effects of low-grade chronic inflammation on skeletal muscle protein metabolism in patients with ckd...
TRANSCRIPT
Effects of low-grade chronic inflammation on skeletal
muscle protein metabolism in patients with CKD
Renal Discoveries Grant Winners Meeting
Lake Bluff December 4-5, 2005
Giacomo GaribottoGenoa University, Division of Nephrology, Dialysis and
Transplantation
Background (1)Background (1)
Several studies have shown a strong association between chronic inflammation and long-term mortality and morbidity in ESRD patients.
Indexes of both nutritional status and physical function are linked to mortality in this population.
The percentage of patients showing evidence of inflammation increases progressively along with the decline in renal function, suggesting that cell release and/or body removal of pro-inflammatory cytokines is altered by uremia and/or treatment.
Background (2)Background (2)
Pathogenic mechanisms linking Pathogenic mechanisms linking chronic kidney disease, chronic kidney disease, inflammation and malnutrition inflammation and malnutrition not completely understood.not completely understood.
Also sAlso sites and mechanisms responsible for the regulation of circulating pro inflammatory cytokines in humans currently poorly known.
Research protocol designed to address the following questions in CKD patients:
1) Does low-grade chronic inflammation affect muscle protein synthesis or degradation and thus the control of net protein balance?
2) Is muscle skeletal energy expenditure increased by microinflammation?
3) Is the response to anabolic hormones blunted by microinflammation?
4) Do peripheral tissues release pro-inflammatory cytokines into the circulation and therefore contribute to the systemic inflammatory response?
Stenvinkel et al 2003
Adipose tissue and muscle-Adipose tissue and muscle-derived proteins known to affect derived proteins known to affect
inflammationinflammation
TNF-α IL-6 IL-1beta leptin adiponectin SAA3 Pentraxin-3 IL-1ra Macrophage
migrator inhibitor factor
TNF-α IL-6 IL.8 IL-10 TNFrsI TNFrsII
Adipocyte Skeletal muscle
IL-6 mRNA is IL-6 mRNA is expressed in resting expressed in resting human muscle and is human muscle and is rapidly increased by rapidly increased by contraction. A release contraction. A release of IL-6 from the legs of IL-6 from the legs (which are mainly (which are mainly composed of skeletal composed of skeletal muscle) has been muscle) has been shown to take place shown to take place during physical during physical exercise or glycogen exercise or glycogen depletion depletion (Febbraio (Febbraio
2004).2004).
Immunostaining for IL-6 In skeletal muscle (Pedersen 2003)
Resting
Exercising
In addition, insulin increases IL-6 In addition, insulin increases IL-6 gene expression in insulin-gene expression in insulin-resistant, but not in healthy resistant, but not in healthy skeletal muscle (Carey 2003) and skeletal muscle (Carey 2003) and Il-6 is released by muscle forearm in obese type 2 diabetic subjects (Corpeleijn JCEM 2005)(Corpeleijn JCEM 2005)
Both reactive oxygen species and Both reactive oxygen species and bacterial infections (Lang 2003) bacterial infections (Lang 2003) can upregulate muscle IL-6, likely can upregulate muscle IL-6, likely because of an activation of nuclear because of an activation of nuclear factor NF kB.factor NF kB.
Aims of the studyAims of the study
To explore the hypothesis that some inflammatory cytokine could be locally produced in skeletal muscle and exported to other tissues.
First, we studied the exchange of cytokines across the forearm in patients with CRF and in hemodialysis-treated patients with ESRD.
Second, we performed an analysis of cytokine protein and mRNA expression in muscle.
Protocols and methods: Protocols and methods: Forearm exchange of Forearm exchange of
cytokinescytokines 31 patients studied. Sixteen patients with 31 patients studied. Sixteen patients with
moderate to severe CKD (estimated moderate to severe CKD (estimated GFR=24±2 ml/min; CRP GFR=24±2 ml/min; CRP 12±312±3 mg/l; HCO3- mg/l; HCO3- 22.0±0.9022.0±0.90 mol/l mol/l); 15 patients were on HD ); 15 patients were on HD (CRP (CRP 35±835±8 mg/l; HCO3- mg/l; HCO3- 23.2±0.9 mmol/l23.2±0.9 mmol/l) ) and studied after approximately 72 to 74 h and studied after approximately 72 to 74 h from the last dialytic treatment.from the last dialytic treatment.
