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

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Growth Plate Cartilage Formation and Resorption Are Differentially Depressed in Growth Retarded Uremic Rats 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 , 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 6 (NX) and, after 14 d, their tibial growth plates were studied and 0.9), longer duration of hypertrophic phase (89.0 6 15.2 h), compared with those of sham-operated rats fed ad libitum and reduced cellular advance velocity (7.4 6 2.2 mm/h) com- (SAL) or pair-fed with NX (SPF). NX rats were growth re- pared with SAL (8.0 6 1.6, 32.1 6 6.7 h, and 11.3 6 2.7 tarded and severely uremic. Growth plate height (mean 6 SD) mm/h) and SPF (7.2 6 1.1, 34.8 6 5.1 h, and 10.1 6 2.5 was much greater (P , 0.05) in NX (868.4 6 85.4 mm) than mm/h). Cell proliferation was no different among the three SAL (570.1 6 93.5 mm) and SPF (551.9 6 99.7 mm) rats as a groups. Because the growth plates of SPF and SAL rats were result of a higher (P , 0.05) hypertrophic zone (661.0 6 89.7 substantially not different, modifications observed in the NX versus 362.8 6 71.6 and 353.0 6 93.9 mm, 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 , cartilage by bone at the metaphyseal end. The two processes 0.05) smaller (12080.4 6 1158.3 mm3) and shorter (34.1 6 2.5 are differentially depressed since cartilage resorption is more mm) than those of SAL (16302.8 6 1483.4 mm3 and 37.8 6 severely lowered than cartilage enlargement and this leads to 2.0 mm) and SPF (14465.8 6 1521.0 mm3 and 36.3 6 1.8 mm). 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 chon- drocyte 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 Morfologı´a y Biologı´a such as growth plate height and daily longitudinal growth rate Celular, Facultad de Medicina, C/Julia´n Claverı´a 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 concentra- tions 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 6 0.04 (SD) mm. 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-29-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 9 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 mm). 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.
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