Growth Plate Cartilage Formation and Resorption Are Differentially Depressed in Growth Retarded Uremic Rats

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J Am Soc Nephrol 10: 971–979, 1999 Growth Plate Cartilage Formation and Resorption Are Differentially Depressed in Growth Retarded Uremic Rats

ANGELES COBO,* JOSEM.L´ OPEZ,´ † EDUARDO CARBAJO,‡ FERNANDO SANTOS,* JESUS´ ALVAREZ,† MARTA FERNANDEZ,´ † and ANA WERUAGA* Departments of *Pediatrics, †Cell Biology, and ‡Anatomy, School of Medicine, University of Oviedo, Oviedo, Asturias, Spain.

Abstract. To characterize the modifications of growth plate in taphyseal bone appeared markedly irregular in NX rats. Kinet- individuals with growth impairment secondary to chronic renal ics of chondrocytes was also modified (P Ͻ 0.05) in the NX failure, young rats were made uremic by subtotal nephrectomy rats, which had lower cell turnover per column per day (5.4 Ϯ (NX) and, after 14 d, their tibial growth plates were studied and 0.9), longer duration of hypertrophic phase (89.0 Ϯ 15.2 h), compared with those of sham-operated rats fed ad libitum and reduced cellular advance velocity (7.4 Ϯ 2.2 ␮m/h) com- (SAL) or pair-fed with NX (SPF). NX rats were growth re- pared with SAL (8.0 Ϯ 1.6, 32.1 Ϯ 6.7 h, and 11.3 Ϯ 2.7 tarded and severely uremic. Growth plate height (mean Ϯ SD) ␮m/h) and SPF (7.2 Ϯ 1.1, 34.8 Ϯ 5.1 h, and 10.1 Ϯ 2.5 was much greater (P Ͻ 0.05) in NX (868.4 Ϯ 85.4 ␮m) than ␮m/h). Cell proliferation was no different among the three SAL (570.1 Ϯ 93.5 ␮m) and SPF (551.9 Ϯ 99.7 ␮m) rats as a groups. Because the growth plates of SPF and SAL rats were result of a higher (P Ͻ 0.05) hypertrophic zone (661.0 Ϯ 89.7 substantially not different, modifications observed in the NX versus 362.8 Ϯ 71.6 and 353.0 Ϯ 93.9 ␮m, respectively). The rats cannot be attributed to the nutritional deficit associated increased size of the growth plate was associated with a greater with renal failure. These findings indicate that chronic renal number of chondrocytes and modifications in their structure, failure depresses both the activity of the growth plate cartilage particularly in the hypertrophic zone adjacent to bone. In this by altering chondrocyte hypertrophy and the replacement of zone, chondrocytes of NX animals were significantly (P Ͻ cartilage by bone at the metaphyseal end. The two processes 0.05) smaller (12080.4 Ϯ 1158.3 ␮m3) and shorter (34.1 Ϯ 2.5 are differentially depressed since cartilage resorption is more ␮m) than those of SAL (16302.8 Ϯ 1483.4 ␮m3 and 37.8 Ϯ severely lowered than cartilage enlargement and this leads to 2.0 ␮m) and SPF (14465.8 Ϯ 1521.0 ␮m3 and 36.3 Ϯ 1.8 ␮m). an accumulation of cartilage at the hypertrophic zone. The interface between the growth plate cartilage and the me-

