Increased Risk of Osteoporosis in Patients With Nephrotic Syndrome. A Nationwide Population-Based Retrospective Cohort Study

Corticosteroid (CS) is commonly used in nephrotic syndrome (NS) and a risk factor for osteoporosis. We evaluated the risk of osteoporosis in patients with nephrotic syndrome using a nationwide population-based dataset. After adjusting for covariates, osteoporosis risk was found to be 3.279 times greater in the NS cohort than in the non-NS cohort, when measured over 11 years after NS diagnosis. Stratication revealed that age older than 18 years, congestive heart failure, hyperlipidemia, chronic kidney disease, liver cirrhosis, and NS-related disease including diabetes mellitus, hepatitis B infection, hepatitis C infection, lymphoma, and hypothyroidism, increased the risk of osteoporosis is when comparing the NS cohort with the non-NS cohort. Additionally, osteoporosis risk was signicantly higher in NS patients with CS use (aHR = 3.397). The risk of osteoporosis in NS patients was positively associated with risk of hip and vertebral fracture (aHR = 2.130 and 2.268, respectively). A signicant association exists between NS and subsequent risk for osteoporosis. Therefore NS patients, particularly those treated with CS, should be evaluated for subsequent risk of osteoporosis. NS versus non-NS incidence rate ratio of osteoporosis was examined. We used the univariate and multivariate Cox proportional hazards regression models with adjustment for age, sex, urbanization, hypertension (HTN), diabetes mellitus (DM), congestive heart failure (CHF), stroke, coronary artery disease (CAD), hyperlipidemia, chronic kidney disease (CKD), liver cirrhosis (LC), obesity, chronic obstructive pulmonary disease (COPD), dementia, postmenopause, hyperparathyroidism, alcohol-attributed disease, NS-related disease (including hepatitis B infection (HBV), hepatitis C infection (HCV), human immunodeciency virus infection (HIV), syphilis, herpes zoster, IgA nephropathy, lymphoma, leukemia, multiple myeloma, neoplasm, amyloidosis, sarcoidosis, SLE, rheumatoid arthritis (RA), Sjögren's syndrome, hyperthyroidism, hypothyroidism, and gestational proteinuria), medication including steroid use, relevant fractures(including hip, wrist, vertebral, and rib fracture), season, city location, urbanization, and level of care to calculate hazard ratio (HR) with a 95% condence interval (CI). Kaplan-Meier curved were used to calculate the cumulative proportion of osteoporosis incidence for both groups, and log-rank test was then employed to test differences between curves. We performed statistical analyses with SAS (V.9.3 for Windows), and a two-tailed P < 0.05 was considered statistically signicant.


Introduction
Nephrotic syndrome (NS) is de ned on the basis of heavy proteinuria, accompanied by hypoalbuminemia, hyperlipidemia, and edema. The mechanidsm of NS can be idiopathic and comprise kidney disease such as minimal-change disease, membranous nephropathy, and focal glomerulosclerosis. Secondary causes include systemic diseases such as diabetes mellitus, systemic lupus erythematosus (SLE), amyloidosis, and cancers, drugs, and infections. [1,2]The complications of NS are divided into two categories: disease-associated and drug-related complications. Both the disease-associated and drug-related complications of NS could be susceptible to osteoporosis. [3] Controversy remained as to whether people with NS have an increased risk of developing osteoporotic fractures. The reported prevalence range from 9-60% of osteoporosis among patients with NS. [4][5][6][7] Some studies have elucidated that NS is correlated with increased risk of osteoporosis, [6]and that the risk of osteoporosis also increases with increasing age, [8] notwithstanding, others have not. [9] It is unclear that the association between osteoporosis prevalence and NS progression. [6] Data on the risk of osteoporosis in patients with NS and corticosteroids (CS) use are scarce. [6] Therefore, this study used the National Health Insurance Research Database (NHIRD) to determine whether NS is a risk factor for osteoporosis and fracture. Further risk in NS patients who do and do not use CS is additionally evaluated.

Data source
The nationwide population study retrieved data from the 2000 and 2010 national health insurance research databases (NHIRD) which is a compsulsory single-payer program delineating practically 99.9% population of the country.
NHIRD contains inpatient and outpatient dataset regarding details of diagnoses based on International Classi cation of Diseases, Ninth Revision, Clinical Modi cation (ICD-9-CM) codes, gender, prescriptions assigned at contracted pharmacies. Researchers can get relevant claims information with scrambled patient identi cation numbers. The Institutional Review Board of Tri-service general hospital approved this study. All methods were performed in accordance with the relevant guidelines and regulations.