Triplicate sets of arterial and venous Triplicate sets of arterial and venous samples taken across the forearm at 20-samples taken across the forearm at 20-min intervals for TNF-min intervals for TNF-αα, , IL-6, IL-10 and IL-IL-6, IL-10 and IL-1 determinations,the postabsorptive state. 1 determinations,the postabsorptive state.
Measure of forearm blood flow. Measure of forearm blood flow. Plasma cytokine levels determined in Plasma cytokine levels determined in
triplicate by ELISA (Diaclone, France)triplicate by ELISA (Diaclone, France)
CKD HDCKD HD Age (yrs) 66±2 67±3Age (yrs) 66±2 67±3 Body weight (Kg) 73±4 68±4Body weight (Kg) 73±4 68±4 BMI (Kg/m2) 26±1 24±1BMI (Kg/m2) 26±1 24±1 Fat-free mass (Kg) 49±2 46± 8Fat-free mass (Kg) 49±2 46± 8 Fat mass (Kg) 25±2 21±2Fat mass (Kg) 25±2 21±2 nPNA (g/kg) 0.90±0.1 1±0.1nPNA (g/kg) 0.90±0.1 1±0.1 albumin (g/dl) 3.5±0.03 albumin (g/dl) 3.5±0.03
3.4±0.13.4±0.1 BUN (mg/dl) 61±5 84±8BUN (mg/dl) 61±5 84±8
TNF-TNF-αα A-V differences A-V differences across the forearm in CRF across the forearm in CRF
and HD patientsand HD patients
0
0,2
0,4
0,6
0,8
1
1,2
ARTERY VEIN ARTERY VEIN
CRF HD
NS NS
pg/ml
Il-6 A-V differences across Il-6 A-V differences across the forearm in CRF and HD the forearm in CRF and HD
patientspatients
0
10
20
30
40
50
60
70
80
90
ARTERY VEIN ARTERY VEIN
CRF HD
pg/ml
P<0.01
P<0.005
Peripheral tissues release IL-6 Peripheral tissues release IL-6 in patients showing evidence of in patients showing evidence of
inflammationinflammation
-16
-14
-12
-10
-8
-6
-4
-2
0
2
pg/m
in.1
00 m
l
Controls(6) CRF (16) HD (15)
AllLow IL-6High IL-6
*
*
*(<5pg/ml)
(>5pg/ml)
0
50
100
-2 18Il-6 release from peripheral tissues
(pg/min.100 ml)
Arter
ial Il-6
(pg
/ml)
R=0.652;p<0.001
Relationship between release Relationship between release of IL-6 from peripheral tissues of IL-6 from peripheral tissues
and plasma CRPand plasma CRP
0
10
20
30
0 20 40 60 80 100 120 140 160
CRP (mg/l)
Il-6
rel
ease
fro
mp
eru
ph
ery
(pg
/min
.100
ml)
R=0.79;p<0.001
O2 uptake
Controls
CKD(all subjects)
CKD(IL-6<5 pg/ml)
CKD (IL-6>5 ...
HD(all subjects)
HD(IL-6<5 pg/ml)
HD(IL-6>5 pg/ml)
0
5
10
15
20
ml/m
in.1
00
ml
Oxygen uptake by the forearm in patients with CKD and in controls
P= NSNS
Co
ntr
ols CK
D(a
ll s
ub
jec
ts)
CK
D(I
L-6
<5
pg
/ml)
CK
D(I
L-6
>5
pg
/ml)
HD
(all
su
bje
cts
)
HD
(IL
-6<
5 p
g/m
l)
HD
(IL
-6>
5 p
g/m
l)
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
Ph
e re
leas
e (n
mo
l/min
.100
ml)
Net protein balance across the forearm in CKD and HD patients
(12) (15) (8) (7) (15) (8) (7)
<0.05NS
Relationship between net Relationship between net protein breakdown and protein breakdown and
forearm IL-6 release in CRF forearm IL-6 release in CRF patientspatients
-4
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
6 8 10 12 14 16 18
Net Protein breakdown (nmol/min .100 ml)
Fo
rearm
IL
-6 r
ele
ase (
pg
/min
.100
ml)
r = 0.3861 p NS
Relationship between net protein Relationship between net protein breakdown and forearm IL-6 breakdown and forearm IL-6
release in HD patientsrelease in HD patients
-4
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
2 4 6 8 10 12 14 16 18 20 22 24
Net Protein Breakdown (nmol/min.100 ml)
fore
arm
IL
-6 r
ele
as
e )
pg
/min
.10
0 m
l)
r = 0.594 p < 0.0194
Protocols and Protocols and Methods:Methods:
Studies on muscle Studies on muscle biopsiesbiopsies Muscle biopsies obtained from rectus Muscle biopsies obtained from rectus
abdominis of 15 “inflamed” ESRD abdominis of 15 “inflamed” ESRD patients (7M-8F, age:69±7 yrs, GFR patients (7M-8F, age:69±7 yrs, GFR 8.4±1 ml/min8.4±1 ml/min ) during the placement ) during the placement of a PD catheter and in healthy of a PD catheter and in healthy subjects (4M-5F age 62±5yrs) during subjects (4M-5F age 62±5yrs) during surgery for abdominal wall hernias.surgery for abdominal wall hernias.