Growth failure is a common manifestation of advanced chronic dogenous growth hormone (GH) considered the main causal renal insufficiency in children. More than 50% of pediatric factor (9,10). Much effort has been devoted in recent years to patients show growth velocities below the third percentile for characterize the alterations induced by renal failure in GH bone age at the beginning of dialysis treatment, and only in a metabolism, either at the pituitary (11–14), hepatic (15,16), or minority is growth velocity above the 50th percentile (1,2). peripheral level (17–19). However, since the study by Mehls et Furthermore, normal growth rate is usually not restored during al. (20) in 1977, little progress has been made in the under- chronic dialysis or even after successful renal transplantation standing of the effect of uremia on the structure and dynamics (3–6). As a result, growth retardation has probably become the of epiphyseal growth plate, which is the effector organ of single most important problem in the clinical management of longitudinal growth. children with chronic renal failure. Longitudinal bone growth results from progressive replace- Pathogenesis of growth retardation in chronic renal failure is ment of growth plate cartilage by osseous tissue at the metaph- not fully understood (7,8). Although the origin of growth yseal ends. Physiologically, the rate and extent of growth for a impairment may be multifactorial, nutritional and hormonal given growth plate is determined by a combination of chon- disorders usually play a major role, with insensitivity to en- drocyte proliferation, matrix production, and increase of chondrocyte volume (21). The few studies reporting data on the characteristics of epiphyseal growth plate in uremic rats have Received June 18, 1998. Accepted October 8, 1998. been restricted mostly to the measurement of bulk parameters Correspondence to Dr. Jose´M.Lo´pez, Department of Morfologıá y Biologıá such as growth plate height and daily longitudinal growth rate Celular, Facultad de Medicina, C/Juliań Claverıá 6, 33006 Oviedo, Asturias, of bone, as assessed by fluorescence labeling (20,22–26). Spain. Phone: 34 98 5103064; Fax: 34 98 5103618; E-mail:JMLOPEZ@ sci.cpd.uniovi.es However, little is known about the effects of uremia on growth 1046-6673/1005-0971$03.00/0 plate chondrocyte activity. By using a methodologic approach Journal of the American Society of Nephrology similar to that described by Hunziker et al. (27,28), the present Copyright © 1999 by the American Society of Nephrology study reports a detailed histomorphometric analysis of the 972 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 971–979, 1999 alterations found in the epiphyseal growth plate of uremic rats fixed in a mixture of 0.5% glutaraldehyde and 4% paraformaldehyde and provides new information on the activity of chondrocytes in 0.1 M sodium cacodylate buffer, pH 7.4, for5hat4°C. After in chronic renal failure. washing in buffer, samples were dehydrated in ascending concentrations of acetone and embedded in Durkupan-ACM. Embedded tissues were cut on a Reicher Ultracut E ultramicrotome Materials and Methods parallel to the tibial vertical axis, with the section angle randomly Animals and Experimental Protocol oriented relative to the horizontal plane for each block. The mean Male Sprague Dawley rats, 22 d old, were housed in individual thickness of the sections was 1.08 Ϯ 0.04 (SD) ␮m. cages and fed a standard 23.9% protein rat chow (AO3, Panlab SL, Barcelona, Spain). After3dofadaptation to the experimental area, Determination of Longitudinal Bone Growth Rate the animals were classified in three groups of five rats each: 5/6 nephrectomized (NX), sham-operated fed ad libitum (SAL), and Sections were examined under a Leitz incident light fluorescence sham-operated pair-fed with NX (SPF). At the beginning of the microscope, and the distance between the zone of vascular invasion experiment, rat weights ranged from 70 to 90 g with a mean weight of and the proximal part of the calcein label was measured with an 82 g. The mean weight of all three groups of rats was not different eyepiece micrometer (30). Measurements were obtained at three un- from one another at this time. biasedly determined locations on each of four sections per animal, and Subtotal nephrectomy or sham operation was performed in two the mean value of these measurements divided by 4 (days) was stages, on day 0 and day 4, as reported previously (11). SPF rats were considered the longitudinal bone growth per day in each animal. The given the same amount of food consumed by NX animals the day volume of newly formed bone per day was estimated by multiplying before. this value by the growth plate area (31). Rats’ body growth was assessed by weight and length gained between day 11 and day 18. Rats and food consumption were weighed Immunocytochemical Identification of daily with an electronic balance Ohaus GT 2001 (Ohaus Scale