A genetic variant of the CC-A genetic variant of the CC-chemokine CCL2 is associated with chemokine CCL2 is associated with renal function decline in PREVENDrenal function decline in PREVEND

Mike W. Zuurman, Inter.Med./Nephrology, UMC Groningen4-1-2006

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Renal function: determinants

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-age

-blood pressure

-smoking

-inflammatory state

-sex?

Sex differences in renal pathologyBreedtestrategie

Park et al. JBC 279, 50:52282- 2004

A murine example:

Females Males

Sex differences in renal pathologyBreedtestrategie

Renal function: determinants

Observed differences between males and females in renal pathologyunderline importance of separating by sex in analyses of renal function

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Sex

Renal function: determinants

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Inflammatory state

1. Albuminuria (proteinuria)

2. CCL2 -2518 pro-inflammatory genotype

Chemokines and their receptors: families

Classified based on position of conserved cystein residuesin the protein structure

Chemokine functions

-Intercellular communication (brain)

-Angiogenesis (f.i. tumour related)

-Orchestration of the immune response * Pro- and anti-inflammatory

-Hematopoiesis

-Organogenesis

-Ovulation, luteal regression

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Chemokines in pathology: renal disease

J Am Soc Nephrol 11:152-176, 2000

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CCL2

-Monocyte chemoattractant protein-1

-CC-chemokine(group of chemokines that are chemoattractants for lymphocytes, monocytes, eosinophils, basophils, but not neutrophils.)

-Pro-inflammatory

-Leukocyte recruitment

-Angiogenesis

Chemokines in pathology: renal diseaseBreedtestrategie

Association between circulating monocyte chemoattractant protein-1 and urinary albumin excretion in nonobese Type 2 diabetic patients, J Diabetes Complications. 2006 Mar-Apr;20(2):98-104.

CCL2 in renal pathology

Monocyte chemoattractant protein-1 promotes the development of diabetic renal injury in streptozotocin-treated mice, Kidney Int. 2006 Jan;69(1):73-80.

Reactive oxygen species-mediated signaling pathways in angiotensin II-induced MCP-1 expression of proximal tubular cells. Antioxid Redox Signal. 2005 Sep-Oct;7(9-10):1261-8.

Renal response to repetitive exposure to heme proteins: chronic injury induced by an acute insult. Kidney Int. 2000 Jun;57(6):2423-33.

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Letendre et al. J Neuroimmunol. 2004 Dec;157(1-2):193-6.

HIV: CCL2 CSF up, plasma trend

CCL2 -2518 A/G functional expression consequences

CCL2 plasma up in AD patients

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Fenoglio et al.Neurobiol Aging. 2004 Oct;25(9):1169-73.

MS: CCL2 plasma trend (P=0.08)

Tissue Antigens. 2004 Jul;64(1):70-3

The polymorphism of monocyte chemoattractant protein-1 is associated with the renal disease of SLE. Kim HL, Lee DS, Yang SH, Lim CS, Chung JH, Kim S, Lee JS, Kim YS. Am J Kidney Dis. 2002 Dec;40(6):1146-52.

Monocyte chemoattractant protein-1 A-2518G gene polymorphism and renal survival of Japanese patients with immunoglobulin A nephropathy. Mori H, Kaneko Y, Narita I, Goto S, Saito N, Kondo D, Sato F, Ajiro J, Saga Clin Exp Nephrol. 2005 Dec;9(4):297-303.

Association of the MCP-1 gene polymorphism A-2518G with carpal-tunnel syndrome in hemodialysis patients. Omori K, Kazama JJ, Song J, Goto S, Takada T, Saito N, Sakatsume M, Narita I, Gejyo F. Amyloid. 2002 Sep;9(3):175-82.

A Monocyte chemoattractant protein-1 (MCP-1) polymorphism and outcome after renal transplantation. Kruger B, Schroppel B, Ashkan R, Marder B, Zulke C, Murphy B, Kramer BK, Fischereder M. J Am Soc Nephrol. 2002 Oct;13(10):2585-9.

