25(OH) D levels assayed.


RESULTS
The prevalence of various mineral bone disease abnormalities were 70% hyper-phosphatemia, 85% hyper-parathyroidism, and 100% low levels of 25 (OH) D among the patients. Estimated GFR correlated negatively with both serum phosphorus, and PTH. Age of the patients ranged from18-76 years with a male to female ratio of 1.7:1. Chronic Glomerulonephritis (CGN), hypertension and diabetes mellitus caused CKD in 75% of the patients. There was no significant decrease in serum calcium levels of patients compared to controls. The patients did not have pathologically raised alkaline phosphatase, although their mean level was significantly higher than that of the control group.


CONCLUSION
Low 25 (OH) D levels (insufficiency/deficiency), hyperparathyroidism, and hyper-phosphatemia were the obvious markers of CKD-MBD in our pre-dialysis patients. These should be evaluated at presentation in these patients.


Introduction
Chronic kidney disease (CKD) is an international public health problem affecting 5-10% of the world population. 1 It is classified according to severity from stages 1-5.Chronic kidney disease mineral bone disorder (CKD-MBD) is a systemic disorder.The broadened concept of CKD-MBD, includes abnormal mineral metabolism (the focus of this study), abnormalities in bone morphology and extra skeletal calcifications.These are closely inter-related and together make major contribution to morbidity, and mortality of patients with CKD.
The metabolic changes that occur in patients with CKD have a profound influence on mineral and bone metab-olism.CKD results in altered levels of serum phosphorus, calcium, vitamin D, PTH, and Fibroblast Growth Factor-23 (FGF-23).Beginning in CKD stage 3, the ability of the kidneys to excrete a phosphorus load is diminished leading to raised phosphorus and PTH, and decreased 1,25 (OH) 2 D levels associated with elevated FGF-23 levels.The conversion of 25 (OH) D to 1, 25 (OH) 2 D is impaired, reducing intestinal calcium absorption.The kidneys fail to respond adequately to PTH which normally promotes phosphaturia and calcium reabsorption; and to FGF-23 which enhances phosphate excretion.There is in addition, at the tissue level a down regulation of vitamin D receptors and resistance to the action of PTH.
Bone abnormalities are found almost universally in patients with CKD stage 5 and in majority of patients with stages 3-5. 2 The biochemical abnormalities form the primary routine indicators in management of CKD-MBD, though there are limitations.Diagnosis for CKD-MBD using the invasive bone biopsy for bone turnover, mineralization, and volume; or arterial, valvular or coronary calcification score using gold standards such as electron beam CT, or multi-slice CT; or measuring bone mineral density (BMD) by Z score of dual energy X ray absorptiometry (DXA), ultrasound or echocardiography to assess tissue calcification, are not necessary in routine clinical practice especially in a developing country as ours.Therefore, changes in serum calcium, phosphorus, PTH, 25 (OH) D, and total alkaline phosphatase were used in this work to assess CKD-MBD in the study group.These changes contribute to increased cardiovascular mortality in affected patients.It is also important to quantify MBD as a complication of CKD even before renal replacement therapy (RRT) and during treatment.Interventions are aimed at controlling hyperphosphataemia, hyperparathyroidism, vitamin D deficiency among others, and include phosphate binding, supplementation with vitamin D analogues, use of calcimimetics, parathyroidectomy, and even kidney transplant.
Their pattern in pre-dialysis and especially in black CKD patients in developing countries is not clear.This study, using the biochemical methods explored the prevalence of these markers in black Ibo patients of South Eastern Nigeria.Excluded were patients on dialysis, taking phosphate binders, calcium supplements, or vitamin D analogues; or having chronic liver disease, and patients with primary hyperparathyroidism or parathyroid surgery.

Statistical analysis
Analysis was performed using Statistical Package for Social Sciences (SPSS) version 20 and results were presented as means and standard deviation.Student t test was used to compare patients' variables with controls.X2 for trend, Spearman's coefficient of correlation as well as multiple regression analysis were used to assess correlations of parameters.A p value of < 0.05 was considered significant.

Results
The baseline characteristics of the study population were summarized in Table 1.There were 85 patients and 83 controls.Majority, 63.1% were males.The subjects were aged between 18 and 86 years.There was no difference between the mean age, body mass index, and serum calcium level of the patients and controls.The weight of the controls was significantly higher than the patients.The mean systolic blood pressure (SBP), diastolic blood pressure (DBP), serum creatinine, serum potassium, phosphate, alkaline phosphatase, and parathyroid hormone were significantly higher in the patients compared to controls.On the other hand, eGFR, serum bicarbonate and 25(OH) D levels were significantly lower in the patients compared to controls.The median serum PTH and 25 (OH) D levels of the patients were 196.8pg/ml and 25nmol/l respectively.
The various features of the patients are shown in Table 2. Chronic Glomerulonephritis, hypertension and Diabetes mellitus were the main causes of chronic kidney     This showed a strong evidence that the odds of vita-min D deficiency increased significantly with increasing CKD stage (table 5).