Corp., Proliferating Chondrocytes Florham Park, NJ). Nose to tip tail length was measured under Semithin sections were etched in 50% sodium ethoxide, pH 12.5, anesthesia on days 0, 11, and 18. On day 18, rats were sacrificed by for 15 min. Etching resulted in both deplastification of the tissues and exsanguination under anesthesia. At that moment, blood was obtained denaturing of the nuclear DNA. After etching, sections were passed for measuring serum concentrations of urea nitrogen, with an auto- through graded alcohols, washed in Tris buffer (0.1 M, pH 7.4), and analyzer Kodak EktachemR (Eastman Kodak, Rochester, NY) and incubated overnight with an anti-BrdU monoclonal antibody (1:20; both tibiae were removed. All animals received intraperitoneal injec- Dakopatts, Glostrup, Denmark) in a moist chamber at 4°C, followed tions of calcein (Sigma, St. Louis, MO) at a dose of 15 mg/kg body by incubation with biotinylated antimouse IgG (1:100, 60 min at room wt and 5-bromo-2Ј-deoxy-uridine (BrdU) (Sigma) at a dose of 100 temperature; Biomeda Corp., Foster City, CA) and streptavidin-per- mg/kg body wt, on day 14 and 1 h before sacrifice, respectively. oxidase complex (45 min at room temperature). The final reaction product was developed by incubation with a solution containing 0.66 Ј Tissue Collection and Processing mM 3,3 -diaminobenzidine and 2 mM H2O2 in 50 mM Tris-HCl, pH After removal, tibia lengths and their frontal and sagittal diameters 7.6. Preparations were lightly counterstained with Gill’s hematoxylin at the level of the upper growth plates were measured with a sliding and mounted. Proliferative activity was quantified by the estimation of mechanical caliper (accuracy 10 ␮m). Frontal and sagittal diameters the labeling index (LI), which was defined as the percentage of were used for estimation of the growth plate area perpendicular to the BrdU-labeled cells within the proliferative stratum. long axis according to the ellipse formula (29). Blocks from proximal tibial growth plates were chosen by a systematic random-sampling Histomorphometry process, as reported by Cruz-Orive and Hunziker (29). Briefly, each Sections were stained with toluidine blue and photographed by growth plate was dissected with the aid of a stereomicroscope into 10 light microscopy at a first low magnification (ϫ230). On these prints, prismatic blocks. Care was taken that sections were oriented parallel growth plates were divided into stem cell, proliferating, and hyper- to the longitudinal axis of tibiae (parallel to the plane of anisotropy). trophic zones according to morphologic criteria (29). The hypertro- Tissue blocks were numbered from 1 to 10, and five of them were phic zone was arithmetically subdivided into an upper (H1) and lower randomly chosen in each tibia. The first two blocks were used for (H2) half. Boundaries of zones were marked on the paper prints. The measuring the rate of longitudinal bone growth, the next two blocks height of the proliferative zone was calculated as the mean of three were used for histomorphometric analysis, and the last one was used different measurements performed at three locations randomly chosen for immunocytochemical identification of proliferating chondrocytes. on each section. The heights of the growth plate and its different zones Tissue blocks for determination of longitudinal growth rate were were estimated by point counting from the height of the proliferative fixed in 40% ethanol for3dat4°Cand, subsequently, dehydrated in zone. ethanol and embedded in Durkupan-ACM (Sigma). Tissue blocks for The volume of the growth plate and the volume of the different histomorphometry were immediately transferred to a 2% glutaralde- zones were estimated by multiplying the growth plate area by the hyde solution in 0.05 M sodium cacodylate buffer, pH 7.4, containing mean growth plate height and the mean height of the different zones, 0.7% ruthenium hexamine trichloride (RHT) (Strem Chemicals, New- respectively. buryport, MA) and maintained in this primary fixative for3hat4°C. From each section, two quadrants from the proliferating zone and They were then washed in isotonic 0.1 M sodium cacodylate buffer one from the hypertrophic zone were subsampled, photographed, and and post-fixed in a solution of 1% osmium tetroxide and 0.7% RHT printed on paper with a final magnification factor of ϫ700. The in cacodylate buffer for2hatroom temperature. Samples were boundaries of the zones were copied from those on low magnification washed in buffer again, dehydrated in ascending concentrations of prints. The following stereologic parameters were determined within acetone, and embedded in Durkupan-ACM. Tissue blocks for immu- each zone: volume fraction of chondrocytes, numerical density of nocytochemical identification of proliferating chondrocytes were chondrocytes, total number of chondrocytes, mean chondrocyte vol- J Am Soc Nephrol 10: 971–979, 1999 Growth Plate Cartilage Alterations in Uremia 973 