Decreased in vitro osteoblast proliferation and low turnover bone disease in nonuremic proteinuric patients

original article

http://www.kidney-international.org & 2007 International Society of Nephrology

Decreased in vitro osteoblast proliferation and low turnover bone disease in nonuremic proteinuric patients CB Dias1, LM dos Reis1, VF Caparbo2, FG Graciolli1, RMA Moyse´s1, RT Barros1, V Jorgetti1 and V Woronik1 1

Renal Division, Department of Internal Medicine, University of Sa˜o Paulo, Sa˜o Paulo, Brazil and 2Rheumatology Division, Department of Internal Medicine, University of Sa˜o Paulo, Sa˜o Paulo, Brazil

Patients with proteinuria, even those with normal glomerular filtration rate, often present abnormal bone histology. We evaluated bone histology and the in vitro proliferation of osteoblasts in samples obtained from 17 proteinuric patients with primary glomerulopathies. Histomorphometric analysis of bone biopsies was performed, and bone fragments were obtained for osteoblast culture, in which we evaluated cell proliferation. In comparison to controls, patients presented lower trabecular bone volume (20.9714.5% vs 26.875.9%; P ¼ 0.0008); lower trabecular number (1.770.2/mm vs 2.070.3/mm; P ¼ 0.004); and greater trabecular separation (475.5796.4 lm vs 368.3786.2 lm, P ¼ 0.0002). We also found alterations in bone formation and resorption: lower osteoid volume (0.970.7% vs 2.071.4%; P ¼ 0.0022); lower osteoid thickness (6.472.8 lm vs 11.573.2 lm; Po0.0001); less mineralizing surface (4.673.1% vs 13.576.0%; Po0.0001); lower bone formation rate (0.0370.04 lm3/lm2/ day vs 0.0970.05 lm3/lm2/day; Po0.0001); and greater osteoclast surface (0.3570.6 vs 0.0570.1%, P ¼ 0.0016). Mean in vitro osteoblast proliferation was lower in patients than in controls (910.27437.1 vs 2261.071121.0 d.p.m./well, P ¼ 0.0016). Serum concentrations of 25-hydroxyvitamin-D3 correlated negatively with proteinuria and positively with in vitro osteoblast proliferation. Our results demonstrate that nonuremic proteinuric glomerulonephritic patients present bone structure disorder, low bone formation and high bone resorption, as well as low osteoblast proliferation. Kidney International (2007) 71, 562–568. doi:10.1038/sj.ki.5002084; published online 17 January 2007 KEYWORDS: proteinuria; glomerulopathy; bone histomorphometry; osteoblasts; vitamin D

Correspondence: CB Dias, Faculdade de Medicina, Universidade de Sa˜o Paulo, Nephrology, Av. Dr Arnaldo 455, 3 andar, sala 3342, 1246903, Rua Ec¸a de Queiroz 58 apto 124 CEP 04011030. Vila Mariana. Sa˜o Paulo – Brasil. E-mail: [email protected] Received 20 February 2006; revised 10 October 2006; accepted 21 November 2006; published online 17 January 2007 562