Immunohistochemical staining for Immunohistochemical staining for human IL-6.human IL-6.
Measurement of IL-6 mRNA in Measurement of IL-6 mRNA in muscle biopsies by semiquantitative muscle biopsies by semiquantitative RT-PCR.RT-PCR.
IL-6/βactin mRNA expression in IL-6/βactin mRNA expression in muscle of control subjects and muscle of control subjects and
ESRD patients ESRD patients
0
0,05
0,1
0,15
0,2
0,25
Exp
ressio
n of
IL-6
/Bac
tin
mR
NA * P=0.018
Control subjects
(IL-6=2±1 pg/ml)
ESRD
(IL-6=12±3pg/ml)
Immunohistochemical staining for IL-6 in skeletal muscle
Control ESRD
IL-6
0
5
10
15
20
25
ESRDControls
*
MyostatinMyostatin Myostatin, a member Myostatin, a member
of TGF-β family of of TGF-β family of signaling moleculessignaling molecules
Myostatin blockade: Myostatin blockade: excessive growth and excessive growth and increased force increased force generation of generation of skeletal muscleskeletal muscle
A role of myostatin in regulation of fiber size and cell survival in adult skeletal muscle.
Hyperexpressed in AIDS wasting
Belgian BlueMutation in myostatin gene
Myostatin/βactin mRNA expression in muscle of
controls and ESRD patients
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
expr
essio
n of
myo
/bac
t mR
NA
* P=0.015
ESRD (IL-6=12±3pg/ml)
Control subjects
(IL-6=2±1 pg/ml)
Muscle myostatin: Muscle myostatin: immunohistochemistryimmunohistochemistry (Rectus (Rectus
abdominis )abdominis )
Control ESRD
*
0
5
10
15
20
25
30
35
ESRDControls
*
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
0 0,1 0,2 0,3 0,4 0,5
mu
scle
myo
stat
in/b
eta-
acti
n m
RN
A
muscle IL-6/beta-actin mRNA
Relationship between IL-6 and Relationship between IL-6 and myostatin gene expressions in myostatin gene expressions in
rectus abdominis muscle of rectus abdominis muscle of ESRD patientsESRD patientsr=0.43, p=0.036
In conclusion, in renal patients with evidence of
microinflammation (I) Peripheral tissues release IL-6 into the circulation and the release of IL-6 from periphery is a major determinant of IL-6 levels.
Net protein breakdown is increased with respect to non-inflamed patients. This appears to be valid for HD but not for CRF patients.
In conclusion, in renal In conclusion, in renal patients with evidence of patients with evidence of
microinflammation (II)microinflammation (II) Il-6 and myostatin protein and gene
expressions are both upregulated in skeletal muscle.
These results suggest that the magnitude of increases in inflammatory cytokines in uremia may be predictive of upregulation of muscle Il-6 synthesis and growth.
Given the possible systemic and local effects of IL-6, peripheral tissues could play the double role of victim and culprit of the inflammatory response in HD patients.
Circulating IL-6
Skeletal muscle
Liver
Atrophy?
Fat cells
Lipolysis
Glicogenolysis
Endothelial damage
IL-1,Ca++,
Glycogen depletion?
Research protocol designed to address the following questions in CKD patients:
1) Does low-grade chronic inflammation affect muscle protein synthesis or degradation and thus the control of net protein balance?
