Laboratory results at baseline and at follow-up (N = 76).

<p>Abbreviations: BMD, bone mineral density; BUN, blood urea nitrogen; C-Ca, serum corrected calcium; CMV, cytomegalovirus; Cr, blood creatinine; DM, diabetes mellitus; HBV, hepatitis B virus; HCV, hepatitis C virus; FN, femoral neck; H hip; LS, lumbar spine; NS, not significant (<i>p</i>>0.05); P, serum inorganic phosphate; T, number of standard deviations above or below the mean value of BMD for sex-matched young adults; TC, serum total cholesterol; TG, serum triglyceride. LS-BMD was significantly greater at follow-up than at baseline. The albumin and calcium levels also decreased significantly, but they remained within normal ranges.</p

Cumulative dose of immunosuppressive agents and bone mineral density change between three bone conditions by WHO.

<p>The patients were divided, according to their baseline DXA, into normal (n = 5), osteopenia (n = 30), and osteoporosis (n = 41) groups. The osteoporosis group received a significantly greater cumulative prednisolone dose than did the osteopenia group (1326.5 mg vs. 724.5 mg; p = 0.005; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048481#pone-0048481-g001" target="_blank">Figure 1A</a>), and the increase in lumbar spine bone mineral density was also significantly greater in the osteoporosis group (0.033 g/cm² vs. 0.009 g/cm²; p = 0.028; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048481#pone-0048481-g001" target="_blank">Figure 1B</a>). The drugs included in the analysis of cummulative immunosuppresive therapy were prednisolone, 5 mg; mycophenolate, 250 mg; tacrolimus, 0.5 mg; sirolimus, 1 mg; and cyclosporine, 100 mg. Abbreviations: L, lumbar spine; H, hipbone; F, femoral neck. *Statistical significance at p<0.05.</p

Baseline and follow-up data for non–alendronate and alendronate patients.

<p>Abbreviations: BMD, bone mineral density; BUN, blood urea nitrogen; C-Ca, serum corrected calcium; Cr, blood creatinine; CMV, cytomegalovirus; DM, diabetes mellitus; FN, femoral neck; H, hip; HBV, hepatitis B virus; HCV, hepatitis C virus; LS, lumbar spine; NS, not significant (<i>p</i>>0.05); P, serum inorganic phosphate; T, number of standard deviations above or below mean BMD of sex-matched young adults; TC, serum total cholesterol; TG, serum triglyceride. NS*, no significance (p>0.05) between non-alendronate patients and alendronage patients. NS<b>^§</b>, no significance (p>0.05) of difference values between non-alendronate patients and alendronage patients. At follow-up, LS-BMD was significantly increased in the alendronate and non-alendronate groups (<i>p</i><0.05). The albumin levels also decreased significantly in patients not taking alendronate sodium, but they remained within normal ranges. The uric acid and creatinine levels were significantly increased at follow-up in patients not taking alendronate sodium, but these also remained within normal ranges.</p

Clinical variables associated with bone mineral density change of the lumbar spine in patients taking (n = 34) and not taking (n = 42) alendronate sodium.

<p>Abbreviations: CMV, cytomegalovirus infection; DM, diabetes mellitus; HBV, hepatitis B virus infection; HCV, hepatitis C virus infection; NS, not significant (<i>p</i>>0.05). Initially, simple linear regression analysis was performed to identify the variables associated with BMD change in patients taking alendronate sodium and those who were not. HCV infection was negatively associated with BMD change of the lumbar spine in patients not taking alendronate. Age at transplantation was positively associated with BMD change in the lumbar spine in patients taking alendronate. In advanced analysis by multiple linear regression, HCV infection was still the risk factor for adverse BMD change of the lumbar spine in patients without alendronate. No clinical variable was a risk factor for predicting BMD change of the lumbar spine in patients taking alendronate. <b><i>β</i></b> (95% CI): Linear regression standardized coefficients (95% confidence intervals).</p

Clinical variables associated with bone mineral density change (N = 76).

<p>Abbreviations: CMV, cytomegalovirus; DM, diabetes mellitus; HBV, hepatitis B virus; HCV, hepatitis C virus; NS, not significant (<i>p</i>>0.05). Initially, simple linear regression analysis was performed to identify the variables associated with bone mineral density (BMD) change in 76 patients. HCV infection and use of alendronate were associated with BMD change of the lumbar spine. Time since transplantation and use of alendronate were associated with BMD change of the hip bone. Age and age at transplantation were associated with BMD change of the femoral neck. In advanced analysis by multiple linear regression, HCV infection was still the risk factor for adverse BMD change of the lumbar spine. <b><i>β</i></b> (95% CI): Linear regression standardized coefficients (95% confidence intervals).</p

Clinical variables associated with bone condition change.