CCL2 -2518 in renal pathology

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Kruger et al. J Am Soc Nephrol. 2002 Oct;13(10):2585-9.

Renal function: determinants

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Inflammatory state

1. Albuminuria (proteinuria)

2. CCL2 -2518 pro-inflammatory genotype

3. Sex (differential regulation of inflammation)

Hypothesis

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Renal function decline is modified by CCL2 -2518 A/G in an albuminuria and sex dependent manner

Research population (1)

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PREVEND (n=8502)General population-basedEnriched for albuminuria

Selection: Succesful genotype, Caucasian descent(n=7504)

Follow-up: 4 years on average

Renal function:BSA adjusted Creatinine clearance (CrCl)based on serum Cr and 2x24h urine concentractions

Renal damage/Inflammation:Urinary albumine excretion (2x24h)

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Research population (2)

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Main outcome parameter:

Renal Function Decline (RFD) : More than 10% decrease in CrCl since baseline

Baseline characteristics by CCL2 -2518 A/G

BreedtestrategieTable 1. Baseline characteristics by CCL2 -2518 A/G genotype

AA AG GG

Males N 1940 1458 292 Age (years) 51 ± 13 51 ± 13 50 ± 13 SBP (mmHg) 134 ± 18 134 ± 19 133 ± 18 DBP (mmHg) 77 ± 9 77 ± 10 77 ± 10

CrCl (ml/min/1.73 m2)

95 ± 21 94 ± 21 94 ± 21

UAE (mg/L/24h) 10.9 (7.1 - 23) 10.6 (6.9 - 22.9) 10.7 (6.8 - 22.9) CRP (mmol/L) 1.2 (0.6 - 2.7) 1.4 (0.6 - 3) 1.1 (0.5 - 2.5) BMI 26 ± 4 26 ± 4 26 ± 3 BSA 2 ± 0.2 2 ± 0.2 2.1 ± 0.2 Smoking (%) 38 36 36 TC (mmol/L) 5.7 ± 1.1 5.7 ± 1.1 5.7 ± 1.1 HDL-c (mmol/L) 1.2 ± 0.3 1.2 ± 0.3 1.2 ± 0.3

ΔCrCl (ml/min/1.73m2)

-2.9 ± 20.5 -1.9 ± 20.6 -2.6 ± 20.8

Females N 1982 1550 282 Age (years) 48 ± 12 48 ± 12 49 ± 13 SBP (mmHg) 124 ± 21 124 ± 20 127 ± 22 DBP (mmHg) 71 ± 9 71 ± 9 71 ± 9 OCC (%) 26 25 24

CrCl (ml/min/1.73 m2)

90 ± 21 91 ± 20 89 ± 20

UAE (mg/L/24h) 8.3 (5.8 - 14.1) 8.5 (5.9 - 14.3) 8.9 (6.1 - 18.3) CRP (mmol/L) 1.4 (0.6 - 3.2) 1.4 (0.6 - 3.2) 1.3 (0.5 - 3.5) BMI 26 ± 5 26 ± 5 26 ± 5 BSA 1.8 ± 0.1 1.8 ± 0.2 1.8 ± 0.2 Smoking (%) 39 38 35 TC (mmol/L) 5.7 ± 1.2 5.6 ± 1.1 5.7 ± 1.2 HDL-c (mmol/L) 1.5 ± 0.4 1.5 ± 0.4 1.5 ± 0.4

ΔCrCl (ml/min/1.73m2)

0.1 ± 18.3 -0.9 ± 18.6 -1.1 ± 19.9

Shown are arithmetic means and standard deviations, median and 25th – 75th percentiles in case of skewed distributions, and group percentages. No characteristics differed significantly between genotype groups.

Table 2. Impaired renal function and renal function decline by genotype

AA AG GG P

Males IRF0 4.2 (81/1907) 6.3 (90/1440) 6.4 (18/283) 0.024

RFD 34.2 (515/1504) 32.7 (370/1130) 29.8 (65/218) n.s. IRF1 6.3 (96/1520) 5.6 (64/1137) 5.4 (12/222) n.s.