2) Is muscle skeletal energy expenditure increased by microinflammation?
3) Is the response to anabolic hormones blunted by microinflammation?
4) Do peripheral tissues release pro-inflammatory cytokines into the circulation and therefore contribute to the systemic inflammatory response?
Protocols and Methods:Protocols and Methods:Studies on forearm muscle Studies on forearm muscle
protein turnoverprotein turnover Study of muscle protein turnover performed Study of muscle protein turnover performed
in the postabsorptive state (Garibotto KI in the postabsorptive state (Garibotto KI 1994).1994).
At 7.00 a.m., a forearm vein cannulated and At 7.00 a.m., a forearm vein cannulated and used for a primed-continuous infusion of used for a primed-continuous infusion of 2H-phenylalanine. 2H-phenylalanine.
Catheters inserted into a brachial artery and Catheters inserted into a brachial artery and in a retrograde manner into the ipsilateral, in a retrograde manner into the ipsilateral, deep forearm vein.deep forearm vein.
Triplicate sets of arterial and venous Triplicate sets of arterial and venous samples taken across the forearm at 20-min samples taken across the forearm at 20-min intervals after 150 min tracer equlibration intervals after 150 min tracer equlibration periodperiod
Measure of blood flow by strain gauge Measure of blood flow by strain gauge plethysmographyplethysmography
Muscle protein turnover rates:Muscle protein turnover rates: CRF (n=21) and HD (n=18) CRF (n=21) and HD (n=18)
patientspatients
-50-40-30-20-10
01020304050
Proteindegradation
Protein synthesis Net proteinbalance
CONTROLS CRF HD
nmol/min.100 ml
Muscle protein turnover rates:Muscle protein turnover rates:inflamed (n=11) vs non-inflamed inflamed (n=11) vs non-inflamed
(n=10)(n=10)CRF patientsCRF patients
-50-40-30-20-10
01020304050
Protein Degradation Protein synthesis Net Protein Balance
INFLAMED NON INFLAMED
NS
NS
NS
Muscle protein turnover rates:Muscle protein turnover rates:inflamed (n=8) vs non-inflamed inflamed (n=8) vs non-inflamed
(n=10) HD patients(n=10) HD patients
-50-40-30-20-10
01020304050
Protein degradation Protein Synthesis Net Protein balance
INFLAMED NON INFLAMED
*
*NS
EFFICIENCY BY MUSCLE PROTEIN TURN EFFICIENCY BY MUSCLE PROTEIN TURN OVER IN HD PATIENTS : INFLAMED VS. OVER IN HD PATIENTS : INFLAMED VS.
NON INFLAMEDNON INFLAMED
MUSCLE
NON-INFLAMED
PS PD
32% lost
MUSCLE
INFLAMED
53% lost
PS PD
Research protocol designed to address the following questions in CKD patients:
1) Does low-grade chronic inflammation affect muscle protein synthesis or degradation and thus the control of net protein balance?
2) Is muscle skeletal energy expenditure increased by microinflammation?
3) Is the response to anabolic hormones blunted by microinflammation?
4) Do peripheral tissues release pro-inflammatory cytokines into the circulation and therefore contribute to the systemic inflammatory response?
Growth Hormone (GH) exerts several physiologic and pharmacologic effects on protein, Na+, K+ and energy metabolism.
Acute effects are caused by GH binding to its own receptors, while chronic changes are mainly due to the release of IGF-I.
Potential interactions of signaling Potential interactions of signaling elements serving the growth hormone elements serving the growth hormone
(GH), IL-6, and IGF-I receptors (Haddad (GH), IL-6, and IGF-I receptors (Haddad
AJP 2004).AJP 2004).