<p>A binary (non-osteoporosis and osteoporosis in follow-up) logistic regression analysis was performed to identify the variables associated with osteoporosis. The dependent variable was non-osteoporosis or osteoporosis. The independent variables were age, sex, DM, smoking, alcohol consumption, age at transplantation, time since transplant, use of immunosuppressive agents and use of Fosamax. Both the use of prednisolone (odds ratio [OR], 5.18; 95% confidence interval [CI], 1.6–16.4; p = 0.005) and the use of Fosamax (OR, 18.75; 95% CI, 5.42–64.76; p<0.001) were associated with the symptoms of osteoporosis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048481#pone-0048481-g002" target="_blank">Figure 2A</a>). In an ordinal logistic regression with multivariate analysis of the change of bone condition (grade 1, changed to the better; grade 2, no change; and grade 3, deterioration, as defined by WHO criteria and clinical variables), after adjusting for age, sex, status of diabetes (DM), smoking, alcohol consumption, time since transplant, age at transplant, and use of prednisolone, the use of Fosamax (OR, −3.115; 95% CI, −5.364 to −0.866; p = 0.007) was found to be associated with a positive prognosis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048481#pone-0048481-g002" target="_blank">Figure 2B</a>).</p

Baseline characteristics of 76 patients.

<p>Abbreviations: n, number of patients; CMV, cytomegalovirus; DM, diabetes mellitus; HBV, hepatitis B virus; HCV, hepatitis C virus.</p

Comparison of patients with (41) and without (35) osteoporosis at presentation and follow-up.

<p>The mean follow-up period was 14 months. In both the non-osteoporosis and the osteoporosis group, the LS-BMD significantly increased. At the end of the period, the cumulative dose of prednisolone and the LS-BMD differential were greater in the osteoporosis group.</p><p>NS*: p>0.05 between the osteoporosis and non-osteoporosis group.</p><p>Abbreviations: LS-BMD, lumbar spine bone mineral density; H-BMD, hip bone mineral density; FN-BMD, femoral neck bone mineral density; T score, number of standard deviations (SD) different from the mean value of the corresponding gender-matched young adult mean BMD. DM, diabetes mellitus; BUN, blood urea nitrogen; Cr, blood creatinine; Ca, serum calcium concentration; P, serum inorganic phosphate level; TC, serum total cholesterol level; TG, serum triglyceride level; NS: not significant, p>0.05.</p

Mesangial cells (MCs) inhibit splenocyte proliferation.

<p>Rat MCs were cultured until passage 5 (P5) and incubated with Con-A stimulated or non-stimulated splenocytes for 72 hours. Cell proliferation was measured by incorporation of tritiated thymidine (³H-TdR). <b>A</b>. Co-culture of WKY MCs and splenocytes resulted in a significant decrease in ConA-stimulated splenocyte proliferation in both 1∶10 and 1∶20 ratio of MC:Splenocyte. **, P<0.001 compared with splenocytes alone. The results are representative of three independent experiments. Cpm, counts per minute <b>B</b>. Co-culture of LEW MCs and splenocytes resulted in a significant decrease in Con-A-stimulated splenocyte proliferation in both 1∶10 and 1∶20 ratio of MC:Splenocyte. **, P<0.001 compared to splenocytes alone. The results are representative of three independent experiments. Cpm, counts per minute.</p

Genome-wide expression analysis by microarrays identifies two distinct transcriptomes in WKY and LEW MCs.

<p><b>A</b>. Dendogram representing the hierarchically clustering using all differentially expressed genes between WKY and LEW MCs in basal (unstimulated) and TNFα-stimulated cells. This shows that the strain (WKY or LEW) and the treatment (basal or TNFα-stimulated) effects cluster in 4 distinct groups. <b>B</b>. Validation of the markedly differentially expressed transcripts (fold change >10) by qRT-PCR. The upper panel shows the top differentially expressed candidates identified by microarray analysis with a false discovery rate (FDR) <0.01. The positive fold change (FC) values designate up-regulation in WKY MCs and negative FC values designate up-regulation in LEW MCs in basal conditions. The lower panel shows qRT-PCR validation for the transcripts showing differential expression in the microarray dataset. n = 4 rats, **, P<0.001. <b>C</b>. KEGG pathway analysis applied to differentially expressed genes between WKY and LEW MCs in basal state (WKY-LEW)_basal and (WKY-LEW)_TNFα identified the DNA replication and the vasculature development pathways as the most significant ones respectively. <b>D</b>. KEGG pathway analysis in LEW and WKY MCs treated with TNFα [(LEW)-(LEW) _TNFα and (WKY)-(WKY)_TNF] identified strain-specific pathways (shown in blue in the LEW and red in WKY) upon TNFα stimulation. Note that the DNA-replication pathway is the most significant one in the WKY MCs following TNFα stimulation.</p