ume, mean matrix volume per chondrocyte, mean projected horizontal Cell Kinetics diameter, mean chondrocyte height, number of chondrocytes in a cell The following parameters were estimated: mean cell turnover per column, and growth fraction. column per day, turnover time, and lineal velocity of advance of The volume fraction of chondrocytes was estimated by using a chondrocytes in the columns (27). transparent screen with a square point lattice spacing of 21 mm The mean cell turnover per column per day was calculated by (equivalent to 30 ␮m) and counting the number of intersections on dividing the daily linear growth rate by the mean cell height in the chondrocytes and on matrix. The volume fraction of chondrocytes in distal stratum (H2). The turnover time of a stratum is the time required each stratum was calculated by dividing the total number of points for the replacement of the total number of cells in that stratum. This over chondrocytes by the total number of points. Total volume of is equal to the period of time each cell spends in this compartment if chondrocytes was then estimated by multiplying this value by the no cell divisions take place. Thus, it can be estimated in the hyper- volume of the stratum. Total volume of matrix was estimated by trophic zone by dividing the total number of cells in the stratum by the deducting the volume of chondrocytes from the volume of the stratum. number of cells leaving the stratum per day. The lineal velocity of The numerical density and total number of chondrocytes were esti- advance of chondrocytes in the columns was estimated at the hyper- mated using the disector method (29) on the first and the fifth sections trophic zone by dividing the height of this zone by the turnover time. of a series of consecutive sections. The number of chondrocytes present in the first section but no longer apparent in the fifth divided Statistical Analyses by the volume of the disector (reference area Ϫ1837 ␮m2 per the Values of each group are given as mean Ϯ SEM. For the growth height of the disector Ϫ4.32 ␮m) gives an estimate of the number of plate data, a mean value for each of the tested parameters was chondrocytes per unit of reference volume. The total number of calculated on a per-animal basis in the group. Then, each animal was chondrocytes was calculated by multiplying this value by the volume considered a sample for statistical purposes. The data obtained were of the stratum. The mean chondrocyte volume and mean matrix shown to follow a normal distribution with homogeneity of variances volume per chondrocyte were estimated by dividing the total volume and independence. Comparison among the three groups was per- of chondrocytes and the total volume of matrix by the total number of formed by ANOVA, using a significance level of 95%, followed by chondrocytes, respectively. The mean projected horizontal diameter the Newman–Keuls multiple range test. of a chondrocyte was defined as the caliper diameter measured perpendicular to the long axis of the bone. The mean chondrocyte height was estimated from the mean profile height. It was defined as the Results length of the intercept bisecting the cell profile measured parallel to Severe renal failure was induced in NX rats as confirmed by the long axis of the bone. Estimators of cell diameters based on shape serum concentrations of urea nitrogen levels 6 to 7 times higher models were not adequate in the present study, since NX animals than those found in SAL and SPF animals (Table 1). Cumu- showed alterations in the shape of hypertrophic chondrocytes and lative food intake of NX rats was 66% of that of SAL and, were not adjusted to a super-egg model. Then, the mean diameters according to the experimental protocol, almost identical to that were obtained by direct measurements of the vertical and horizontal of SPF animals. NX animals were growth-etarded as shown by axes of 500 chondrocyte profiles cut centrally (through the nucleus) low gains in body weight and length and less tibial length and per animal (27). The number of cells in a vertical cell column was longitudinal growth rate than control animals (Figure 1). obtained by dividing the height of the stratum by the mean chondrocyte height (29). Due to the bias involved in its estimation, this Under the light microscope, the growth plates appeared parameter was also determined directly by counting the number of bigger and more irregular in NX than SPF and SAL groups cells within complete column profiles in the section plane (27). Both (Figures 1 and 2). Stereologic parameters describing the fea- procedures yielded similar results. Finally, the growth fraction was tures of growth plate and chondrocytes are given in Tables 2 estimated by dividing in a column the number of chondrocytes at the and 3. The height of the entire growth plate was markedly proliferative zone by the total number of chondrocytes (27). greater in NX than control rats. Growth plate enlargement was