Ethics statement
Patient consent was not required for us to access the NHIRD because of encrypted patient personal information system to protect patient privacy.
Institutional Review Board of the Tri-Service General Hospital approved the study. (TSGHIRB No. 2-105-05-082). Study participants Figure 1 depicted the details of study design and speci c patient characteristics including inclusion and exclusion criteria. During 2000-2010, 26,614 patients who had been diagnosed with NS (ICD-9-CM code 581) during 2000-2010 been selected in our study. The cohort was con ned to at least two NS diagnoses during ambulatory visits or patients who had received at least one NS diagnosis during an inpatient visit. Furthermore, the cohort was circumscribed to patients with ICD-9codes assigned by a nephrologist with appropriate validation.
To ensure the accuracy of the data, we only included cases if they received 2 osteoporosis diagnoses for ambulatory visits or 1 diagnosis in inpatient care and the ICD-9 code was assigned by orthopedists and receiving at least one BMD examination were included in the osteoporotic group. The index date was selected as the date of the rst clinical visit for NS. Exclusion criteria were as follows: diagnosis with NS before 2000, osteoporosis (ICD-9-CM code 733) before the index date, incomplete data, and unknown gender. Strati cation analysis employed with the ratio of NS to non-NS patients maintained at 1:4 according to age, sex and index date. Using these criteria, 106,456 non-NS patients were identi ed.

Outcome
The main outcome was a discharge diagnostic claim of osteoporosis (ICD-9-CM code 733) after at least one BMD examination and orthopedist validation. For each participant, follow-up duration was estimated from the index data of osteoporosis diagnosis of uncensored subjects and until date of health insurance policy termination (mostly due to death) or Dec.31,2010 for those who were censored. Aside from analysis of baseline comorbidities, baseline sociodemographic characteristics, we also adjusted environmental factors according to urbanization level and the use of CS.