Vitamin D3 is a leading factor for local and systemic control of bone remodeling. It affects the activity of both boneforming osteoblasts and bone-resorbing osteoclasts by modulating the synthesis/signaling of growth factors and cytokines.1 Disorders of the vitamin D system can promote an uncoupling of bone turnover, with various presentations of clinical bone disease. In a bone histomorphometry study involving vitamin D system was shown that, in nonnephrotic vitamin D-deficient transgenic rats, bone mineral density decreases and trabecular separation increases.2 In another study, cultured osteoblasts obtained from osteoporotic patients and stimulated with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) presented less response than osteoblasts obtained from normal controls.3 Patients with glomerular diseases and proteinuria present excessive urinary excretion of various vitamin D metabolites and their binding proteins.4–6 In addition, even when the glomerular filtration rate (GFR) is near normal, hypocalcemia and intestinal malabsorption of calcium, as well as reduced serum vitamin D metabolites, are frequently identified.7–9 Therefore, these patients can experience significant biological consequences of bone metabolism and abnormal bone histology. Some data show normal bone histology in most such patients,9,10 osteomalacia in 17–56.7% and even mixed bone disease in 7–50%.11–13 Such discrepant data might be explained by the inclusion of patients at different stages of the disease and by the corticosteroid treatment given to some of the patients studied. The identification of bone disease is particularly relevant to patients with nephrotic syndrome, as the incidence of treatment-resistant and progressive glomerular disease, both leading causes of chronic kidney disease (CKD), is increasing. This makes such patients vulnerable to developing uremic bone disease. Nevertheless, the histological bone alterations identified in patients with nephrotic syndrome do not consistently correlate with serum concentrations of divalent ions or calciotropic hormones. Levels of parathyroid hormone (PTH) are near normal, despite low serum ionized calcium levels,12,14 serum phosphorus concentration is usually normal,12 and calcitriol remains normal in most patients.12,14 Therefore, the mechanisms that result in altered Kidney International (2007) 71, 562–568

original article

CB Dias et al.: Histology studies in nonuremic proteinuric patients

In the histomorphometric analysis, comparing mean patient values to those of controls, we found that bone structure was altered, the study group patients presenting lower (trabecular bone volume, 20.9714.5 vs 26.875.9%; P ¼ 0.0008), lower (trabecular number, 1.770.2/mm vs 2.07 0.3/mm; P ¼ 0.004) and greater (trabecular separation, 475.5796.4 mm vs 368.3786.2 mm; P ¼ 0.0002). In one patient, the trabecular bone volume was so low (o11%) that it constituted an increased fracture risk. That patient also presented the lowest level of serum 25(OH)D3 (5.02 ng/ml). In addition, most indices of bone formation were significantly lower in patients compared to controls: (osteoid volume, 0.970.7% vs 2.071.4%; P ¼ 0.0022); (osteoid thickness, 6.472.8 mm vs 11.573.2 mm; Po0.0001); (mineralizing surface, 4.673.1% vs 13.576.0%; Po0.0001); and bone formation rate (0.0370.04 mm3/mm2/day vs 0.0970.05 mm3/mm2/day; Po0.0001). As for bone resorption, eroded surface was similar in both groups, however 41% of the study group patients presented

a

Serum creatinine (mg/dl) GFR (ml/min/1.73 m2) Serum albumin (g/dl) Ionized calcium (mg/dl) Serum phosphorus (mg/dl) iPTH (pg/ml) 25(OH)D3 (ng/ml) 1,25(OH)2D3 (pg/ml) Proteinuria (g/day) Calciuria (mg/kg/day)

Mean7s.d. 1.070.2 87.3721.9 2.371.0 5.070.2 4.170.7 26.3715.6 18.4710.3 32.1717.2 7.574.5 0.470.5

Range 0.7–1.7 56–120 0.96–4.5 4.6–5.4 3.1–5.9 11.0–62.0 5.0–37.7 12.2–67.7 1.5–17.3 0.05–1.7

Reference values 0.5–1.2 80–120 3.5–5.0 4.8–5.4 2.3–4.6 11–60 8.9–46.7 15.9–55.6 o0.2 2.571.2 (22)

GFR, glomerular filtration rate; iPTH, intact parathyroid hormone; 25[OH]D3, 25-hydroxyvitamin D3; 1,25[OH]2D3, 1,25-dihydroxyvitamin D3. Kidney International (2007) 71, 562–568

0.5 1.0 1.5 Calciuria (mg/kg/day)

2.0

c Serum albumin (mg/dl)

Characteristic

r=– 0.54 P=0.02

10

0 0.0

Table 1 | Biochemical characteristics of the patients studied (n=17)

b5

20

Serum albumin (g/dl)

