The role of bone local GH-IGF system in CKD and its modulation by non-pharmacologic approaches

BSF 4th Annual Report (BSF Grant # 2007067) ("Bone disease in chronic kidney disease: the role of local GH-IGF system and its modulation by non-pharmacologic approaches").

D. Landau, Y. Segev & R. Rabkin. Departments of Pediatrics (DL) and Microbiology (YS), Ben Gurion University. Department of Medicine (RR), Stanford University.

Project #1: Decreased epiphyseal growth plate growth hormone receptor signaling in chronic kidney disease related growth retardation Abstract: Linear growth retardation in children with CKD has been ascribed to growth hormome (GH) insensitivity. This resistant state has been attributed to impaired GH signaling through the JAK2/STAT5 pathway in liver and skeletal muscle leading to reduced insulin-like growth factor I (IGF-I). Since there is no information on the impact of CKD on GH signaling in bone, we set out to investigate whether alterations in GH signaling might contribute to CKD induced linear growth retardation. Partially nephrectomized (CKD) and pair-fed control 20 day old rats were followed for 2 weeks. Serum GH and IGF-I, as well as tibial epiphyseal growth plate (EGP) histomorphology, IGF-I, GH receptor (GHR) and its signaling molecules were measured. CKD rats were growth retarded. Serum GH did not change, yet serum IGF-I levels were decreased. The EGP hypertrophic zone was wider and vascularization at the primary ossification center was decreased in CKD. Also EGP VEGF mRNA and immunostainable IGF-I and VEGF levels were reduced. EGP GHR and STAT5 protein levels were unchanged, while JAK2 was reduced. Despite comparable GH and GHR levels in CKD and controls, relative STAT5 phosphorylation was significantly depressed in CKD. Of note, the mRNA of SOCS2, an inhibitor of GH signaling, was increased. In conclusion, linear growth impairment in CKD can in part be explained by impaired long bone EGP GHR signaling through the JAK2/STAT5 pathway, an abnormality that may be caused by an increase in SOCS2 expression.

Project #2: Impaired renal growth hormone JAK/STAT5 signaling in chronic kidney disease Background: Treatment with recombinant human growth hormone (GH) has been accepted as standard therapy for short stature in children with chronic kidney disease (CKD). However, concerns have been raised on the potential renal fibrogenic effects of GH. There is no information regarding the renal GH receptor (GHR) signaling pathway in CKD.

Methods: Subtotal nephrectomized (CKD) and pair-fed sham operated control (C) juvenile rats were followed for 2 weeks. A single intravenous GH bolus or vehicle was provided prior to euthanasia.

Results: CKD rats showed reduced body weight and longitudinal growth. Remnant kidney showed glomerular hypertrophy and early interstitial fibrosis without inflammatory infiltration. Circulating GH levels were unchanged and expression of kidney IGF-1 mRNA in CKD was unaltered. Kidney GHR mRNA and protein levels were reduced and basal phosphorylation of JAK2 and STAT5 was significantly impaired. However, high dose GH administration normalized STAT5 phosphorylation. Basal renal IL6 mRNA and phosphorylation of its downstream signaling molecule STAT3 were increased as was the product of its action, the suppressor of cytokine signaling 3 (SOCS3) mRNA.

Conclusions: Despite unaltered circulating GH levels, remnant kidneys of uremic growth retarded juvenile rats show impaired basal signaling along the GH activated JAK2/STAT5 signaling pathway. This may well be a consequence of the reduced GHR level and the inhibitory effect of the increase in IL-6 mediated SOCS3 expression. This renal GH insensitivity, if present in humans, may protect against the potential adverse renal effects of modest but not high dose GH administration in CKD patients.

Project #3: Effects of endurance training on bone formation in a rat model of chronic kidney disease (CKD) related growth retardation Objectives: CKD in children is associated with suppressed body growth. We have recently reported on suppressed epiphyseal growth plate (EGP) growth hormone receptor (GHR) signaling and reduced local IGF-I in a rat model of CKD related growth retardation. Physical activity has been previously shown to increase expression of IGF-I signaling in muscles of rats with CKD, but the effects of this intervention on bone tissue have not been investigated yet. The purpose of this study was to examine the effects of endurance training on CKD related bone disease.

Methods: Twenty-day old / 50g male rats underwent a 2 step subtotal nephrectomy (Nx) or sham surgeries. The animals were divided into 2 running groups: control (Cr) and CKD (CKDr) (treadmill running, 20 m/min, 0.5 hr/day, 5 days/wk,) and 2 non running groups: control (C) and CKD. Food delivery was equal for all groups. The intervention lasted 4 weeks. We monitored somatic growth levels, kidney function and proximal tibiae EGP histomorphometry.

Results: Growth retardation (both longitudinal and weight gain) was well noticed in CKD Vs C. Exercise caused an increase in longitudinal growth and tibial length in Cr Vs C rats. However, CKDr rats did not grow better than CKD. CKDr rats consumed less food than CKD but grew to the same extent, hinting for better food efficiency. Total EGP width was increased in CKD Vs C, mostly at the expense of the hypertrophic zone. This effect was suppressed in CKDr Vs CKD. EGP cells were better organized in CKDr and showed less necrotic areas than CKD. Type II collagen was increased in CKD and decreased in CKDr. IGF-I mRNA and immunostainable IGF-I were increased in CKDr Vs CKD.

Conclusions: Endurance training did not rescue CKD somatic growth retardation, but longitudinal bone formation was better organized, associated with increased EGP IGF-I, supporting its positive role in bone health.

Project #4: The role of SOCS- 2 in uremic GH resistance Resistance to GH contributes to growth retardation and muscle wasting in chronic kidney failure (CRF). In part, the resistance is due to a post-receptor defect in GH-stimulated STAT5a/b phosphorylation and nuclear translocation, a pathway required for normal body growth. Since SOCS2 expression is increased in liver and muscle of rats with CRF we proposed that over-expression of this key negative regulator of GH signaling induced by GH and pro-inflammatory cytokines, might be one cause of the resistance to GH stimulated body growth. To test this thesis we studied mutant SOCS2 deficient C57BL/6J-hg/hg (hg = high growth) mice 3 weeks post-surgically induced CRF and compared them with SOCS2 intact normal (N) C57BL/6J mice. Sham operated (SO) mice served as controls. Non-uremic SOCS 2 mutants have high body growth (HG) and increased sensitivity to GH. At 8 weeks of age HG mice weighed significantly more than N mice, 21±0.7 vs 17.7±0.05g (p