Statistical analysis
We rst compared distribution of age, sex, urbanization, comorbidities and CS use between NS and non-NS cohorts, which were examined using χ2 test. We then calculated the incidence density of osteoporosis (per 10 5 person-years). NS versus non-NS incidence rate ratio of osteoporosis was examined. We used the univariate and multivariate Cox proportional hazards regression models with adjustment for age, sex, urbanization, hypertension (HTN), diabetes mellitus (DM), congestive heart failure (CHF), stroke, coronary artery disease (CAD), hyperlipidemia, chronic kidney disease (CKD), liver cirrhosis (LC), obesity, chronic obstructive pulmonary disease (COPD), dementia, postmenopause, hyperparathyroidism, alcohol-attributed disease, NS-related disease (including hepatitis B infection (HBV), hepatitis C infection (HCV), human immunode ciency virus infection (HIV), syphilis, herpes zoster, IgA nephropathy, lymphoma, leukemia, multiple myeloma, neoplasm, amyloidosis, sarcoidosis, SLE, rheumatoid arthritis (RA), Sjögren's syndrome, hyperthyroidism, hypothyroidism, and gestational proteinuria), medication including steroid use, relevant fractures(including hip, wrist, vertebral, and rib fracture), season, city location, urbanization, and level of care to calculate hazard ratio (HR) with a 95% con dence interval (CI). Kaplan-Meier curved were used to calculate the cumulative proportion of osteoporosis incidence for both groups, and log-rank test was then employed to test differences between curves. We performed statistical analyses with SAS (V.9.3 for Windows), and a two-tailed P < 0.05 was considered statistically signi cant. Figure 1 shows that the 28,772 patients enrolled between January, 2000 and December, 2010 as inclusion criteria.
After strati cation, the risk of osteoporosis notably increased independent of age status, except in patients younger than 18 years (aHR was highest in the 30-39 years group and lowest in the ≧ 60 years group). The risk of osteoporosis also increased independent of CHF, hyperlipidemia, CKD, LC. We further evaluated the risk of osteoporosis strati ed by NS etiology subtypes. A signi cantly higher risk of osteoporosis was observed in patients with DM, HBV, HCV, lymphoma and hypothyroidism. Additionally, osteoporosis risk was signi cantly higher in NS patients with corticosteroid (CS) use (aHR = 3.397). The risk of osteoporosis in NS patients was positively associated with risk of hip and vertebral fracture (aHR = 2.130 and 2.268, respectively). (Table 3) Loss of a variety of plasma proteins (vitamin D -binding protein) and minerals in the urine, as well as steroid therapy can have great impact on bone integrity. [10] In a prospective study with 30 NS and 30 control patients conducted by Mohamed GB et al, compared to the control patients, the NS patients demonstrated a signi cantly lower level of serum OPG and parameters of bone formation (alkaline phosphatase and osteocalcin),conversly, a signi cantly higher 24-hour urinary Ca. [11] The bone lesions in these patients can be attributed to Vitamin D de ciency and elevated blood levels of PTH.
In the NS cohort, we observed that an age older than 18 years, and especially within the 30-39 years group, had the highest risk of osteoporosis after strati cation. In a prospective study by Gulati et al., the authors observed that only about 22% of children with idiopathic NS developed osteoporosis. [6] The authors also concluded older age at onset, lower total calcium intake (P < 0.0001), and greater cumulative steroid dose were the main predictive factor for a low BMD (P < 0.0001), (P = 0.005). A study by Hegarty et al. found that adult survivors of childhood minimal change nephrotic syndrome have signi cantly reduced forearm trabecular volumetric BMD, placing them at increased fracture risk at this site, indicating a delayed effect of osteoporosis in NS cohorts. [8] Besides, because in the NHIRD we use BMD to de ne osteoporosis, which may be less accurate than bone mineral content measurements, especially in children, this could have led to an underestimation of the number of patients in the NS cohort who progressed to osteoporosis. [11] DM is often associated with low levels of insulin, higher glucose levels with higher advanced glycation end-products which precipitate in collagen leading to reduce bone strength. The indirect effect of glycosuria with hypercalciuria leads to decreased levels of calcium in the body and poor bone quality, which hastens bone loss. [12] In ammatory cytokines, such as interleukin-1, interleukin-6 and tumor necrosis factor-alpha induced by chronic HBV infection which increase RANKL to stimulate osteoclast formation and bone resorption. Additonally, tumor necrosis factor-alpha can inhibit osteoblast differentiation and promote osteoblast apoptosis. Chronic HCV infection can also induce interleukin-6, which activates osteoclast to increase bone resorption. The combined effects of the aforementioned in ammatory cytokines can eventuating in coupling of decreased bone formation, and increased bone resorption to diminish the BMD. [13][14] Although the relationship between lymphoma and osteoporosis is unclear, osteopenia and osteoporosis in untreated NHL patients are regarded as a common nding. [15] Mild hypothyroidism is usually observed in NS patients, and results from losses of T4, free T4, T3, free T3, and TBG into the urine. [16] It is proposed that hypothyroidism is correlated with increased risk of fractures with unknown mechanism. Hypothyroidism causes general hypometabolism, with subsequent lowering of bone formation and resorption. The subsequent reduced calciuria may lead to decreased serum osteocalcin and alkaline phosphatase, with elevated parathyroid hormone, which may be a proposed mechanism for osteoporosis. [17] In our study, we observed a prominent deleterious effect of CS on osteoporosis. (aHR = 3.397; 95% CI = 3.101-3.623; P < 0.001). In table 3, we observed that the osteoporosis risk was not increased in our pediatric NS cohort (aged younger than 18 years) after strati cation. We further evaluated the role of CS in pediatric NS (aged younger than 18 years), using non-NS and non-CS as a reference, and found that use of CS signi cantly increased the risk of osteoporosis in both the NS cohort (aHR = 1.478; 95% CI = 1.009-1.803; P = 0.042) and the non-NS cohort (aHR = 1.315; 95% CI = 1.001-1.698; P = 0.049) (Fig. 3).
It is reported that after 12 months' use of more than 7.5 mg/day of prednisone can lead to trabecular bone loss. [18] Glucocorticoid-induced osteoporosis is caused by decreased bone formation and increased bone resorption through the coupling of bone resorption, gastrointestinal calcium absorption and renal tubule excretion imbalance. [19] In NS, CS have been reported to disturb the function of osteoblasts by decreasing the expression of RANKL soluble decoy receptor-OPG, and in contrast, activate the osteoclast by increasing the expression of M-CSF and RANKL. [19][20][21] There are several limitations to the present study despite the strength of this cohort study which comprise a large number of Asian patients. First, some adaptable risk factors such as body mass index, dietary habits and family history are not available in NHIRD. Laboratory results such as urine protein level, serum lipid and serum albumin are lacking.
Histopathologic changes involved in NS, speci cally minimal change disease, membranous nephropathy and focal segmental glomerulosclerosis results are not accessible. Second, bone densitometry results was unavailable thus put validating the diagnosed of osteoporosis in a di cult situdation. Neverheless, diagnostic accuracy was strengthen by limiting the study population to patients with at last one BMD examination and medical care for osteoporosis for more than 2 separate visits. Those physicians who enter the ICD-9 codes of osteoporosis were request to enter accurately subject because of big nes for incorrect entries. Finally, potential biases related to adjustment for confounding variables existed results in a lower quality of statistical analysis of data derived from a retrospective cohort study.