Aim of the studyAim of the study
To evaluate if the microinflammatory state associated with uremia causes a resistance to the acute actions of GH regarding K+ and amino acid metabolism
Patients and Methods (I)Patients and Methods (I) 16 patients with advanced chronic renal failure
(Creat.clear 10-16 ml/min). No history or evidence of infection, liver disease or
neoplasia Calorie intake about 28-32 Kcal/kg; protein intake
0.8-1.1 g/kg Eight patients with evidence of peripheral vascular
or cardiovascular disease and CRP > 10 mg/l on three sequental determinations (Group A)
Eight patients with CRP levels persistently in the normal range (< 10 mg/l) (Group B)
Study performed also in 6 healthy volunteers (5M/1F) (Controls)
Patients and Methods(II)Patients and Methods(II)ParametParamet
ererGroupGroup
AAGroupGroup
BB
Age Age (yrs)(yrs) 60± 460± 4 63± 563± 5
gendergender 8M/8M/1F1F
8M/1F8M/1F
BMIBMI(Kg/m(Kg/m22))
23±323±3 23± 423± 4
nPNA nPNA (g/kg)(g/kg)
0.85±30.85±3 0.83±40.83±4
[[HCOHCO33]] (mmol/l)(mmol/l)
22±322±3 23± 223± 2
Creat Creat ClearClear(ml/min/(ml/min/1.731.73mm22))
10± 310± 3 8± 38± 3
ProceduresProcedures Study performed in the postabsorptive, overnight fasted state. Study performed in the postabsorptive, overnight fasted state. Arterialized samples for the measure of plasma hormones, KArterialized samples for the measure of plasma hormones, K++ and and
amino acid levels drawn from a dorsal hand vein at the baseline (at amino acid levels drawn from a dorsal hand vein at the baseline (at –15 and 0 min) and at 30-min intervals during a 300-min primed-–15 and 0 min) and at 30-min intervals during a 300-min primed-continuous infusion of rhGH (0.6 U) (0.7 mU/min/Kg) continuous infusion of rhGH (0.6 U) (0.7 mU/min/Kg) (Genotropin®, Pharmacia; Stockholm, Sweden) in the (Genotropin®, Pharmacia; Stockholm, Sweden) in the contralateral arm.contralateral arm.
rhGH
-30 0 60 120 180 240 300 min
sampling
MethodsMethods
Measure of blood amino acids and Measure of blood amino acids and plasma potassium, Caplasma potassium, Ca++++, acid-base as , acid-base as well as GH well as GH (chemiluminescent IRMA assay (chemiluminescent IRMA assay and Immunofunctional GH) and Immunofunctional GH) (Strasburger(Strasburger JCEM JCEM
1996)1996)..
Measure of plasma insulin, cortisol and Measure of plasma insulin, cortisol and proinflammatory cytokines (Il-6, Il-1, proinflammatory cytokines (Il-6, Il-1, TNF-α, TNFrs-1).TNF-α, TNFrs-1).
0
20
40
60
80
100
120
140
160
180
0 30 90 180 240 270
minutes
GH
leve
ls [m
g/l]
GH IRMA group A
GH IRMA group B
IFGH group A
IFGH group B
GH infusion
Effects of rhGH infusion on GH levels
3
5
7
9
11
13
15
0 30 60 90 120 150 180 210 240 270
minutes
pla
sm
a i
ns
uli
n (
U/L
)
Group A Group B Controls
*
Effects of rhGH infusion on plasma insulin
rhGH
NS
3
3,5
4
4,5
5
5,5
6
0 30 60 90 120 150 180 210 240 270
minutes
pla
sma
po
tass
ium
[m
Eq
/l]
Group A Group B Controls
* * **
*
*****
Effects of rhGH infusion on plasma K+
rhGH
NS
20
21
22
23
24
25
0 30 60 90 120 150 180 210 240 270
minutes
[HC
O3]
[m
Eq
/l]
Group A Group B Controls
*
* * *
Effects of rhGH infusion on plasma [HCO3-]
rhGH
* * * * *
NS
NS
200
220
240
260
280
300
320
340
360
0 30 60 90 120 150 180 210 240 270
minutes
BC
AA
[m
mo
l/l]
Group A
Group B
Controls
* * **
*
GH infusion
Acute effects of GH infusion on BCAA levels
* **
* *
**
Relationships between GH-induced Relationships between GH-induced changes in plasma Kchanges in plasma K++ at 180 min and at 180 min and
selected parametersselected parameters
VariableVariable rr PPBMIBMI -0.07-0.07 NSNS
ageage 0.3610.361 0.0150.015
InsulinInsulin -0.279-0.279 NSNS
CRPCRP 0.5150.515 0.040.04
Il-1Il-1 -0.276-0.276 NSNS
Il-6Il-6 0.5430.543 0.0250.025
TNF-TNF-αα 0.2000.200 NSNS
TNFrs1TNFrs1 0.5940.594 0.0250.025
PTHPTH 0.150.15 NSNS
Relationships between GH-induced changes in Relationships between GH-induced changes in plasma essential amino acids at 180 min and plasma essential amino acids at 180 min and
selected parametersselected parameters
VariableVariable RR PP
BMIBMI -0.65-0.65 <0.001<0.001ageage 0.300.30 NSNS
InsulinInsulin -0.30-0.30 NSNS
CRPCRP 0.5690.569 0.050.05
Il-1Il-1 -0.276-0.276 NSNS
Il-6Il-6 0.580.58 0.050.05
TNF-TNF-αα 0.2300.230 NSNS
TNFrs1TNFrs1 0.360.36 NSNS
Crear clearCrear clear -0.88-0.88 <0.001<0.001
Conclusions (I)Conclusions (I)
RhGH infusion causes a significant RhGH infusion causes a significant decrease in Kdecrease in K++ levels, with correction of levels, with correction of hyperkalemia in non-inflamed patients hyperkalemia in non-inflamed patients with CRF. This response is maximal after with CRF. This response is maximal after three hours. The absolute decline in Kthree hours. The absolute decline in K++ levels is similar to that observed in levels is similar to that observed in healthy subjects.healthy subjects.
