Serum Uric Acid and Urinary pH as Risk Factors of CKD...

1

Aug. 26, 2017 Semmelweis University

Sadayoshi Ito

Tohoku University, SENDAI

Serum Uric Acid and Urinary

pH as Risk Factors of CKD and CVD

The 24th Budapest Nephrology School

COI

NON

Urine of the first grade students of Tohoku University

Pink Urine syndrome

Student Check-up (n=4940) 216(4.4%) with PUS ・Cloudy orange urine ・Pink sediment after centrifugation (Uric acid precipitation)

Ogawa S, et al: Clin Exp Nephrol 2015:19:822-9

All subjects (−) (+) p

n 4940 4724 216

Gender (M / F) 3651 / 1289 3478 / 1246 173 / 43

% Male (%) 73.91 73.62 80.09 0.031

Age (years) 18.40 ± 1.14 18.40 ± 1.15 18.36 ± 0.74 0.496

BMI (kg/m2) 21.38 ± 3.00 21.33 ± 2.96 22.48 ± 3.56 <0.001

U-pH 5.97 ± 0.44 5.98 ± 0.44 5.75 ± 0.37 <0.001

SBP (mm Hg) 128.2 ± 15.7 128.0 ± 15.8 132.4 ± 16.6 0.0002

DBP (mm Hg) 73.15 ± 11.05 73.03 ± 11.05 76.00 ± 10.75 0.0001

HR (bpm) 88.97 ± 15.86 88.81 ± 15.78 92.64 ± 17.10 0.0014

Mean ± SD.

PUS is associated with high BMI, low UpH, higher BP and higher Heart rate

Ogawa S, et al: Clin Exp Nephrol 2015:19:822-9

In the logistic regression analysis, the presence or absence of UPS was used as the dependent variable, and gender, U-pH, BMI, age, SBP, DBP, and HR were used as the independent variables. U-pH (β = -1.392, p<0.0001), BMI (β= 0.085, p<0.0001), and HR (β = 0.011, p = 0.019) were extracted as independent factors.

Ogawa S, et al: Clin Exp Nephrol 2015:19:822-9

vs. U-pH p

n 300 -

Gender (M/F) 200 / 100 -

BMI (kg/m2) 23.4 ± 5.2 −0.35 0.011

SBP (mm Hg) 129.7 ± 19.3 −0.27 0.024

DBP (mm Hg) 75.7 ± 11.1 −0.20 0.030

WC (cm) 78.0 ± 13.2 −0.40 0.005

HR (bpm) 93.2 ± 17.7 0.01 0.863

CPR (μg/day) 21.5 ± 1.7 −0.26 0.022

AGT (ng/day) 1615.4 (110.9–74597.9) −0.47 <0.001

TBARS (µM/day) 4.3 ± 1.4 −0.70 <0.001

MG (nM/day) 1237.0 ± 498.6 −0.44 <0.001

U-Na (mM/day) 130.5 ± 31.0 −0.44 <0.001

U-pH 6.0 ± 0.6 - -

Mean ± SD, median (range). CPR: urinary C-peptide excretion

Characteristics of the 300 participants and the relationship between various parameters and urinary pH.

Ogawa S, et al: Clin Exp Nephrol 2015:19:822-9

Amadri 3-deoxyglucosone (DG)

Metabolism of MG

Glucose

Glyceraldehyde

3-Phosphate

pyruvate

TCA

F-1,6-bP Dihydroxyacetone

Phosphate (DHAP)

Methylglyoxal

(MG)

G-3P dehydrogenase;

S-D-lactoyl

glutathione

Gly-I

D-lactate

GSH

Gly-II

Hemithioacetyl

Glyoxylase system

AngII, RAGE

ROS

(a)

(b)

ESR spectrum

H2O2 (10mM)

MGO (3.9 mM)

Hydroxyl radical

（Nakayama M, et al. Redox Rep 12; 125-133, 2007)

(c)

MnO MnO

H2O2 + MGO (3.9 mM)

undetermined Carbon-centered radical

Methyl radical CH3・

Hydroxyl radical

C

3 H C

O O

H

C

Very reactive aldehyde

In the multiple-regression analysis, U-pH was used as the dependent variable, and gender, BMI, age, SBP, DBP, HR, WC, as well as excretion of urinary CPR, angiotensinogen, methylglyoxal, TBARS, and Na were used as the independent variables. Only TBARS (β = -0.421, p<0.0001) and angiotensinogen (β= 0.000, p<0.0001) were extracted as independent variables.