Table 1. Serum urea nitrogen, cumulative food intake, and body and bone growth data in the three groups of ratsa

Parameter SAL SPF NX

SUN (mg/dl) 17.4 Ϯ 2.7 14.4 Ϯ 2.2 105.8 Ϯ 46.1b,c Food intake (g) 168.7 Ϯ 26.2 107.9 Ϯ 13.6b 111.0 Ϯ 17.2b Weight gain (g) 60.5 Ϯ 11.2 23.2 Ϯ 14.3b 42.0 Ϯ 11.9b,c Length gain (cm) 3.8 Ϯ 0.7 3.5 Ϯ 0.4 2.3 Ϯ 0.9b,c Tibial length (mm) 31.9 Ϯ 0.9 31.6 Ϯ 0.4 29.7 Ϯ 0.7b,c Tibial width at GP level (mm2) 39.8 Ϯ 4.2 27.7 Ϯ 3.1b 37.2 Ϯ 2.2c Longitudinal growth rate (␮m/d) 304.0 Ϯ 13.6 266.0 Ϯ 19.9b 189.6 Ϯ 21.5b,c Volume of formed bone (mm3/d) 12.1 Ϯ 1.8 7.4 Ϯ 1.3b 7.1 Ϯ 1.1b

a Data are given as mean Ϯ SD. SAL, control rats fed ad libitum; SPF, control rats pair-fed with NX; NX, 5/6 nephrectomized rats; SUN, serum urea nitrogen; GP, growth plate. b P Ͻ 0.05 compared with SAL. c P Ͻ 0.05 compared with SPF. 974 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 971–979, 1999

Figure 1. Parallel sections of the proximal tibial growth plates of sham-operated rats fed ad libitum (SAL) (a) and 5/6 nephrectomized (NX) rats (b) viewed by bright-field microscopy after toluidine blue staining (left) and by incident-light fluorescence microscopy (right). The distance between the lower border of the growth plate and the fluorochrome-labeled front appears clearly longer in SAL than in NX rats. Figure 1C is a higher magnification of the proximal end point of the calcein front in the metaphysis of a control rat. Magnification: ϫ25 in A and B; ϫ110 in C. Figure 2. Light micrographs of vertical semithin sections of the proximal tibial growth plate of SAL rats (a), sham-operated rats pair-fed with NX (b), and NX rats (c). Growth plate in NX rats appears greater than in any of the control groups. Toluidine blue stain. Magnification, ϫ105. J Am Soc Nephrol 10: 971–979, 1999 Growth Plate Cartilage Alterations in Uremia 975

Table 2. Stereologic estimators of the upper tibial growth plate in the three groups of ratsa

Parameter SAL SPF NX

Growth plate height (␮m) global 570.1 Ϯ 93.5 551.9 Ϯ 99.7 868.4 Ϯ 85.4b,c resting zone 42.8 Ϯ 19.5 27.6 Ϯ 3.1 27.7 Ϯ 7.6 proliferating zone 164.5 Ϯ 10.7 171.3 Ϯ 10.7 180.2 Ϯ 22.1 hypertrophic zone 362.8 Ϯ 71.6 353.0 Ϯ 93.9 661.0 Ϯ 89.7b,c Growth plate volume (mm3) global 22.6 Ϯ 4.2 15.3 Ϯ 4.0b 32.3 Ϯ 3.8b,c resting zone 1.7 Ϯ 0.7 0.8 Ϯ 0.2 1.0 Ϯ 0.4 proliferating zone 6.5 Ϯ 0.7 4.7 Ϯ 1.8 6.7 Ϯ 1.1 hypertrophic zone 14.4 Ϯ 3.1 9.8 Ϯ 3.6 24.6 Ϯ 3.8b,c Volume fraction of chondrocytes (%) resting zone 21.0 Ϯ 6.0 16.2 Ϯ 2.9 21.0 Ϯ 3.6 proliferating zone 34.4 Ϯ 6.0 27.8 Ϯ 2.9 32.7 Ϯ 4.5 upper hypertrophic zone 54.5 Ϯ 4.9 53.1 Ϯ 8.9 51.6 Ϯ 7.2 lower hypertrophic zone 67.0 Ϯ 3.6 61.2 Ϯ 12.3 62.7 Ϯ 8.7 Numerical density of chondrocytes (no. of cells ϫ 103/mm3 of tissue) resting zone 196.5 Ϯ 36.9 170.0 Ϯ 39.8 220.0 Ϯ 23.0 proliferating zone 264.6 Ϯ 30.2 223.7 Ϯ 23.0 253.6 Ϯ 22.6 upper hypertrophic zone 62.3 Ϯ 5.4 59.1 Ϯ 10.1 57.9 Ϯ 8.3 lower hypertrophic zone 41.1 Ϯ 5.1 42.3 Ϯ 6.9 51.9 Ϯ 7.4b Total number of chondrocytes (ϫ103) resting zone 334.1 Ϯ 65.5 136.0 Ϯ 32.9b 220.0 Ϯ 23.0b,c proliferating zone 1719.9 Ϯ 43.2 1051.4 Ϯ 114.9b 1699.1 Ϯ 137.3c upper hypertrophic zone 448.6 Ϯ 38.9 289.6 Ϯ 55.0b 712.2 Ϯ 107.1b,c lower hypertrophic zone 295.9 Ϯ 34.2 207.3 Ϯ 41.8b 638.4 Ϯ 90.8b,c

