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

*