CYSTINOSIS RESEARCH FOUNDATION PROGRESS REPORT VITAMIN D AND MUSCLE WASTING IN NEPHROPATHIC CYSTINOSIS FINAL REPORT Overview Nephropathic cystinosis is a lysosomal storage disorder leading to chronic kidney disease (CKD) in children. Muscle wasting is a common complication of nephropathic cystinosis. The underlying mechanism of the muscle wasting in cystinosis is not well understood. We have shown that there is vitamin D deficiency, of both 25(OH)D3 and 1,25(OH)2D3, in Ctns-/- mice which demonstrate cachexia (decreased appetite and increased metabolic rate) and muscle wasting. We have preliminary data showing 25(OH)D3 supplementation leads to amelioration of these abnormalities in Ctns-/- mice. We hypothesize that vitamin D deficiency signals through the pro-inflammatory cytokine IL-6 resulting in aberrant energy homeostasis, which leads to cachexia and muscle wasting. The results of this proposal will lead to novel therapy for muscle wasting in cystinosis, for which there is no known effective treatment. Therapy may be in the form of vitamin D supplements [especially 25(OH)D3 which is not routinely prescribed. Amelioration of muscle wasting and function will improve quality of life and perhaps even survival in patients with nephropathic cystinosis. Hypothesis 1) Vitamin D deficiency is important in the pathogenesis of cachexia and muscle wasting in nephropathic cystinosis. 2) The mechanism of vitamin D deficiency causing cachexia and muscle wasting in nephropathic cystinosis involves inflammatory cytokine IL-6 resulting in aberrant energy homeostasis. Strategy Three specific aims have been proposed to test our hypothesis. 1) To examine the differential effects of 25(OH)D3 and 1,25(OH)2D3 early supplementation on preventing abnormalities in energy homeostasis, muscle mass regulation and muscle function in Ctns-/- mice. 2) To examine the differential effects of 25(OH)D3 and 1,25(OH)2D3 late supplementation on reversing abnormalities in energy homeostasis, muscle mass regulation and muscle function in Ctns-/- mice. 3) To examine the roles of the pro-inflammatory cytokine IL-6 in the pathogenesis of vitamin D deficiency associated cachexia and muscle wasting in Ctns-/- mice. Result 25(OH)D3 and 1,25(OH)2D3 improve weight gain, basal metabolic rate and energy efficiency in Ctns-/- mice We performed the following experiments. Twelve-month old male Ctns-/- mice were treated with 25(OH)D3 (n=5, 1,000 IU/kg/24h), 1,25(OH)2D3 (n=7, 40 ng/kg/24h) or vehicle (n=9, normal saline) and compared with wild-type (WT) mice (n=7) treated with vehicle. Ctns-/- + vehicle mice were fed ad libitum while other groups of mice were pair-fed to Ctns-/- + vehicle mice (Figure 1C). At the end of 6-week experiment, mice were sacrificed and serum chemistry indicated that Ctns-/- mice were uremic as serum creatinine and BUN levels were elevated in Ctns-/- mice versus controls (Figure 1A and 1B). 25(OH)D3 and 1,25(OH)2D3 normalized food weight gain, basal metabolic rate and energy efficiency in Ctns-/- mice (Figure 1D, 1E and 1F). 1 200 180 160 140 120 100 Basal metabolic rate (ml/hr per kg) D 3 2.5 2 1.5 1 0.5 0 F 0.02 Energy efficiency (g/g) 0.015 4250 4000 3750 3500 3250 3000 # 0.01 WT + Vehicle Ctns-/- + VitD1,25 0 # Ctns-/- + VitD25 0.005 Ctns-/- + Vehicle WT + Vehicle Ctns-/- + VitD1,25 Ctns-/- + VitD25 # Ctns-/- + Vehicle Weight change (g) # WT + Vehicle # # Ctns-/- + VitD1,25 # # Ctns-/- + VitD25 100 80 60 40 20 0 # Ctns-/- + Vehicle B Serum BUN (mg/dL) 2 1.6 1.2 0.8 0.4 0 E Accumulated food intake (g) C Serum crea nine (mg/dL) A Figure 1: Serum chemistry, accumulated food intake, weight change, basal metabolic rate and energy efficiency of mice at the end of 6-week study. 12-month old male Ctns-/- mice were treated with VitD25 (n=5, 1,000 IU/kg/24h), VitD1,25 (n=7, 40 ng/kg/24h) or Vehicle (n=9, normal saline) and compared with WT mice (n=7) treated with vehicle. Ctns-/- + vehicle mice were fed ad libitum while other groups of mice were pair-fed to Ctns-/- + vehicle mice. Date are expressed as mean ± SEM. # P < 0.05. 