a Data are given as mean Ϯ SD. Abbreviations as in Table 1. b P Ͻ 0.05 compared with SAL. c P Ͻ 0.05 compared with SPF. mainly due to an increased height of the hypertrophic zone, and mainly as a result of flattened hypertrophic chondrocytes ex- this resulted from a greater number of chondrocytes in this tending through the zone of the primary spongiosa (Figure 4, a zone rather than an increase in cell size or extracellular matrix. and b). Within the metaphyseal bone, chondrocytes were well Moreover, the chondrocytes of the lower half of the hypertro- preserved and located (either isolated or in small clusters) in phic zone were smaller (less volume) and shorter (less height) close proximity to osteoblasts and capillary vessels (Figure 4c). in NX animals. Interestingly, in SAL rats chondrocyte volume In addition, the pattern of the invading capillary sprouts found and profile height increased by 86 and 23% from the upper to in control animals—a single capillary profile per terminal the lower hypertrophic layer, whereas in NX rats these in- lacunar compartment—was clearly disrupted in NX rats. As a creases were reduced to 36 and 10%, respectively. By contrast, result, the vascular front could be hardly recognized at the light the proliferative layer showed little variation in NX rats com- microscope in these animals. pared with SPF and SAL rats. There were no differences in the Modifications of chondrocyte phenotype were accompanied height or the morphologic features of the chondrocytes in this by changes in chondrocyte kinetics (Table 4). The BrdU- zone. Furthermore, the distribution pattern of BrdU-labeled labeling index and the columnar growth fraction, as estimators cells was not modified in NX rats (Figure 3). of cell proliferation, were no different among the three groups The interface between the growth plate cartilage and the of animals. However, the cell turnover per column was signif- metaphyseal bone appeared markedly irregular in NX rats, icantly impaired by uremia, since seven to eight cells left the

Figure 3. 5-Bromo-2Ј-deoxy-uridine (BrdU)-labeled cell proliferation in the proximal tibial growth plate of SAL (a) and NX (b) rats. Sections counterstained with hematoxylin. Magnification, ϫ210. Figure 4. Sections through the lower growth plate and upper metaphyseal portion of SAL (a) and NX (b and c) rats. Growth plate from NX rats is more irregular and presents chondrocytes with small size appearing organized into vertical columns of flattened cells resembling those of the proliferative zone (arrows). Magnification: ϫ170 in a and b; ϫ450 in c. 976 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 971–979, 1999

Table 3. Chondrocyte structural parameters in the three groups of ratsa

Parameter SAL SPF NX

Mean cell volume (␮m3) proliferating zone 1300.1 Ϯ 51.4 1242.7 Ϯ 155.4 1289.5 Ϯ 104.2 upper hypertrophic zone 8747.2 Ϯ 474.9 8984.4 Ϯ 1335.2 8911.5 Ϯ 590.5 lower hypertrophic zone 16302.8 Ϯ 1483.4 14465.8 Ϯ 1521.0 12080.4 Ϯ 1158.3b,c Mean matrix volume per cell (␮m3) proliferating zone 2479.1 Ϯ 381.7 3227.4 Ϯ 487.2 2653.9 Ϯ 597.7 upper hypertrophic zone 7302.7 Ϯ 1343.7 7935.4 Ϯ 2668.7 8358.8 Ϯ 2300.2 lower hypertrophic zone 8029.7 Ϯ 1803.6 7418.9 Ϯ 2616.9 7186.6 Ϯ 2016.7 Mean projected horizontal diameter (␮m) proliferating zone 17.0 Ϯ 0.2 17.7 Ϯ 0.7 17.0 Ϯ 0.4 upper hypertrophic zone 24.6 Ϯ 0.7 25.8 Ϯ 1.6 24.8 Ϯ 1.8 lower hypertrophic zone 30.5 Ϯ 2.0 29.2 Ϯ 2.0 27.9 Ϯ 2.5 Mean height of cell profile (␮m) proliferating zone 8.6 Ϯ 0.2 8.4 Ϯ 0.4 8.6 Ϯ 0.2 upper hypertrophic zone 30.6 Ϯ 1.6 31.1 Ϯ 2.0 30.8 Ϯ 2.5 lower hypertrophic zone 37.8 Ϯ 2.0 36.3 Ϯ 1.8 34.1 Ϯ 2.5b,c No. of chondrocytes in a cell column global 29.8 Ϯ 4.5 31.0 Ϯ 6.5 41.4 Ϯ 8.7b,c proliferating zone 19.1 Ϯ 2.2 20.4 Ϯ 4.2 21.0 Ϯ 4.0 upper hypertrophic zone 5.9 Ϯ 1.3 5.7 Ϯ 1.6 10.7 Ϯ 2.7b,c lower hypertrophic zone 4.8 Ϯ 0.9 4.9 Ϯ 0.7 9.7 Ϯ 2.0b,c