Low urinary pH may be associated with activation of RAAS and ROS

Ogawa S, et al: Clin Exp Nephrol 2015:19:822-9

Intracellular pH

Acid overload Sulfate, FFA

Urine citrate

Pathogenesis of Pink Urine Syndrome

Ogawa S, et al: Clin Exp Nephrol 2015:19:822-9

Fatty acid

β-oxidation

Ammonium

production

Fatty acids Glutamine

Fatty acyl CoA

Glutamate

Propionyl-Co-A

(for odd chain

fatty acids)

Acetyl-Co-A

Isocitrate

Citrate α-Ketoglutarate

Krebs

cycle Oxaloacetate

Malate

Fumarate

Succinyl-Co-A

Succinate

NH4+

NH4+

Metabolic syndrome

(Bobulescu IA: Curr Opin Nephrol

Hypertens 19; 393-402, 2010)

Urine pH and metabolic derangements

Low urinary pH: Obesity, type 2 DM, uric acid stone

The diurnal variation in urine acidification differs between normal individuals and uric acid stone formers.

(Cameron M, et al: Kidney Int 81:1123-30, 2012)

Acid – base balance

Acid load – Buffer = UNAE

UNAE=urinary net acid excretion

Food (protein） Reabsorption Metabolism

HCO Others

3 -

H+ NH3

NH4 +

MCD

(Kamel KS, et al: Q J Med 98; 57-68, 2005)

Urine pH is determined by H+ and NH3 secretion

H + A - ・

(TA)

TA+ UNAE= NH4 +

TA: Titratable Acid (amount of NaOH to bring urine pH to 7.4)

Thin

descending

limb

Glutamine HCO3 - Renal vein

Na+ NH4 +

NH4 +

Cortex

Medulla

Competes

with K+

NH4 +

Na+ ， Cl-

1

2 3

(Kamel KS, et al: Q J Med 98; 57-68, 2005)

NH4+ NH3 + H+

Ammonium production and recycling in the kidney

Healthy volunteers (HVs) Uric acid stone formers (UASFs)

Gender: M/F 6/3 9/1

Age (years) 52.8±13.1 57.0±8.2

Weight (kg) 85±19 109±19*

Height (cm) 171±10 172±6

Body mass index (kg/m2) 28.5±4.3 36.9±6.8*

SUA (mg/dl) 6.0±1.6 8.0±1.5*

Bicarbonate (mEq/l) 27.0±1.3 26.1±3.3

Venous pH 7.41±0.02 7.40±2.02

Abbreviations: F, female; M, male.

Demographic

*P<0.05, t-test (Cameron M, et al: Kidney Int 81; 1123-1130, 2012)

Controlled diet

(Cameron M, et al: Kidney Int 81; 1123-1130, 2012)

Urine pH: controlled diet

7.5

7.0

6.5

6.0

5.5

5.0

4.5

08 12 16 20 00 04 08

HV UASF

Uri

nar

y p

H

Time Meals are indicated by black boxes.

(Cameron M, et al: Kidney Int 81; 1123-1130, 2012)

60

50

40

30

20

10

0

Tota

l uri

c ac

id, m

g/h

08 12 16 20 00 04 08

Time

HV UASF

08 12 16 20 00 04 08

Time

HV UASF

25

20

15

10

5

0 Un

dis

soci

ated

uri

c ac

id, m

g/h

Urate excretion

a b

(a)Total (urate＋undissociated uric acid), (b)Undissociated uric acid excretion in urine. Black boxes represent meals.