Discussion
The individuals studied were all Ibo Nigerians, of the black race: an adverse risk factor for kidney diseases. 3hey were mostly middle aged as against the geriatric CKD patients seen in developed countries.Studies in the past in Nigeria, and in most sub-Saharan African countries, showed that kidney diseases occur in relatively younger age group. 4,5,6Most of the patients had pallor (83.5%) even at stage 3 disease, most also had peripheral edema (70.6%) and half of them had other symptoms of uremia at first presentation.Haemoglobin estimation would have given a more objective estimate of those that are pale.However, this was not included in our protocol as it has no direct bearing on the objectives of the study.Majority of our patients present late, (50.6%) presented at stage 4 in this study.Late presentations/ referrals contributed to poor outcomes in our CKD patients.This finding is similar to other studies that noted late presentation and severe illness at first presentation. 5,7,8,9The predominant causes of ESRD in Africa are essentially CGN and hypertension, with both sharing the lead in most study series. 6This study found CGN the predominant cause of CKD.However, a rise of the percentage contribution of diabetes has been noted in various centers, consistent with urbanization and improvement in the living standards in African countries. 10Diabetes was the 3rd most common cause of CKD in this study, accounting for 20% of cases.

Markers of CKD-MBD
Studies have shown that decreases in calcitriol levels occurred in patients with early CKD and preceded increases in serum PTH levels. 11These are in agreement with this study in which none of the patients had sufficiency of 25 (OH) D (from FGF-23 inhibition), while about 15% of them had normal levels of PTH.
Though most of these patients (76%) were not in end stage renal disease, there was still a high prevalence of these markers of CKD-MBD.This may explain the high mortality rates even in earlier stages of CKD.This study showed low levels of vitamin D, with high rates of hyperparathyroidism and hyperphosphataemia.
In SEEK study 12 , where pre-dialysis patients were similarly studied, it was found that blacks had significantly lower levels of 25(OH) D but higher levels of calcium, phosphorus and PTH.This high secondary hyperparathyroidism (SHPT) and 25(OH) D deficiency occurs early in the course of CKD, irrespective of age, gender, diabetes mellitus, eGFR, calcium and phosphorus.Some studies have suggested that this high prevalence might be due to blacks having reduced calcium sensing capacity, 13 or due to greater skeletal resistance to PTH among blacks. 14e patients and controls had normal mean serum calcium levels which were not significantly different between them.Again, in the CHOICE study, 15 serum calcium level at the commencement of dialysis was 2.34 mmol/L, similar to values in our study.Llach et al, 16 also found from their work that serum calcium, phosphorus, and ALP are usually normal, until late in the course of CKD.Our study like the one by Gutierrez et al 12 has shown that raised PTH and phosphorus levels, do occur with normal calcium and low vitamin D levels, especially among blacks.In-fact calcium is said to be frequently but not consistently abnormal with reduced GFR. 17Since the results in our study are cross sectional, causal relationships between dependent variables (kidney function) and independent variables (PTH, Vitamin D metabolites, calcium and phosphorous) cannot be defined.No longitudinal data has been reported here, African Health Sciences Vol 15 Issue 3, September 2015 thus relationships between the variables and different eGFR levels may change over time in different individuals.
There was a 69.4% hyperphosphataemia in this study.This high prevalence is similar to those found in Benin, Nigeria by Onyemekeihia et al 18 (79%) and Sanusi et al 19 at Ife, Nigeria (60%).The high prevalence of hyperphosphataemia occurs despite the fact that Africans are likely to have lower daily intake of dairy products and other sources of calcium and phosphorus. 12This might be due to renal resistance to the phosphaturic stimulus of raised PTH seen in blacks. 17ere is currently a phosphate-centric paradigm for the pathophysiology and therapy of CKD.In our environment, serum phosphorus is routinely assayed in patients.There is, the tendency to treat hyperphosphataemia with available calcium containing phosphate binders inaddition to vitamin D analogues.Also patients are dialyzed with high calcium containing dialysate.Without concomitant assays of serum PTH, vitamin D, and FGF-23, this practice no longer enjoys a favorable review, and predisposes patients to Adynamic bone disease (ABD) which worsens vascular calcification type of MBD as noted, in the work by Sanusi et al. 18 Also, calcium in these compounds has a stimulatory effect on the secretion of FGF-23 which further sustains the MBD.This however is not the case with Sevelamer or Lanthanum carbonate. 20ough ALP levels, were significantly higher in patients than controls, absolutely high levels of total ALP (>95U/L) were insignificant in both groups and showed that this affordable assay is not a valid surrogate marker for CKD-MBD in pre-dialysis patients.The bone fraction (bALP), however might be.Raised ALP has been shown to have a linear and incremental association with both all cause and cardiovascular disease mortality 21 because it reduces the pyrophosphate/phosphate ratio.Pyrophosphate is an inhibitor of vascular calcification.
All patients in this study had low 25(OH) D levels.The study showed that majority of the patients (62.4%) had 25 (OH) D deficiency, worse in stage 5 than 3, and the remaining 37.6% had insufficiency, more in stage 3 than 5.The progressive deficiency in the stages was found to be significant by X2 for trend.The Gutierrez et al 12 study found low levels of 25(OH) D as well, in Blacks.
Other studies done in USA 22,23 and UK 24 showed similar findings.Wolf et al 23 found 78% of patients they studied, had low 25(OH) D i.e. <75nmo/L, with blacks being more deficient than whites.Gonzalez et al, 22 found that 86% of 43 CKD patients they studied had inadequate 25(OH) vitamin D. Kosmadakis et al, 24 studied stages 3 and 4 CKD patients, found 25(OH) D insufficiency with values not much different in the 2 stages (39.8±24versus 38.3±22.3nmol/L).This feature was similar to our findings.The finding of low 25 (OH) D in our study is significant considering the increased allcause mortality noted in patients with deficiency as was documented by Mehrota et al. 25 They studied 3011 predialysis patients, found that those with 25(OH) D levels <37.5nmol/L had increased risk for all-cause mortality, compared to those with vitamin D sufficiency.
Levin et al, 11 found a high SHPT prevalence of 56% in mainly stages 3 and 4 pre-dialysis patients.The mean PTH levels were noted to be 71.7±59.08,and 185.5±159.88pg/mlrespectively.They did not study stage 5 disease patients.The higher prevalence and higher values in our study could be explained by various factors.The patients in this study were younger and purely blacks and both factors have been documented as causes of high SHPT. 26Furthermore, our study also included stage 5 CKD patients.PTH level > 300pg/ml is associated with Blacks, long duration of ESRD and varies inversely with age. 27Similar to this index study, Levin el al 11 found no correlation between hyperparathyroidism and serum calcium, and ALP especially in stage 3 disease.This underscores the need to assay PTH levels for diagnosis of MBD early in CKD as calcium, ALP, and phosphorus may be normal even with elevated PTH.
It was instructive that as expected, both hyperparathyroidism and hyperphosphataemia correlated inversely with eGFR, even in multiple regression analysis.However, that vitamin D levels did not correlate with eGFR could be explained by its inhibition caused early by increased FGF-23 in the course of MBD.Unfortunately FGF-23 was not assayed in the study.

