Zoledronic Acid

Male Cy/+ rats, Han:SPRD rats with autosomal dominant polycystic kidney disease, and their non-affected (normal) littermates were used for this study. Male heterozygous rats (Cy/+) develop characteristics of CKD (azotemia) around 10 weeks of age which progresses to terminal uremia by about 40 weeks. This animal model spontaneously develops all three manifestations of CKD-MBD: biochemical abnormalities, extra skeletal calcification, and abnormal bone(10 (link),11 (link)).
At 25 weeks of age, animals were assigned to treatment groups. In the CKD (Cy/+) animals, this age represents approximately 30-40% of the kidney function of the normal littermates. This was chosen to simulate late stage 3 CKD, a stage at which there is elevated PTH, yet normal calcium and phosphorus levels, and a stage at which clinical practice guidelines do not recommend treatment with bisphosphonates(8 ). The CKD treatment groups (n=10 per group) were given 1) a single subcutaneous (SQ) dose of vehicle as control (CTL) and normal deionized drinking water, 2) a single SQ dose of zoledronic acid (ZOL) (20 μg/kg body weight) and normal deionized drinking water, 3) no injection but administered 3% calcium gluconate (3% Ca) in the drinking water, or 4) Zoledronic acid plus 3% Ca in the drinking water (Ca + ZOL). The calcium gluconate group was used to simulate calcium administration as a phosphate binder. In addition, we studied age-matched normal (NL) littermate animals (n = 10) to determine if treatments normalized bone manifestations or extra skeletal calcification. All animals were fed a casein diet (Purina AIN-76A; 0.53% Ca and 0.56% P) during the experiment which has been shown to produce a more consistent kidney disease in this model(10 (link)). Two weeks prior to the end of the study, all animals were given an intraperitoneal injection of calcein (1% concentration, 0.1mL/100g body weight); a second injection was given 10 days later. At 35 weeks of age all animals were euthanized by an overdose of sodium pentobarbital. All procedures were reviewed and approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee.

Male Cy/+ rats, a Han:SPRD rat with autosomal dominant polycystic kidney disease, and their non-affected (normal) littermates were used for this study. As shown previously, male heterozygous rats (Cy/+) develop characteristics of CKD (azotemia) around 10 weeks of age and the condition progresses with age [12 (link), 17 (link)–19 (link)]. We have found that at 20 weeks of age, the urinary creatinine clearance in the Cy/+ rats is 50 % of normal animals, and at that stage the PTH and FGF23 are twice normal, and the 1,25(OH)2D levels are half normal with no difference in blood calcium or phosphate levels. Furthermore, the kidney klotho expression is less than half of normal supporting that CKD-MBD is quite apparent at 20 weeks [12 (link), 17 (link)–19 (link)]. The secondary hyperparathyroidism progresses with slowly progressive kidney deterioration such that at 34 weeks of age, the animals show evidence of arterial and cardiac calcification, severe osteitis fibrosa cystica, and hyperphosphatemia consistent with all manifestations of CKD-MBD [18 (link)].
At 25 weeks of age, animals were assigned to treatment groups. In the CKD (Cy/+) animals, this age represents approximately 30 % of the kidney function of the normal littermates, and was chosen to simulate late stage 3, early stage 4 CKD, a stage at which there is elevated PTH yet normal calcium and phosphorus levels. In humans, bisphosphonates are not approved for clinical use at this level of GFR (and below) due to a lack of clinical studies. Normal animals (n=15) were given a single intraperitoneal dose of saline vehicle (VEH, 0.3 ml) or zoledronic acid (ZOL, 100 µg/kg body weight). CKD animals (n=25) were given a single intraperitoneal dose of VEH or ZOL (either 20 or 100 µg/kg body weight) or administered 3 % calcium gluconate in the drinking water. Both ZOL doses have been shown to be effective in preserving bone volume in ovariectomized rats when given as a single dose [20 (link), 21 (link)]. The calcium gluconate group was used as a positive control to simulate calcium administration as a phosphate binder, which in CKD has been shown to reduce bone remodeling [22 (link)]. All animals were fed a casein diet (Purina AIN-76A; 0.53 % Ca and 0.56 % P) during the experiment to increase phosphorus availability which has been shown to produce a more consistent kidney disease in this model [18 (link)]. Two weeks prior to the end of the study, all animals were given an intraperitoneal injection of calcein (10 mg/kg body weight); a second injection was given 10 days later. At 30 weeks of age all animals were euthanized by an overdose of sodium pentobartibal. The 5-week study duration was chosen as it approximates one remodeling cycle in a rat [23 ] and is prior to the time when Cy/+ animals begin to become excessively sick from kidney disease complications.
At necropsy, blood and urine were collected by cardiac and bladder puncture, respectively. The paired kidneys were dissected, wet weights obtained, and then embedded in paraffin for qualitative evaluation. The right femur was wrapped in saline-soaked gauze and frozen for imaging and mechanical testing. Left tibiae were placed in 10 % neutral buffered formalin for 48 h and then changed to 70 % ethanol for histological processing. All procedures were reviewed and approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee.

Animals and surgical procedures were handled according to guidelines of the UCLA Chancellor’s Animal Research Committee. Thirty-five 4-month old C57BL/6J male mice (Jackson Laboratories, Bar Harbor, ME, USA) received intraperitoneal (IP) injections of veh (endotoxin free water) or 200 µg/kg zoledronic acid (BP group) three times per week for 1 week prior to periapical lesion induction. The dose of 200 µg/kg is approximately 3 fold higher than the oncologic zoledronic acid dose of 66 µg/kg [32 (link)], and in the range of experimental BP doses used in other animal models [27 (link), 29 (link)]. We chose to use this higher dose to increase the incidence of ONJ in our animals, since there appears to be a dose-dependent effect of BPs on ONJ incidence in humans [33 (link), 34 (link)].
Mice were anesthetized with isoflurane and mounted on a jaw retraction board. Pulpal exposure of the left first and second mandibular molars was performed utilizing a size 1/4 round bur, avoiding furcal perforation (Fig 1A, taken at sacrifice) as described [35 (link), 36 (link)]. Exposed teeth were left open to the oral environment. Vehicle or ZA IP injections continued for an additional 7 weeks, following the same protocol of 200 µg/kg ZA at three times per week. We elected to inject the animals three times per week for the eight weeks of the experiment, to mimic the monthly injections in humans given the estimation that 17 days of a rodent life correspond to one human year [37 (link)]. Thus, the animals received a total of 24 ZA injections, which corresponds to 2 years of treatment for a cancer patient [38 (link)]. The mean time to onset of ONJ in patients treated with ZA is approximately 18 months [39 (link)].
At the end of the experiment, animals were sacrificed, mandibles were removed, placed in 4% paraformaldehyde for 48 hours and stored in 70% ethanol. Seventeen veh and 18 BP treated animals were utilized.