Females IRF0 5.6 (108/1941) 5.6 (85/1505) 7 (19/273) n.s. RFD 27 (413/1530) 30.6 (358/1170) 31.3 (67/214) n.s.

IRF1 6.2 (96/1544) 5.5 (65/1190) 5.9 (13/219) n.s.

IRF: impaired renal function (CrCl < 60 ml/min/1.73m2); RFD: more than 10% decline in CrCl during follow-up.

Renal function and change by genotype

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Breedtestrategie -RFD +RFD P

Males Age (yrs) 50 +/- 12 52 +/- 13 <0.05

SBP (mmHg) 132 +/- 17 135 +/- 19 <0.05 Smoking (%) 34.5 37.8 0.08 BMI 26.2 +/- 3.5 26.3 +/- 3.4 n.s. TC (mmol/L) 5.7 +/- 1.1 5.6 +/- 1.1 n.s. HDL-c (mmol/L) 1.16 +/- 0.3 1.15 +/- 0.3 n.s. (V)LDL-c (mmol/L) 4.5 +/- 1.2 4.5 +/- 1.1 n.s.

TGL (mmol?L) 1.6 +/- 1.2 1.6 +/- 1.1 n.s. CrCl (ml/min/1.73m2) 91.3 +/- 18.6 103.2 +/- 20.9 <0.05 delta CrCl (ml/min/1.73m2) 8 +/- 14.5 -23.5 +/- 13.4 <0.05 lnlnUAE 0.9 +/- 0.3 1 +/- 0.4 <0.05 MA (%) 15 17.7 0.07 CRP (mmol/L) 2.3 +/- 3.8 2.7 +/- 4.6 <0.05

CCL2 -2518 (%) n.s. AA 52 54.2 AG 40 38.9 GG 8 6.8 CCL2 -2518 (%) n.s. AG+GG 48 45.8

Females Age (yrs) 48 +/- 12 49 +/- 12 <0.05 SBP (mmHg) 123 +/- 19 125 +/- 22 <0.05 Smoking (%) 35.8 39 0.1 BMI 25.8 +/- 4.7 26.1 +/- 4.6 n.s. TC (mmol/L) 5.6 +/- 1.2 5.7 +/- 1.1 n.s. HDL-c (mmol/L) 1.51 +/- 0.4 1.46 +/- 0.4 <0.05

(V)LDL-c (mmol/L) 4.1 +/- 1.3 4.2 +/- 1.2 n.s. TGL (mmol?L) 1.2 +/- 0.7 1.3 +/- 0.9 <0.05 CrCl (ml/min/1.73m2) 87 +/- 17 99.5 +/- 21.9 <0.05 delta CrCl (ml/min/1.73m2) 8 +/- 12.9 -21.3 +/- 13 <0.05 lnlnUAE 0.8 +/- 0.3 0.8 +/- 0.3 <0.05 MA (%) 7.7 10.6 0.01

CRP (mmol/L) 2.6 +/- 4.4 3.2 +/- 5.5 <0.05 CCL2 -2518 0.08 AA 53.8 49.3 AG 39.1 42.7 GG 7.1 8 CCL2 -2518 0.027

AG+GG 46.2 50.7

Baseline characteristics by RFD

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Table 3. Logistic regression model of Renal Function Decline

OR P

Age (yrs) 1.03 <0.0001 BMI 0.97 <0.01

Smoking (Yes) 1.15 <0.05 SBP (mmHg) 1.01 <0.05 CrCl0 (ml/min/1.73m2) 1.04 <0.0001 ltUAE 1.34 <0.01 CRP (mmol/L) 1.02 <0.01

ltUAE: double natural logistic transformed UAE (mg/L/24h).