(Cameron M, et al: Kidney Int 81; 1123-1130, 2012)

Urinary Net Acid Excretion

10

8

6

4

2

0

-2 08 12 16 20 00 04 08

Time

Uri

nar

y n

et a

cid

ex

cret

ion

, mEq

/h

HV UASF

Black boxes represent meals.

Acid load despite identical meal

• Increased endogenous acid production: lactate, keto-acids, et

• Increased alkali loss in the intestine • Increased absorption of exogenous

organic acid from intestinal bacterial sources

(Maalouf NM, CJASN 5; 1277-1281, 2010)

Proportion of NAE excreted as ammonium (NH4+/NAE) in nine volunteers with type 2 diabetes (T2DM;●) and 16 age- and BMI-matched

volunteers without type 2 diabetes (Controls; ♦) evaluated while consuming a fixed metabolic diet. Horizontal bars indicate means for each

group. NH4+/NAE was significantly lower in patients with type 2 diabetes than in control subjects (0.70 ± 0.12 versus 0.94 ± 0.36; P < 0.01).

Results represent the mean of two 24-hour urine collections for each participant

P<0.01 1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0

Controls

(N=16)

T2DM

(N=9)

U N

H4

+/N

AE

7.0

6.5

6.0

5.5

5.0

4.5

24-h

r U

pH

P<0.01

Controls

(N=16)

T2DM

(N=9)

Low Urine pH and Ammonia Excretion type 2 DM

Urine pH and renal-vascular damages in real world

• Significance of urine pH in DM has not been elucidated

• Ogawa followed up 400 DM patients from 2003 to 2013.

• Fifty patients were lost mostly due to Great East Japan Earthquake

• Specialist’s cares: best possible treatments

The study subjects’ demographic and clinical data at the baseline and endpoint.