2 25(OH)D3 improves body composition and muscle function in Ctns-/- mice Body composition was analyzed twice, prior to the initiation of the study and at the end of 6week study, using EchoMRI technique. Muscle loss (lean mass and percentage of lean mass) was clearly evident in Ctns-/- mice as compared to controls (Figure 2C and 2D). 25(OH)D3 treatment normalized lean mass and percentage of lean mass in Ctns-/- mice. In vivo muscle function, as assessed by rotarod activity and forelimb grip strength, was also restored in Ctns-/- mice (Figure 2E and 2F). Differential effects of 25(OH)D3 versus 1,25(OH)2D3 treatment in Ctns-/- mice was observed. Percentage of lean mass and forelimb grip strength in 1,25(OH)2D3-treated Ctns-/- mice was still lower than controls (Figure 2E and 2F). 26 25 # 170 150 35 # # 30 20 WT + Vehicle 25 Ctns-/- + VitD1,25 # # 80 75 190 40 95 85 210 F 100 90 230 Ctns-/- + VitD25 WT + Vehicle Ctns-/- + VitD1,25 Ctns-/- + VitD25 # # 250 Ctns-/- + Vehicle Lean mass (%) D 14 13 12 11 10 9 Ctns-/- + Vehicle Fat mass (%) B Rotarod ac vity (s) 0 27 Grip strength (g per 100 g body mass) 1 28 WT + Vehicle 2 29 Ctns-/- + VitD1,25 3 Ctns-/- + VitD25 4 E 30 Ctns-/- + Vehicle C 5 Lean mass (g) Fat mass (g) A Figure 2: Body composi on and muscle func on in mice. Ctns-/- mice were compared with WT mice. Date are expressed and analyzed as in Figure 1. # P < 0.05. 3 Protein content (pg/ml) 25(OH)D3 and 1,25(OH)2D3 normalize pro-inflammatory cytokine mRNA level in skeletal muscle in Ctns-/- mice Ctns-/- + Vehicle Ctns-/- + VitD1,25 We measured IL-1α, IL-6 and TNF-α mRNA expression in Ctns-/- + VitD25 WT + Vehicle extracted gastrocnemius muscle. 25(OH)D3 and 1,25(OH)2D3 ameliorated IL-1α, IL-6 and TNF-α mRNA 20 levels in Ctns-/- mice (Figure 3). # 25(OH)D3 and 1,25(OH)2D3 improve the mRNA level of muscle regenerative and muscle proteolytic molecules in skeletal muscle in Ctns-/- mice We measured mRNA levels of key genes regulating muscle regeneration (Pax-3, Pax-7, myogenin, MyoD and IGF-I) as well as muscle degradation pathways (myostatin, atrogin and MuRF-1). 25(OH)D3 treatment normalized Pax-3, Pax-7, IGF-I, myostatin and MuRF-1 mRNA level while 1,25(OH)2D3 normalized Pax-3 and IGF-I mRNA level in Ctns-/- mie (Figure 4). # 15 # 10 5 0 IL-1α Rela ve mRNA levels (arbitrary units) 3 Ctns-/- + VitD1,25 Ctns-/- + VitD25 WT + Vehicle TNF-α Inflamma on Figure 3: Pro-inflammatory cytokine protein contents in gastrocnemius muscle. Results were analyzed and expressed as in Figure 1. # P < 0.05. 4 Ctns-/- + Vehicle IL-6 # # # # ## # 2 1 0 # Pax-3 # # # # # # ## # Pax-7 Myogenin MyoD IGF-I Muscle regenera on Myosta n Atrogin MuRF-1 Muscle degrada on Figure 4: mRNA levels of key genes in muscle regenera on and degrada on in gastrocnemius muscle. Results were analyzed and expressed as in Figure 1. # P < 0.05. TA fiber area (μm2) Soleus muscle fiber area (μm2) 25(OH)D3 and 1,25(OH)2D3 improve muscle fiber size in Ctns-/- mice We next examined effects of vitamin Ctns-/- + Vehicle D supplements on skeletal muscle Ctns-/- + VitD25 WT + Vehicle fiber size. Soleus and tibias anterior (TA) were harvested. These muscles A B were chosen as they represent 2000 2000 essentially, the extremes of muscle 1500 1500 # # # types in the mouse. The samples were sectioned and labeled with an 1000 1000 anti-laminin antibody to outline single 500 500 cells. Fiber area was quantified. Both soleus and TA fiber cross-sectional 0 0 area was significantly lower in Ctns-/mice (Figure 5A and 5B). 25(OH)D3 Figure 5: Soleus and TA muscle average fiber area in each group of mice. ameliorated the effects of cystinosis Results were analyzed and expressed as in Figure 1. # P < 0.05. 