a Data are given as mean Ϯ SD. Abbreviations as in Table 1. b P Ͻ 0.05 compared with SAL. c P Ͻ 0.05 compared with SPF.

Table 4. Chondrocyte kinetic parameters in the three groups of ratsa

Parameter SAL SPF NX

Labeling index (%) 18.5 Ϯ 4.0 13.9 Ϯ 1.1 15.0 Ϯ 4.0 Growth fraction (%) 64.1 Ϯ 10.7 65.8 Ϯ 11.9 50.7 Ϯ 10.7 Cell turnover per column per day 8.0 Ϯ 1.6 7.2 Ϯ 1.1 5.4 Ϯ 0.9b,c Duration hypertrophic phase (hours) 32.1 Ϯ 6.7 34.8 Ϯ 5.1 89.0 Ϯ 15.2b,c Cellular advance velocity (␮m/hour) 11.3 Ϯ 2.7 10.1 Ϯ 2.5 7.4 Ϯ 2.2b,c

a Data are given as mean Ϯ SD. Abbreviations as in Table 1. b P Ͻ 0.05 compared with SAL. c P Ͻ 0.05 compared with SPF.

columns every day (one chondrocyte every 3.4 to 3 h) in SPF tissue at the epiphyseal/metaphyseal interface in a process and SAL rats, whereas this turnover fell to approximately five called ossification. A strict coordination between the processes cells per day (one chondrocyte every 4.8 h) in the NX group. of cartilage enlargement, cartilage resorption, and osseous tis- This decrease in cell turnover was accompanied by a longer sue formation at the metaphyseal end is required for normal duration of the hypertrophic phase, which was extended from, bone growth. The results presented here indicate that both approximately, 33 h in both control groups to nearly 90 h in production of cartilage and ossification were significantly NX rats. Finally, the lineal velocity of advance of chondrocytes slowed down in uremic rats. Furthermore, the two processes in the columns was significantly decreased in NX rats when were differentially depressed in the sense that cartilage resorp- compared with control animals. tion/bone deposition was affected to a higher degree than cartilage formation. This led to a disequilibrium that resulted in Discussion an increased growth plate height as a result of accumulation of Elongation of long bones is the result of the interplay of two cartilage at the hypertrophic zone. This finding cannot be coupled processes: continual and vectorial production of car- attributed to the nutritional deficit associated with renal failure tilage by the growth plate and replacement of cartilage by bone since the growth plates of SPF rats were not substantially J Am Soc Nephrol 10: 971–979, 1999 Growth Plate Cartilage Alterations in Uremia 977 different from those of SAL animals and, by contrast, growth and volume during chondrocyte hypertrophy (22,43). In agree- plates of SPF and NX rats differed markedly. ment with these findings, retardation of growth in our uremic Earlier studies analyzing the height of epiphyseal growth rats was associated with a significant decrease of both cell plates in uremic rats have yielded no uniform results, since, height and cell volume at the distal hypertrophic zone (Table compared with control animals, increased (20,26), decreased 3). Matrix volume per cell or cell proliferation did not signif- (22,32), and unchanged (23–25) height has been reported. This icantly change. Thus, it may be concluded that chronic renal discrepancy may be explained by two factors. First, a system- failure caused a profound alteration of the chondrocyte hyper- atic random-sampling method is necessary to obtain unbiased trophy process. measurements, because growth plate is a somewhat twisted Cell size and shape are generally maintained and modulated disc whose height shows perceptible regional differences (29). by components of the cytoskeletal system. However, it has This is especially important in the presence of uremia, because been reported that chondrocyte shape and volume are highly the borderline between the distal end of cartilage and the dependent on the composition of the extracellular matrix, proximal end of bone in nephrectomized rats is rather ill- which despite being elastic and compressible is also stiff, and defined. The degree of renal failure obtained in the different thus provides a firm supportive coat around the chondrocyte studies may also play a role. Unpublished data from our group (27,44). As a result, changes in cell shape and volume during indicate that growth plate modifications described in the hypertrophy are normally coupled to degradative and synthetic present report are not found in mildly uremic rats. It is of note processes in the