Vitamin D and Muscle Wasting in Nephropathic Cystinosis

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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 32C 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
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