Baseline (2003-4) Endpoint (2012-13) p

Age (years) 53.7 ± 13.6 - -

Gender (M/F) 157 / 193 - -

Duration (years) 11.8 ± 7.8 - -

BMI (kg/m2) 25.5 ± 5.0 25.5 ± 5.0 0.802

WC (cm) 91.9 ± 13.9 91.9 ± 13.6 0.983

UpH 6.0 ± 0.8 6.2 ± 2.6 0.201

PG (mg/dL) 129.0 ± 48.0 119.4 ± 19.2 0.734

HbA1c (%) 6.9 ± 1.1 6.5 ± 0.5 0.0042

SBP (mmHg) 134.5 ± 18.6 128.0 ± 10.9 < 0.0001

DBP (mmHg) 80.1 ± 10.3 76.4 ± 7.2 < 0.0001

HR (bpm) 73.0 ± 14.1 70.4 ± 8.5 < 0.0001

TC (mg/dL) 188.3 ± 34.5 192.8 ± 25.7 0.257

TG (mg/dl) 120.6 ± 70.2 114.8 ± 52.1 0.583

HDL-C (mg/dL) 55.7 ± 15.8 57.5 ± 15.3 0.019

BUN (mg/dL) 19.6 ± 9.9 25.5 ± 16.7 < 0.0001

eGFR (ml/min/1.73 m2) 72.7 ± 19.8 55.5 ± 16.2 < 0.0001

ACR (mg/g Cre) 24.8 (0.0-4735.6) 55.1 (3.8-4999.5) < 0.0001

UA (mg/dL) 6.2 ± 1.7 6.5 ± 1.4 0.046

ALT (U/L) 23.6 ± 22.6 26.9 ± 15.2 < 0.0001

AST (U/L) 25.2 ± 16.0 25.7 ± 9.6 < 0.0001

K+ (mEq/L) 4.4 ± 0.5 4.5 ± 0.5 0.676

Na+ (mEq/L) 140.8 ± 2.3 139.9 ± 2.6 0.0142

Cl- (mEq/L) 103.6 ± 3.2 105.4 ± 4.6 0.0002

TP (g/dL) 7.2 ± 0.5 7.1 ± 0.3 0.0018

Hb (g/dL) 12.8 ± 1.7 13.1 ± 1.3 0.0030

Plt (104/μL) 22.5 ± 6.6 18.0 ± 10.7 0.0003

WBC (103/μL) 6.2 ± 1.8 6.1 ± 1.5 0.241

PWV (cm/s) 1573.5 ± 718.7 1612.3 ± 423.2 0.283

ABI 1.1 ± 0.1 1.1 ± 0.2 0.211

IMT (mm) 0.7 ± 0.3 1.3 ± 1.1 < 0.0001

8-OHdG (ng/mg Cre) 9.7 ± 4.5 -

The study subjects’ demographic and clinical data at the baseline and endpoint.

Baseline (2003-4) Endpoint (2012-13) p

Age (years) 53.7 ± 13.6 - -

Gender (M/F) 157 / 193 - -

BMI (kg/m2) 25.5 ± 5.0 25.5 ± 5.0 0.802

UpH 6.0 ± 0.8 6.2 ± 2.6 0.201

HbA1c (%) 6.9 ± 1.1 6.5 ± 0.5 0.0042

SBP (mmHg) 134.5 ± 18.6 128.0 ± 10.9 < 0.0001

HR (bpm) 73.0 ± 14.1 70.4 ± 8.5 < 0.0001

eGFR (ml/min/1.73 m2) 72.7 ± 19.8 55.5 ± 16.2 < 0.0001

ACR (mg/g Cre) 24.8 (0.0-4735.6) 55.1 (3.8-4999.5) < 0.0001

UA (mg/dL) 6.2 ± 1.7 6.5 ± 1.4 0.046

PWV (cm/s) 1573 ± 718 1612. ± 423 0.283

ABI 1.1 ± 0.1 1.1 ± 0.2 0.211

IMT (mm) 0.7 ± 0.3 1.3 ± 1.1 < 0.0001

8-OHdG (ng/mg Cre) 9.7 ± 4.5 -

DM 10-year follow-up study (n=350)

Despite of good glycemic and BP control, obesity, renal and vascular damages were not halted. ΔGFR 17/10 years

Correlations between %IMT, %PWV, %eGFR, Δ log ACR and baseline parameter.

IMT PWV eGFR ACR

% IMT % PWV % eGFR ΔL ACR

r p r p r p r p

UpH -0.37 < 0.01 -0.12 0.01 0.23 < 0.01 -0.16 0.04

BMI 0.16 0.02 0.09 0.35 -0.17 0.05 0.13 0.05

UA 0.11 0.04 -0.06 0.57 -0.29 < 0.01 -0.10 0.08

HbA1c 0.01 0.95 -0.02 0.78 -0.12 0.08 -0.05 0.71

eGFR -0.17 0.01 -0.10 0.04 -0.01 0.80 -0.15 0.04

ACR 0.02 0.59 0.31 < 0.01 -0.26 < 0.01 -0.28 < 0.01

SBP 0.06 0.38 0.06 0.42 -0.07 0.12 0.16 0.04

HR 0.09 0.26 -0.01 0.83 -0.02 0.58 0.04 0.74

Acidic urine and uric acid were associated with renal-vascular damages

Ogawa S et al :BMJ Open Diabetes Res Care. 2015 Jun 30;3(1):

%IMT Coefficient Std. Err p 95% Conf. Interval

UpH -55.332 10.712 0.000 -76.403 -34.260

BMI 1.228 2.410 0.611 -3.513 5.970

UA -4.982 4.477 0.267 -13.788 3.824

eGFR -0.160 0.369 0.664 -0.887 0.566

A multiple regression analysis that used the %IMT as the dependent variable and factors that individually correlated with %IMT as independent variables.