The group of patients studied consisted of 11 men and six women. The mean age was 37.479 years (range, 19–55 years). The results of the biochemical analysis are shown in Table 1. Twelve of the 17 patients presented pronounced proteinuria (mean, 9.474.0 g/day) and low serum albumin (mean, 1.770.5 g/dl). The remaining five patients presented non-nephrotic proteinuria (mean, 2.871.2 g/day) and normal serum albumin (mean, 3.670.8 g/dl). In two patients, the concentration of ionized calcium was below normal. In another two patients and five patients, respectively, concentrations of 25-hydroxyvitamin D3 (25(OH)D3) and 1,25(OH)2D3 were below normal.

Bone histomorphometry

4 r=0.5 P= 0.03

3 2 1 0 0.0

0.5 1.0 1.5 Calciuria (mg/kg/day)

2.0

d 5 4

r=0.80 P<0.0001

Proteinuria (g/day)

RESULTS

Urinary excretion of calcium was lower than in the general population22 and correlated negatively with proteinuria (r ¼ 0.52; P ¼ 0.03; Figure 1a) and positively with serum albumin (r ¼ 0.5; P ¼ 0.02; Figure 1b). Serum concentrations of 25(OH)D3 were found to correlate positively with serum albumin (r ¼ 0.80; Po0.0001; Figure 1c) and negatively with proteinuria (r ¼ 0.4; P ¼ 0.04; Figure 1d). Comparing the 12 most proteinuric patients with the five least proteinuric, we observed that serum levels of 25(OH)D3 were lower in the former than in the latter (13.475.6 ng/ml vs 30.579.1 ng/ml; P ¼ 0.0003).

Proteinuria (g/day)

bone metabolism and bone histology in patients with proteinuric glomerular diseases, before the drop in GFR, are still unclear despite strong evidence implicating vitamin D deficiency. Recently, some cytokines and interleukins (ILs) have been identified as modulators of bone remodeling.15,16 High serum levels of tumor necrosis factor-a, IL-2, IL-4, and IFN-g have been described in nephrotic patients with active renal disease, whereas remission is characterized by downregulation of these cytokines.17–19 It is well known that the osteoblast-related interaction between the receptor activator of nuclear factor-kappaB ligand and its soluble receptor triggers an osteoclast signaling pathway that induces osteoclast formation, fusion, activation and survival, leading to bone resorption and bone loss.20 Freundlich et al.21 showed that serum from patients with nephrotic syndrome and normal GFR stimulates the activity of normal human osteoblasts in culture and up regulate their expression of receptor activator of nuclear factor-kappaB ligand. There have been no other studies involving the culture of osteoblasts obtained from patients presenting glomerular diseases without renal failure. Therefore, the principal aim of our study was to evaluate the in vitro proliferation of osteoblasts obtained from patients with nonuremic proteinuric primary glomerular diseases. Bone histology studies were also performed.

3 2 1 0

10 20 30 25(OH)D3 (ng/ml)

40

20 r=– 0.4 P= 0.04 10

0

10 20 30 25(OH)D3 (ng/ml)

40

Figure 1 | Correlations between nephrotic ‘status’ and calciuria, as well as between nephrotic ‘status’ and serum 25(OH)D3. (a) Calciuria and proteinuria, (b) calciuria and serum albumin, (c) 25(OH)D3 and serum albumin, (d) 25(OH)D3 and proteinuria. 563