4 in soleus and TA muscle fiber size while 1,25(OH)2D3 normalized TA fiber size in Ctns-/mice. Rela ve mRNA levels (arbitrary units) 25(OH)D3 and 1,25(OH)2D3 suppress inflammasome gene expression We measured skeletal muscle mRNA expression of pro-IL-1β Ctns-/WT and NLRP3 in mice. mRNA expression was normalized in IL-1β NLRP3 Ctns-/-+VitD25 mice while still higher in Ctns-/-+VitD1,25 # 6 # mice relative to controls 4 # # (Figure 6). 2 0 + Vehicle + VitD25 + VitD1,25 + Vehicle + Vehicle + VitD25 + VitD1,25 + Vehicle We further examined the antiinflammatory effects of 25(OH)D3 and 1,25(OH)D3 on bone marrow derived Ctns-/WT Ctns-/- WT macrophages (BMDMs) isolated from cold sensitive 6: mRNA levels of IL-1β & Figure 7: FCAS bone marrow derived NLRP3 gain of function Figure N L R P 3 i n fl a m m a s o m e i n macrophages were cultured at 32°C with mutant mice, a highly NLRP3 gastrocnemius muscle. # P < 0.05. 25(OH)D3 (A) and 1,25(OH)D3 (B). IL-1β secre on was measured by ELISA (n=2, specific model in which mild performed in triplicate). hypothermia results in direct NLPR3 inflammasome activation and IL-1β release. BMDMs were pre-treated with 25(OH)D3 and 1,25(OH)D3 prior to incubation at 32C overnight and IL-1β release from treated cells was compared to untreated cells demonstrating a dose dependent reduction (Figure 7). This data supports our hypothesis that 25(OH)D3 and 1,25(OH)D3 suppress inflammation at the level of the NLRP3 inflammasome. IL-6 gene deletion fails to normalize energy homeostasis in Ctns-/- mice Upregulation of IL-6 is an importance cause of cachexia in CKD. Skeletal IL-6 protein content was elevated in Ctns-/- mice but was normalized by 25(OH)D3 and 1,25(OH)D3 supplements. We investigated the potential beneficial effects of IL-6 deletion in Ctns-/- mice by generating Ctns-/-IL-6-/- double KO mice. Ctns-/-IL-6-/- and Ctns-/- mice were compared to age- and sex-appropriated WT controls. No difference in body mass was observed in all groups at the age of 9-months (Figure 8A). Linear growth retardation was observed in both Ctns-/- and Ctns-/-IL-6-/- mice relative to controls (Figure 8B and 8C). Elevated basal metabolic rate and decreased energy efficiency was also observed in Ctns-/-IL-6-/- and Ctns-/mice but not in controls starting from 4-months of age (Figure 8D and 8E). Rotarod activity was impaired in Ctns-/-IL-6-/- and Ctns-/- mice but not in controls at 9-month (Figure 8F). 5 Ctns-/B n.s 30 20 10 0 n.s n.s 9 n.s n.s n.s 4000 # # # # 3500 3000 # 8 7 n.s 1.5 n.s P<0.05 # # # 1 0.5 0 F 0.1 n.s 0.05 n.s n.s # # # 2500 1-month 4-month 9-month 2 n.s E Energy efficiency Basal metabolic rate (ml/hr/kg) D C 10 n.s Femur length(cm) n.s Nose to base of tail (cm) Body mass (g) 40 0 1-month # 4-month 9-month Ctns-/-IL-6-/- Rotarod ac vity (s) A Ctns-/-IL-6-/- WT 250 n.s n.s n.s 225 200 # # 175 150 1-month 4-month 9-month Ctns-/- Figure 8: Comparison of phenotype of double KO, and WT mice. Number of mice ≥ 6 in each me point. All mice were fed ad libitum. Results are expressed as mean ± SEM. # P < 0.05. We are investigating the signaling pathways associated with skeletal muscle wasting in Ctns-/mice. Our results show that gene expression for several transcripts associated with myogenesis and skeletal regeneration (Pax-3, Pax-7, Myogenin, MyoD and IGF-I) is significantly decreased in gastrocnemius muscle of Ctns-/- mice. In contrast, expression of muscle proteolytic genes, Myostatin, Atrogin-1 and MuRF-1, are significantly increased in Ctns-/- mice. Muscle lysate protein levels of inflammatory cytokines (IL-1, IL-6 and TNF-) are increased in Ctns-/- mice. We are measuring whether 25(OH)D3 and 1,25(OH)2D3 treatment will influence the expression of those molecules implicated in muscle wasting in Ctns-/- mice. We are preparing a manuscript for submission. We appreciate the financial support from the Cystinosis Research Foundation. Sincerely, Robert Mak 6