The sensitivity to GH regarding amino The sensitivity to GH regarding amino acids is delayed in non-inflamed patients acids is delayed in non-inflamed patients with CRF; however, the overall response with CRF; however, the overall response is similar to controls.is similar to controls.
Conclusions (II)Conclusions (II) The sensitivity to GH regarding both KThe sensitivity to GH regarding both K++ and and
amino acid metabolism is blunted in amino acid metabolism is blunted in patients with chronic kidney disease patients with chronic kidney disease showing evidence of inflammation.showing evidence of inflammation.
In these patients responses regarding In these patients responses regarding potassium metabolism are predicted by age, potassium metabolism are predicted by age, CRP, plasma Il-6 and TNFrs-1 levels. CRP, plasma Il-6 and TNFrs-1 levels. Responses regarding amino acid metabolism Responses regarding amino acid metabolism are predicted by BMI, CRP, Il-6 and residual are predicted by BMI, CRP, Il-6 and residual renal function.renal function.
These data are consistent with a block in GH These data are consistent with a block in GH signaling caused by age and signaling caused by age and microinflammation.microinflammation.
Does the human kidney Does the human kidney remove IL-6 from the remove IL-6 from the
circulation in humans?circulation in humans?
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
(ng/
min
.1.7
3 m
2)
KIDNEY SPLANCHNIC ORGANS
*
Inter-organ exchange of IL-6Inter-organ exchange of IL-6(n=6 patients undergoing(n=6 patients undergoing
venous catheterizations for diagnosis)venous catheterizations for diagnosis)
A-V gradient: 7%
A-V gradient: 16%
*
Removal of IL-6 by splanchnic organs+ kidney Removal of IL-6 by splanchnic organs+ kidney accounts foraccounts for ~ ~50% of the calculated50% of the calculated (Mohamed (Mohamed
–Alì JCEM 1997)–Alì JCEM 1997)IL-6 production in humansIL-6 production in humans
0
1
(mm
ol/
min
)
Kidney+Splanchnic removal Released by adipocytes
Studies on GH sensitivityStudies on GH sensitivity Antonella Barreca, Antonella Barreca, Francesco Minuto (DiSEM)Francesco Minuto (DiSEM) Genoa UniversityGenoa University
Forearm balance studiesAntonella Sofia Rodolfo RussoValeria Cappelli
Massimiliano Di MartinoAlice Tarroni
Muscle Biopsies:Stefano SaffiotiFranco De Cian
Francesca Aloisi
Muscle molecolar biologyVanessa Procopio
Daniela Verzola
Homocysteine/IL-6Maria Rita Sala
Barbara VillaggioAlessandro Valli
Leptin/granulocytesTomaso BarrecaFranco Dallegri
Luciano Ottonello(DiMI)
Genoa University
Supported by The Supported by The Baxter/ISN Extramural Baxter/ISN Extramural
Program 2002Program 2002
Truncal fat mass as a contributor to Truncal fat mass as a contributor to inflammation in end-stage renal inflammation in end-stage renal disease (Axelsson, AJCN 2004)disease (Axelsson, AJCN 2004)
-1,8
-1,6
-1,4
-1,2
-1
-0,8
-0,6
-0,4
-0,2
0
0,2
35 55 75 95 115 135 155
TNFrs-1 (pg/ml)
pla
sma
po
tass
ium
[m
Eq
/L]
Relationship between GH-induced changes in plasma K+ and basal TNFrs-1
levels
R=0.59;p<0.025
In conclusion, in patients with In conclusion, in patients with CKD:CKD:
Prevalence of inflammation and altered nutrition increase progressively along with the decline in GFR.