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CB Dias et al.: Histology studies in nonuremic proteinuric patients

higher eroded surface, compared of controls. Mean osteoclast surface was significantly higher in the study group (0.3570.6 vs 0.0570.1%, P ¼ 0.0016) (Table 2). The five patients presenting the lowest levels of proteinuria also presented bone alterations in comparison to the controls: altered bone structure, as evidenced by greater trabecular separation (434.1727.6 mm vs 368.3786.2 mm) and lower trabecular bone volume (21.871.7% vs 26.875.9%); and lower bone formation, as evidenced by lower osteoid volume (8.473.8 mm vs 11.573.2 mm), lower mineralizing surface (6.573.8% vs 13.576.0%) and lower bone formation rate (0.0470.03 mm3/mm2/day vs 0.0970.5 mm3/mm2/day). Owing to the limited sample size, it was not possible to draw statistical comparisons between these patients with mild proteinuria and controls. Osteoblast culture

In patient samples, there was less osteoblast proliferation (910.27437.1 d.p.m./well) than in control samples (2261.071121.0 d.p.m./well) (P ¼ 0.0016; Figure 2). Although cell proliferation was not found to correlate with any histomorphometric parameter, it did correlate positively with serum levels of 25(OH)D3 (r ¼ 0.6; P ¼ 0.02; Figure 3). We found no difference between proteinuric patients and controls in terms of alkaline phosphatase activity in osteoblasts.

The lower bone formation observed could indicate low osteoblast function related to the vitamin D insufficiency. This lower osteoblastic activity, releases osteoclasts to unregulated activity, resulting in uncoupling of bone resorption – formation, thereby contributing to the bone structure disorder. These results were obtained in nephrotic as well as in nonnephrotic patients, thereby allowing joint analysis. Our findings could hardly be attributed to the effects of the corticosteroids used to treat nephrotic syndrome, as only a few of the patients (n ¼ 5) used corticosteroids, and as those patients had taken the corticosteroids for a period of only 2–3 months and had discontinued their use 24–84 months before the bone biopsy. No differences were found between the two groups in terms of biochemical characteristics or bone histomorphometry, with or without corticosteroids. Data in the literature show that bone mineral density, as measured by densitometry analysis, is decreased in nephrotic patients.23,24 Although these findings could not be isolated from corticosteroid or cyclosporine administration, bone histomorphometry studies of nephrotic patients have shown increased bone resorption11–13 and decreased mineralizing surface,11 similar to that seen in patients with CKD. However, in contrast to CKD patients, nonuremic nephrotic patients consistently present normal serum phosphorus concentrations, normal serum iPTH concentrations and no metabolic

DISCUSSION

Variable

Patients

Controls

20.974.5 122.8724.0 475.5796.5 1.770.2

26.875.9 131.6727.0 368.3786.2 2.070.3

0.0008 NS 0.0002 0.0045

Formation OV/BV (%) Ob.S/BS (%) O.Th (mm) OS/BS (%) MS/BS (%) MAR (mm/day) BFR (mm3/mm2/day) Aj.AR (mm/day) MLT (days)

0.970.7 2.171.8 6.472.8 8.975.1 4.673.1 0.770.3 0.0370.04 0.4870.4 21.7715.6

2.071.4 1.371.6 11.573.2 11.777.1 13.576.0 0.6570.12 0.0970.05 0.570.2 22.572.5

0.0022 NS o0.0001 NS o0.0001 NS o0.0001 NS NS

Resorption ES/BS (%) Oc.S/BS (%)

3.173.9 0.3570.6

2.071.3 0.0570.1

Structure BV/TV (%) Tb.Th (mm) Tb.Sp (mm) Tb.N (n/mm)

P-value

NS 0.0016

Aj.AR, adjusted apposition rate; BFR, bone formation rate; BV/TV, trabecular volume; ES/BS, eroded surface; MAR, mineral apposition rate; MLT, mineralization lag time; MS/BS, mineralizing surface; NS, nonsignificant; Ob.S/BS, osteoblast surface; Oc.S/BS, osteoclast surface; OS/BS, osteoid surface; O.Th, osteoid thickness; OV/BV, osteoid volume; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness. Control values from references.40,41

564

Incorporation of thymidine (dpm/well)

Table 2 | Histomorphometric parameters

3000

2000

1000

0

*

Patients n =13 *P=0.0016

Controls n=5

Figure 2 | Osteoblast proliferation measured by incorporation of thymidine.