Logistic regression model for RFD

Note that in this model sex and CCL2 -2518 genotype are not independent factors

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Multivariable fractional polynomial model for RFD

Table 4. Multivariable fractional polynomial model for RFD

RFD OR Std. Err. z P [95% Conf.Interval]

Age 1.004287 0.000387 11.1 0 1.003529 1.005046 SBP 1.004076 0.001975 2.07 0.039 1.000213 1.007954 BMI 9771328 0.008494 -2.66 0.008 0.960625 0.993924 Smoking 1.16629 0.077666 2.31 0.021 1.023583 1.328893 frCrCL_1 3.08E-07 9.67E-07 -4.77 0 6.50E-10 0.000146 frCrCl_2 202957.3 339270.3 7.31 0 7664.907 5374058 ltUAE 1.101698 0.201094 0.53 0.596 0.770357 1.575552 CRP 1.016894 0.007196 2.37 0.018 1.002888 1.031096 Sex 0.874998 0.217463 -0.54 0.591 0.537599 1.42415 CCL2 -2518 G+ 0.524823 0.137238 -2.47 0.014 0.314362 0.876186 sex*ltUAE 1.168397 0.315529 0.58 0.564 0.68821 1.983626 sex*CCL2 2.5841 0.91073 2.69 0.007 1.295135 5.155887 ltUAE*CCL2 1.890192 0.498371 2.41 0.016 1.127397 3.169092 CCL2*sex*ltUAE 0.438736 0.166384 -2.17 0.03 0.20864 0.922591

A-hom ozygotes G-ca rriers

1 2 3 4 5 6 7 8 9 10

Tentiles of UAE (transformed)

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Tentiles of UAE (transformed)

Multivariable fractional polynomial model for RFD

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Correlations

1 ,719 **

,000

166593808,9 86494005,816

21093,164 13681,431

7899 6323

,719 ** 1

,000

86494005,816 144101986,2

13681,431 22739,780

6323 6338

Pearson Correlation

Sig. (2-tailed)

Sum of Squares andCross-products

Covariance

N

Pearson Correlation

Sig. (2-tailed)

Sum of Squares andCross-products

Covariance

N

Mean Urinary AlbuminExcretion (UAE)

Mean Urinary AlbuminExcretion (UAE)

Mean UrinaryAlbuminExcretion

(UAE)

Mean UrinaryAlbuminExcretion

(UAE)

Correlation is significant at the 0.01 level (2-tailed).**.

Correlations nc1 vs nc2

Correlations

1 ,528 **

,000

7349 5766

,528 ** 1

,000

5766 5832

Pearson Correlation

Sig. (2-tailed)

N

Pearson Correlation

Sig. (2-tailed)

N

Creatinine clearance(ml/min/1,73m**2)

Creatinine clearance(ml/min/1,73m**2) NC2

Creatinineclearance

(ml/min/1,73m**2)

Creatinineclearance

(ml/min/1,73m**2) NC2

Correlation is significant at the 0.01 level (2-tailed).**.

UAE

CrCl

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Males AA AG GG

ORN

case/controlOR P N OR P N Overall P

CrCl (measured) 1 81/1826 1.5 0.01 90/1350 1.53 0.113 18/265 0.025

CG 1 69/1853 0.92 0.67 48/1397 0.58 0.2 6/279 0.435

MDRD 1 40/1896 0.86 0.56 26/1428 0.66 0.44 4/286 0.67

Females

CrCl (measured) 1 108/1833 1.01 0.92 85/1420 1.3 0.35 19/254 0.645

CG 1 103/1848 0.84 0.28 68/1453 1.12 0.564 17/261 0.381

MDRD 1 18/1956 1.36 0.37 19/1522 0.39 0.36 1/279 0.364

Impaired renal function – univariate Measured versus predicted

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Logistic regression Number of obs = 5306 LR chi2(10) = 329.43 Prob > chi2 = 0.0000Log likelihood = -2841.3476 Pseudo R2 = 0.0548------------------------------------------------------------------------------cock_del~c_b | Odds Ratio Std. Err. z P>|z| [95% Conf. Interval]-------------+---------------------------------------------------------------- Iage___1 | 1.006379 .0004285 14.94 0.000 1.00554 1.007219 Icock__1 | 4.571734 .8421515 8.25 0.000 3.186269 6.55963 Icock__2 | .0035736 .0018236 -11.04 0.000 .0013144 .0097159 sex | 2.029337 .5176993 2.77 0.006 1.230853 3.345817 ccl22518_g | .8152031 .2230756 -0.75 0.455 .4768035 1.393773 Ilnln__1 | 1.394434 .2640953 1.76 0.079 .9620257 2.021199 Iint___1 | .7489486 .2049527 -1.06 0.291 .4380437 1.280521int_SEX_CCL2 | 1.304824 .469326 0.74 0.459 .6447441 2.640685 Iint_a_1 | 1.356773 .3682877 1.12 0.261 .7969918 2.309727 Iint_b_1 | .7595832 .2913697 -0.72 0.473 .3581473 1.610976------------------------------------------------------------------------------