Both proxies for inflammation and nutrition are associated to a worse outcome.
The question whether inflammation contributes to atherosclerotic cardiovascular disease and dialysis causes inflammation remains in part unanswered.
Selective alterations can be ascribed to individual cytokines. Anorexia is best accounted for TNF-α levels, while Il-6 appears to be the best predictor for resistance to anabolic factors (EPO and Growth Hormone) and hypoalbuminemia.
In conclusion, in patients with In conclusion, in patients with CKD (II):CKD (II):
Besides circulating cells and endothelia, somatic cells (adipocytes and muscle cells) are also a source of inflammatory cytokines.
In this regard, skeletal muscle appear to be both culprit and victim of the inflammatory processes.
All these data are consistent with a different concept of malnutrition, which is based not only on reduced nutrient intake but on overall dysregulation, involving both nutritional and non-nutritional (immune, endocrine, circulatory) effects.
-1,8
-1,6
-1,4
-1,2
-1
-0,8
-0,6
-0,4
-0,2
0
0,2
-2 -1,5 -1 -0,5 0 0,5 1
pla
sm
a p
ota
ss
ium
[m
Eq
/L]
Relationship between GH-induced changesin plasma K+ and [HCO3 ]
Delta HCO3
r=0.597;p<0.015
-1,8
-1,6
-1,4
-1,2
-1
-0,8
-0,6
-0,4
-0,2
0
0,230 35 40 45 50 55 60 65 70 75 80
Age [yrs]
Cha
nges
in p
lasm
a K
+ [m
Eq/
l]
r = 0.56 ; p < 0.015
Group A Group B
Relationship between GH-induced changesin plasma K+ and age
Introduction (3)Introduction (3) Myostatin, a member of TGF-β family of
signaling molecules (Mc Pherron, Nature 1997) acts as a negative regulator of skeletal muscle mass.
Myostatin blockade results in excessive growth and increased force generation of skeletal muscle (Tobin 2005)
A role of myostatin in regulation of fiber size and cell survival has been shown to occur in adult skeletal muscle (Tobin 2005).
Myostatin is hyperexpressed in AIDS wasting (Cadavid, 1998)
-1,8
-1,6
-1,4
-1,2
-1
-0,8
-0,6
-0,4
-0,2
0
0,2
0 5 10 15 20 25 30
C-reactive protein [mg/l]
Ch
ang
es in
pla
sma
po
tass
ium
[m
Eq
/l]
r = 0.46 ; p < 0.04
Relationship between GH-induced changesin plasma potassium and CRP level
IL-6IL-6 An endocrine cytokine Major effector of the acute-phase response Released by fat (adipocytes+macrophages)
accounts for 30% of circulating IL-6 If infused, it causes muscle atrophy,
lipolysis and worsens atherosclerosis (Huber 1999)
Predicts outcome in the elderly (Harris AJM 1999) and in HD patients (Pecoits-Filho NDT 2002, Rao AJKD 2005)
Predicts myocardial infarction in healthy humans (Ridker, Circulation 2000)
IntroductionIntroduction Although most of the circulating IL-6 is secreted from Although most of the circulating IL-6 is secreted from
activated mononuclear cells, adipocytes (Mohamed–activated mononuclear cells, adipocytes (Mohamed–Alì JCEM 1997) and skeletal muscle (Febbraio MA Alì JCEM 1997) and skeletal muscle (Febbraio MA FASEB 2002) are also a possible source of this FASEB 2002) are also a possible source of this cytokine. cytokine.
IL-6 mRNA is expressed in resting human muscle and IL-6 mRNA is expressed in resting human muscle and is rapidly increased by contraction. A release of IL-6 is rapidly increased by contraction. A release of IL-6 from the legs (which are mainly composed of skeletal from the legs (which are mainly composed of skeletal muscle) has been shown to take place during physical muscle) has been shown to take place during physical exercise or glycogen depletion exercise or glycogen depletion (Febbraio 2004).(Febbraio 2004).