Conclusion
The findings of this study suggest that MBD as a complication is common in our CKD patients.Raised PTH, low 25(OH) D, and raised phosphorus levels were the most prevalent markers, even in this population of African Health Sciences Vol 15 Issue 3, September 2015 pre-dialysis patients.Total alkaline phosphatase was not found to be a good surrogate marker for CKD-MBD in our predialysis patient population.It did not correlate with early markers -serum PTH and vitamin D. This cheap MBD marker may therefore not be ideal in pre-dialysis CKD patients.Majority of our patients presented, or were referred late.Clinical features of MBD in CKD were poor guides to the presence of MBD in our pre-dialysis patients.It therefore may not be proper for practitioners to wait for symptoms and signs to manifest.

Limitations of the study
Due to the cross-sectional nature of this study, patients were assessed only at presentation.There is a chance of changes in CKD stages estimation on further patient follow up.There are also diurnal variations in the metabolites assayed, some variations occur with meals, and different coefficients of variation of assays exist.Bone fraction (bALP) ALP and FGF-23 levels could not be assayed in this study.
Eighty five adult CKD patients (in stages 3-5) and 83 healthy control subjects presenting for the first time at the renal clinic and medical outpatient clinic of the University of Nigeria Teaching Hospital (UNTH), Ituku/ Ozalla, Enugu, between December 2010 and May 2011, were recruited consecutively for the study.Ethical clearance was obtained from Ethics Committee of UNTH, Enugu.All subjects were physically assessed, and had serum PTH, 25 (OH) D, Calcium, Phosphorus, total alkaline phosphatase, Bicarbonate, Potassium, and Creatinine measured.Glomerular filtration rate (eGFR) was estimated based on 4 variable MDRD formula.PTH and 25 (OH) D were analyzed using ELISA based International Immuno-Diagnostics-USA; PTH EIA (Lot # 1386) and Vitamin D 25 (OH) EIA (Lot K2110-100722) kits for iPTH and 25 (OH) D assays respectively.The measurement of serum 25 (OH) D is regarded as the best measure of vitamin D status, because of its long half-life of approximately 3 weeks i.e. the storage form of the vitamin.The active form 1, 25 (OH)2D3(-Calcitriol) has short half-life of 4-6 hours.