Incorporation of thymidine (dpm/well)

The results of the present study demonstrate that nonuremic proteinuric glomerulonephritis patients present bone structure disorder, low bone formation, and high bone resorption.

1500

1000

500 r =0.6 P =0.02 0

10

20 30 25(OH)D3 (ng/dl)

40

Figure 3 | Correlation between osteoblast proliferation, as measured by incorporation of thymidine, and serum levels of 25(OH)D3. Kidney International (2007) 71, 562–568

CB Dias et al.: Histology studies in nonuremic proteinuric patients

acidosis,12 as was seen in our patients. In addition, whereas serum levels of 25(OH)D3 are frequently reduced during active nephrotic syndrome as a result of urinary losses,6 those of calcitriol often remain normal even in patients with alterations seen in bone densitometry findings.14 Therefore, none of the principal biochemical characteristics of CKD bone disease are present in nonuremic nephrotic patients. Hosogane et al.2 demonstrated that non-nephrotic vitamin D-deficient rats present lower trabecular number and greater trabecular separation, similar to the findings of the present study. The same authors also found an increase in osteoid tissue, leading to osteomalacia, a situation not observed in our study. The osteomalacia found in other studies11,12 might have been related to serum levels of 25(OH)D3 that were lower than those observed in the present study. Chan et al.25 studied nephrotic rats and found no osteomalacia, attributing the fact to the short duration of the nephrotic syndrome. The calciuria observed in our patients was found to correlate negatively with proteinuria and positively with serum albumin. In addition, the calciuria values obtained were, on average, lower than those seen in a normal Brazilian population.22 Other authors have presented similar results regarding calciuria in nephrotic patients.8 It should be borne in mind that various factors, such as excretion of sodium, as well as serum levels of iPTH and 1,25(OH)2D3, are involved in urinary excretion of calcium.8,26 In our patients, calciuria was not found to correlate with sodium excretion (data not shown) or serum iPTH. Therefore, the pathogenesis of hypocalciuria in proteinuric patients remains undefined. However, it is known that increased 25(OH)D3 induces hypercalciuria, whereas decreased 25(OH)D3 induces hypocalciuria.27,28 In this sense, our findings regarding hypocalciuria are consistent with a 25(OH)D3 deficit. Concerning vitamin D metabolism, urinary losses have been documented in previous studies.25 In our study, despite the fact that we did not measure urinary excretion of vitamin D metabolites, such losses must be present, as evidenced by the fact that serum concentration of 25(OH)D3 correlated positively with serum albumin and negatively with proteinuria. Serum levels of 25(OH)D3 were lower in heavy proteinuric patients than in non-nephrotic patients. Concentrations of 1,25(OH)2D3 were normal in 70% of our patients. The explanation for a finding of normal serum 1,25(OH)2D3 levels in patients with 25(OH)D3 deficiency is twofold. First, PTH inhibits the enzyme responsible for the degradation of 1,25(OH)2D3 in inactive metabolites such as 24-hydroxylase.2 Second, as serum concentrations of 25(OH)D3 are 1000 times greater than those of 1,25(OH)2D3, even after drops of up to a third, serum levels of 25(OH)D3 might be sufficient to maintain normal levels of 1,25(OH)2D3.2,26 In a recent review, Nordin and Need29 concluded that there is a biphasic relationship between serum concentrations of 25(OH)D3 and those of 1,25(OH)2D3. Therefore, as 25(OH)D3 is a precursor of 1,25(OH)2D3, serum levels of the former correlate positively with those of Kidney International (2007) 71, 562–568