surrounding matrix (45,46). In our NX rats, the that a severe degree of uremia is needed to induce sustained matrix volume per chondrocyte was not significantly modified growth failure in subtotally nephrectomized rats. Otherwise, when compared with control rats. However, the velocity of growth retardation is rather due to the period of acute renal advance of chondrocytes at the hypertrophic zone of the failure that follows the second stage nephrectomy than to growth plate was slowed down in uremic rats (Table 4). Such chronic uremia itself (33). Finally, another source of variation movement is not passive but requires vectorial degradation of might be the time period of nephrectomy, since it varies in the matrix at the metaphyseal cell pole and resynthesis at the different studies. epiphyseal cell pole. Thus, our results indicate that renal failure It is also of note that the only measurement of growth plate impairs matrix remodeling at the hypertrophic zone of growth height provides little information on how a disease is interfer- cartilage. ing with the metabolism of growth cartilage, and interpretation Our study extensively describes the modifications induced of this finding may be misleading. In the above-quoted report, by uremia at the long bone growth plate level and reveals that Mehls et al. (20) stated that the increased height of tibial increased height of growth plate results from a disequilibrium growth plate of uremic rats was reminiscent of vitamin D between cartilage formation and cartilage resorption, which is deficiency. However, in vitamin D-deficient rickets, the chon- related to significant changes in the dynamics and morphology drocytes of the hypertrophic zone have been described to be of chondrocytes. Unfortunately, our study does not provide any characteristically much bigger than those of aged-matched insight into the mechanisms underlying the defective matura- control animals (34), whereas the hypertrophic chondrocyte tion process of growth plate chondrocytes found in uremic rats. volume of the NX rats presented here was significantly reduced It is tempting to speculate that substances that modulate cell in relation to the two groups of sham-operated rats with normal hypertrophy and whose metabolism is altered in uremia might renal function (Table 3). be responsible for this effect. Very little is known about the The rate and extent of growth for a given growth plate is influence of uremic state on the effect of the several endocrine, determined by a combination of chondrocyte proliferation, paracrine, and autocrine factors that act differentially on chon- matrix synthesis, and cell-controlled phenotype modulation drocytes according to their stage of differentiation (21,47,48– (chondrocytic hypertrophy) (21). The relative contribution of 50). Low levels of mRNA insulin-like growth factor-1 in each of these processes to longitudinal bone growth is not well chondrocytes of proliferative and hypertrophic zones of the established, since it may vary significantly at different ages and growth plate (24) and resistance to GH and insulin-like growth in different species (35). Chondrocyte proliferation has been factor-1 in primary cultures of epiphyseal chondrocytes (51) classically considered as the major modulator of growth have been reported in uremic rats. Further investigations are (36,37). However, in some disorders, growth rate may be clearly needed to clarify how chronic renal failure interferes severely impaired in the presence of normal cell proliferation at with the activity of the epiphyseal growth plate and the normal the growth plate (28,38). Furthermore, the division rates of maturation of their chondrocytes. proliferating chondrocytes in all cartilage plates of a given animal have been found to be fairly constant (39–41). In Acknowledgment addition, a positive correlation between the size of chondro- This research was supported by MEC (Direccio´n General de cytes in the terminal hypertrophic zone and longitudinal Ensen˜anza Superior PM96-0110). growth rate has been demonstrated in both physiologic and pathologic conditions (27,28,38,42). Thus, it is now generally References considered that chondrocyte proliferation tends to be preserved 1. 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