In addition, it has been recently observed that insulin In addition, it has been recently observed that insulin increases IL-6 gene expression in insulin-resistant, increases IL-6 gene expression in insulin-resistant, but not in healthy skeletal muscle (Carey 2003). Both but not in healthy skeletal muscle (Carey 2003). Both reactive oxygen species and bacterial infections (Lang reactive oxygen species and bacterial infections (Lang 2003) can upregulate muscle IL-6, likely because of 2003) can upregulate muscle IL-6, likely because of an activation of nuclear factor NF kB.an activation of nuclear factor NF kB.
-1,8
-1,6
-1,4
-1,2
-1
-0,8
-0,6
-0,4
-0,2
0
0,2
0 10 20 30 40 50 60 70 80 90
IL-6 [pg/ml]
Ch
an
ges i
n p
lasm
a K+ [
mE
q/l
]
r = 0.54 ; p < 0.03
Group A Group B
Relationship between GH-induced changes in plasma K+ and basal Il-6 levels
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
2 4 6 8 10 12 14 16 18 20 22 24
NP ALL
IL-6
AL
L
r = 0.395 p < 0.0508
-28
-25
-22
-19
-16
-13
-10
-7
-4
-1
2
5
0 10 20 30 40 50 60 70
IL-6 ART HD
IL-6
RE
LE
AS
E H
D
-4
-3
-2
-1
0
0 5 10 15 20 25 30 35 40 45
IL-6 ART CFR
IL-6
RE
LE
AS
E C
R
r = 0.69 p<0.04
r = -0.2856 p<0.28
Relationship between IL-6 Relationship between IL-6 release by the forearm and release by the forearm and
arterial IL-6 in CRF patientsarterial IL-6 in CRF patients
-4
1
6
11
16
21
26
0 5 10 15 20 25 30 35 40 45
Arterial IL-6
IL-6
RE
LE
AS
E b
y t
he
fo
rea
rm r = -0.2856 p=NS
Relationship between IL-6 Relationship between IL-6 release by the forearm and release by the forearm and arterial IL-6 in HD patientsarterial IL-6 in HD patients
-4-202468
1012141618202224262830
0 10 20 30 40 50 60 70
Arterial IL-6
IL-6
re
lea
se
by
th
e f
ore
arm
r = 0.69 p<0.04
IL-6 is produced by human skeletal muscle
during physical activity
IL-6Glycogen depletion
LipolysisGluconeogenesis
Arterial interleukin-6 (IL-6) concentration (Arterial interleukin-6 (IL-6) concentration (toptop), ), hepatosplanchnic vein-arterial (hv-a) IL-6 concentration hepatosplanchnic vein-arterial (hv-a) IL-6 concentration
((middlemiddle), and net hepatosplanchnic IL-6 uptake ), and net hepatosplanchnic IL-6 uptake ((bottombottom) before (0 min) and during 120 min of ) before (0 min) and during 120 min of
semirecumbent cycling at 62 ± 2% of maximal oxygen semirecumbent cycling at 62 ± 2% of maximal oxygen uptake (Febbraio AJP 2003)uptake (Febbraio AJP 2003)
IL-6 is released by forearm IL-6 is released by forearm muscle in insulin-resistant muscle in insulin-resistant
obese subjects obese subjects (Corpeleijn JCEM 2005)(Corpeleijn JCEM 2005)
-3
-2,5
-2
-1,5
-1
-0,5
0
Ins.Res Normaltolerance
IL-6 exchange
*
R2 p Net Protein balance
0.08 NS
O2 uptake
0.02 NS
CO2 release
0.24 0.04
Relationships between the percent IL-6 Relationships between the percent IL-6 enrichment in the forearm vein and other enrichment in the forearm vein and other
variables in CKD patientsvariables in CKD patients
Efficiency of muscle protein Efficiency of muscle protein turnover in patients with CKD turnover in patients with CKD
(NB/PD)(NB/PD)
0
5
10
15
20
25
30
35
40
45
50
Recycled Phe
Controls CRF CAPD HD HD HD CRP>10 mg Malnourished CRP>10 mg/l
*