original article

the latter. However, when concentrations of 25(OH)D3 are low, the correlation with 1,25(OH)2D3 becomes a negative one owing to the fact that serum levels of 1,25(OH)2D3 are maintained by the effect of PTH. Concerning serum values for vitamin D, there is still considerable debate regarding normal values for 25(OH)D3, a metabolite measured in order to evaluate stores of vitamin D.30 Thomas et al.31 studied unselected general medical inpatients and demonstrated that serum concentrations of 25(OH)D3 lower than 15 ng/ml correlated with high serum concentrations of PTH. Other authors have found that serum concentrations of 25(OH)D3 above 30 ng/ml were adequate, showing that lower concentrations favored an increase in PTH.32 If this higher cutoff point is used, the majority of our patients presented 25(OH)D3 concentrations that were below normal, or rather, 25(OH)D3 deficiency, which could explain the low osteoblastic activity observed. However, other factors, such as ILs, which are bone metabolism modulators, must be present. Elevated serum and urinary activity of various cytokines, including IL-1, IL6, IL-2, IL-4, and tumor necrosis factor-a, has been identified in patients with nephrotic syndrome, particularly during clinical activity.18 Most of these pro- and anti-osteoclastogenic cytokines act primarily through the osteoblasts by altering levels of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin, the balance of which determines osteoclast formation.33 Some cytokines, such as IL-6, also have direct osteoclast effects.33 Concerning patient osteoblast culture, we have shown decreased osteoblast proliferation when compared to controls. However, there was no difference between patients and controls in terms of alkaline phosphatase production. In addition, it has been shown that osteoblast proliferation correlates positively with serum concentrations of 25(OH)D3 in age- and gender-matched patients, confirming the important role that 25(OH)D3 plays in osteoblast proliferation. Data in the literature concerning osteoblast culture are scarce and are restricted to CKD patients, among which those with adynamic bone diseases present decreased osteoblast proliferation, as would be expected.34,35 Recently, Freundlich et al.21 incubated normal osteoblasts with sera collected from children with nephrotic syndrome and observed increases in alkaline phosphatase and receptor activator of nuclear factor-kappaB ligand in the supernatant, correlating these findings with an increase in osteoblast activity. Their data incubating normal osteoblasts with nephrotic sera is divergent from the osteoblast culture data obtained for our patients. In conclusion, the results of our study show that nonuremic proteinuric glomerulonephritis patients develop alterations in bone structure, low bone formation and high bone resorption, and that these changes are unrelated to corticosteroid use. Patients showed a ‘state’ of vitamin D deficiency that correlated with protein excretion and low calcium excretion. Culture of osteoblasts obtained from these patients showed decreased osteoblast proliferation, which was 565

original article

in concordance with the histomorphometric data. In addition, osteoblast proliferation in culture correlated with serum levels of 25(OH)D3 from age- and gender-matched patients. These disturbances may represent important contributing factors to the histological bone abnormalities present in patients with protracted glomerular diseases before any decline in GFR. Our study breaks new ground in the use of osteoblast culture in patients with proteinuria. However, further studies are needed in order to identify other factors that might be responsible for the decreased osteoblast proliferation seen during glomerular proteinuric disease. MATERIALS AND METHODS Subjects Between March 2001 and March 2004, 17 adult patients with idiopathic glomerulonephritis, all under follow-up treatment at the Glomerulonephritis Unit of the University of Sa˜o Paulo Hospital das Clı´nicas, were evaluated. Kidney biopsies revealed that 12 patients had membranous glomerulonephritis, one had membranoproliferative glomerulonephritis, three had focal segmental glomerulosclerosis, and one had immunoglobulin A nephropathy. Patients were enrolled in our protocol and submitted to a bone biopsy at 38741 months (8–156 months) after being diagnosed with glomerulopathy. At the time of their enrollment, all the patients had proteinuria in excess of 1.5 g/day, GFR greater than 55 ml/min/1.73 m2, and serum creatinine lower than 1.8 mg/dl. All male patients enrolled (n ¼ 11) were younger than 55 years of age (range, 19–55 years), and all female patients (n ¼ 6) were younger than 40 years (range, 28–45 years). Patients currently treated with calcium supplements, anticonvulsants, oral anticoagulants, or other drugs affecting bone metabolism, were excluded, as were menopausal women and patients with thyroid disease. Ten of the patients had never been treated with corticosteroids or other immunosuppressive drugs. Two of the seven remaining patients had never used any corticosteroids except cyclosporin and mycophenolate mofetil for a short period of time. The other five had taken 1 mg/kg/day of prednisone for 2–3 months, but the prednisone had been discontinued 24–84 months before the bone biopsy. During the study thiazide diuretics were on hold for 3 weeks before the bone biopsy in order to collect serum and urine for biochemical analysis. All patients were pre-labeled with oral tetracycline (20 mg/kg/day for 3 days) administered for two separate periods, 10 days apart. During the protocol, patients were on a free medication prescription including beta-blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, or loop diuretics. Informed consent was obtained from all patients in advance, and protocols were approved by the ethical committee of the governing institution. Biochemical analysis The GFR (ml/min/1.73 m2) was estimated with the Modification of Diet in Renal Disease Study formula as follows: GFR ¼ 186  (serum creatinine1.154)  (age0.203)  (0.742 if female)  (1.21 if black).36 Serum ionized calcium, serum phosphorus and serum albumin were measured using established methods. Proteinuria and calciuria were 566