Deviance: 5682.695.

Multivariable fractional polynomial model for RFD (Cockroft)

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AAAGGG

CCL2 SNPolymorphism -2518

Ba rs s how % of Cases = 1 ,00

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De Novo IRFBreedtestrategie

De novo Cockroft < 60 NC2 :------------------------------------------------------------------------------cock_2_de~60 | Odds Ratio Std. Err. z P>|z| [95% Conf. Interval]-------------+---------------------------------------------------------------- Iage___1 | 1.138783 .0110555 13.39 0.000 1.117319 1.160658 sex | 1.399462 .2379566 1.98 0.048 1.00283 1.952965 ccl22518_g | .947831 .156412 -0.32 0.745 .6859063 1.309776 UAE15 | 1.413706 .2426113 2.02 0.044 1.009907 1.97896------------------------------------------------------------------------------Deviance: 1181.832.

------------------------------------------------------------------------------cock_2_de~60 | Odds Ratio Std. Err. z P>|z| [95% Conf. Interval]-------------+---------------------------------------------------------------- Iage___1 | 1.138128 .011092 13.28 0.000 1.116594 1.160077 sex | 1.758046 .5305301 1.87 0.062 .9731076 3.176139 ccl22518_g | .9569597 .3532386 -0.12 0.905 .4641871 1.972851 UAE15 | 1.852891 .586917 1.95 0.052 .9959224 3.447262int_SEX_CCL2 | .8027043 .3740964 -0.47 0.637 .322001 2.001031intUAE15_SEX | .3144433 .1678408 -2.17 0.030 .1104576 .8951366intUAE15_C~2 | .7700184 .3677037 -0.55 0.584 .3020163 1.963233intUAE15_S~2 | 4.583692 3.334675 2.09 0.036 1.101436 19.07531------------------------------------------------------------------------------Deviance: 1174.213.

UAE 15

UAE15*CCL2*SEX

Conclusions

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Mild to severe renal function decline (RFD) in the general populationis associated with a complex interaction between gene and environment

Inflammation plays a role in RFD as suggested by the observed interaction

Results are suggestive of an interaction between the CCL2 promoter SNP and albuminuria as an independent predictor of RFD and de novo IRF, that is different in males and females.

The exact nature of this relationship is unclear.

Discussion (1)

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Limitations:

Definition of renal function decline

There is no golden standard for estimation of renal function decline. Due to the nature of the analyses, a three-way interaction including a SNP, there’s a possible conflict between biological relevance and statistical power. A decline of more than 10% is aplausible estimate, since it will separate “normal” decline from putative pathological decline.

Exactness of CrCl-measurements

Regression to the mean and technical/compliance errors during collection of urine to estimate CrCl provide a statistical challenge. deltaCrCl shows a normal distrbution curve with CrCl-increase matching decrease in proportion. However, correlation of the two points is at a very low.

Discussion (2)

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Limitations:

Follow-up time and population

The observed univariate association of genotype with impaired renal function (<60 ml/min) in males is not found at the second visit. However, a large number of people that were at CV/renal risk at baseline did not return for the second visit. Thus, a high risk group was lost to follow-up.

A period of 4 years was the ultimate goal between first and second visit, yet there’s individual variety in actual time between first and secondVisit. Correcting our results for actual follow-up time did not alter the results.

Theoretical Discussion (2)Breedtestrategie

Exit of participants during period NC1-NC2 bij BP class, AHT and IRF (<60ml)

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