CB Dias et al.: Histology studies in nonuremic proteinuric patients

determined in 24-h urine samples. For urinary calcium detection, a colorimetric assay that detects all urinary calcium (Roche/Hitachi system with o-cresolphthalein) was utilized. Standard radioimmunoassay was used to measure serum 25(OH)D3 and 1,25(OH)2D3.37 A PTH-specific radioimmunoassay (ELSA-PTH; CIS Bio International, Gif-sur-Yvette, France) was used to measure iPTH. Bone histomorphometry Biopsy samples of approximately 1.5 cm in length were obtained from the anterior iliac bone using an electric trephine (Gaulthier Medical, Rochester, MN, USA) with a 7.5-mm internal diameter. The patients were under local anesthesia and light sedation. Undecalcified bone fragments were submitted to the usual processing and to histological studies.38 Sections (5 mm in thickness) were stained with toluidine blue for static and structural parameters using the Osteomeasure software (Osteometrics Inc., Atlanta, GA, USA). From the same material, 10-mm unstained slices were obtained for analysis of dynamic parameters under a microscope with ultraviolet light. The mean bone area was 38449715723 mm2. The parameters analyzed were in accordance with the standards set by the American Society of Bone and Mineral Research.39 The histomorphometric parameters analyzed were as follows: bone structure, as defined by measuring trabecular bone volume (%), trabecular number (n/mm), trabecular separation (mm) and trabecular thickness (mm); bone formation, as defined by measuring osteoid volume (%), osteoid thickness (mm), osteoid surface (%), osteoblast surface (%), all of which were measured on stained sections, as well as mineralizing surface (%), bone formation rate (mm3/mm2/day), mineral apposition rate (mm/day), adjusted apposition rate (mm/day), and mineralization lag time (days), all of which were measured on unstained sections under ultraviolet light; and bone resorption, as defined by measuring eroded surface (%) and osteoclast surface (%). Normal values for the parameters evaluated were determined as described by Dos Reis et al.40 from our laboratory and by Melsen et al.41 Gender- and age-matched controls (n ¼ 33) were selected from a large bone histomorphometry database compiled from the analyses of iliac biopsies of healthy individuals submitted to autopsy after an early demise. Most were victims of gunshot wounds, knife wounds, trauma, or traffic accidents and were not known to suffer from any disease. In addition, none were users of anticonvulsant drugs, corticosteroids, or any medication known to interfere with bone metabolism. The mean age was 3675.4 years, and gender distribution was 1.2 M:1.0 F, both comparable to the study group data. The controls described by Melsen et al.41 were obtained from 41 volunteers, 12 males aged 23–56 years (mean, 32 years), and 29 females aged 19–53 years (mean, 29 years). Osteoblast culture Bone cell populations were isolated from the trabecular bone surfaces of all biopsies. The trabecular bone was minced into small (1-mm3) fragments that were washed extensively with phosphatebuffered saline to remove adherent bone marrow cells. The bone explants were first cultured in Petri polystyrene plates (Nalge Nunc, Naperville, IL, USA) with Dulbecco’s Modified Eagle’s Medium, supplemented with antibiotics (100 mg/ml of penicillin, 100 mg/ml of streptomycin, and 2.5 mg/ml of amphotericin B) and 10% fetal bovine serum, then incubated at 371C in a humidified atmosphere of 5% CO2. Cell outgrowth from the bone surfaces occurred after a Kidney International (2007) 71, 562–568

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CB Dias et al.: Histology studies in nonuremic proteinuric patients

mean of 13 days. At confluence on the mesh, adherent cells were dissociated with a combination of 0.2% trypsin and 0.02% ethylenediaminetetraacetic acid (Instituto Adolfo Lutz, Sa˜o Paulo, Brazil) and cultured again in 25-cm2 flasks. At each new confluence, cells were again dissociated, after which they were cultured in 24-well plates for evaluation of cell proliferation and 6-well plates for determination of total alkaline phosphatase activity. Control group samples for normal osteoblast culture were obtained from cadaveric donor organs (n ¼ 5; mean age, 38710.6 years; gender distribution, four M:one F) and were collected in parallel with the study period. As the phenotypic expression of human bone cells has been shown to decrease after multiple passages,42 we studied these cells in the second passage. The cell population obtained from the trabecular bone surface exhibited an osteoblast phenotype, as evidenced by the production of alkaline phosphatase and the mineralization of extracellular matrix.43

4.

5.

6. 7.

8.

9. 10.

Cell proliferation On day 9 of culture in the 24-well plates, 5 mCi/ml of 3H-thymidine (Amersham Pharmacia Biotech Brasil Ltda, Sa˜o Paulo, Brazil) were added to each well, and the culture was continued for an additional 2 h. Subsequently, the thymidine was removed, the cells were washed twice with phosphate-buffered saline, and DNA was precipitated with 5% trichloroacetic acid suspended in 300 ml of 0.2 N NaOH and 0.3% sarcosyl. The cell lysates (100 ml) were aliquoted using liquid scintillation. To incorporate 3H-thymidine into DNA, six aliquots were measured using a Beckman beta scintillation counter model LS6200 (Beckman Instruments, Palo Alto, CA, USA).

11.

Alkaline phosphatase activity At confluence into the 6-well plates, the wells were washed twice with ice-cold phosphate-buffered saline and scraped, and 500 ml of iced water were added. The material was sonicated for 30 s and centrifuged for 30 min at 2000 r.p.m. In the supernatant, alkaline phosphatase activity was determined on p-nitrophenyl phosphate substrate, and the results are expressed as IU/l/mg of protein.

16.

Statistical analysis Except where otherwise indicated, results are expressed as mean7s.d. For comparisons between groups, either Student’s unpaired t-test or Mann–Whitney test was used, as appropriate. Correlations between two variables were determined using the Pearson coefficient (for normally distributed data) or the Spearman correlation coefficient (for nonparametric data). Values of Po0.05 were considered statistically significant.

12.

13.

14.

15.

17. 18.

19.

20.

21.

22.

23.

ACKNOWLEDGMENTS

We thank Wagner Domingues, Jose´ Edvanilson Gueiros, Carolina Lara Neves, and Daniela Guimara˜es for their expert technical assistance. Financial support provided by the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP, Foundation for the Support of Research in the state of Sa˜o Paulo; Grant no. 00-111450).

24.

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27.

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