UpH is the only parameter

Ogawa S et al :BMJ Open Diabetes Res Care. 2015 Jun 30;3(1):

New-onset CKD

• Logistic regression analysis for new-onset CKD (eGFR < 60 ml/min/1.73 m2) : acidic urine was an independent risk factor (p = 0.001), with an odds ratio of 0.522777 (Ref UpH < 6: 95% CI: 0.3526216 - 0.77504).

• In those who developed CKD, low UpH was also an independent risk factor in multiple regression analysis that used % change in eGFR as a dependent variable (β = 2.365226, p = 0.036).

Ogawa S et al :BMJ Open Diabetes Res Care. 2015 Jun 30;3(1):

UpH Coefficient Std. Err p 95% Conf. Interval

8-OHdG -0.083 0.009 < 0.001 -0.101 -0.064

BMI 0.005 0.013 0.707 -0.020 0.030

UA -0.053 0.229 0.021 -0.098 -0.008

eGFR 0.006 0.002 0.001 0.003 0.010

ACR 0.000 0.000 0.002 0.000 0.000

HR -0.005 0.002 0.034 -0.009 0.000

A multiple regression analysis using UpH as the dependent variable and factors that correlated with UpH at baseline as independent variables.

Low UpH is associated with high ROS and serum UA Ogawa S et al :BMJ Open Diabetes Res Care. 2015 Jun 30;3(1):

Intracellular pH

Acid overload Sulfate, FFA

Urine citrate

Pathogenesis of Pink Urine Syndrome

Ogawa S, et al: Clin Exp Nephrol 2015:19:822-9

H++HCO3-←H2O+CO2 (3)

CO2

Glucose gluconeogenesis glycolysis

Phosphoenolpyruvate

PEPCK (2)

starvation, insulin resistance diabetes mellitus

Serine, Cysteine, Threonine, Glycine, Alanine Tryptophan

Pyruvate

Pyruvate carboxylase CO2

Oxaloacetate

Pyruvate dehydrogenase

Acetly-CoA

Citrate

α-ketoglutarate

Fumarate

Succinyl-CoA

Tyrosine, Phenylalanine

Isoleucine, Methionine, Valine, Threonine

Glutamata Glutamine

H+

NH4+

NH3

Histidine, Proline, Hydroxyproline, Arginine

TCA cycle

(4)

(4)

(4)

(5) NH3

mitochondrion

PEPCK: Phosphoenolpyruvate carboxykinase

Glycogenic amino acids

TCA cycle: tricarboxylic acid cycle

(1)

(Ogawa S, et al: Tohoku J Exp Med 239; 103-110, 2016)

The relationship, under acidotic conditions, between glucose metabolism and amino acids use in the renal proximal tubules.

malate shuttle

Oxaloacetate

• Studied the relationship between

urine pH and urinary amino acids.

• Multiple regression analysis shows

that low urinary pH can be

explained by only low urinary

glutamate excretion (β = 0.0387, p<

0.0001)

• Glutamate is reabsorbed, but not

utilized for ammonia production.

• Where did it go?

Nrf2 (Nuclear factor-erythroid related factor-2 )

ROS

Kobayashi A. et al. (2004) Mol. Cell Biol. 24:7130-7139.

NQO1

keap 1

Glutathione

Keap 1 and Nrf2 as defense mechanism for Stress

Low Urine pH Oxidative stress marker

近位尿細管にNrf2は発現・活性化しているのか？ Nrf2 Nrf2 Nrf2

NQO1 NQO1 NQO1

Diabetic N IgA Miminal Change

S Ogawa et al JSN 2014

Oxidative Stress and renal amino acid metabolism

Glucose

Phosphoenolpyruvate

Pyruvate

Oxaloacetate

Fumarate

Succinyl-CoA

α-ketoglutarate

Glutamate

Glutamine

H+ NH3

H+ NH3

CO2 Lactate

Pentose phosphate pathway (PPP) Nucleotide

Uric acid

Glu

tam

ino

lysi

s

Glutathione

A

B

C

Cysteine

Nrf2 OS Glycogenic amino acids

Relations between synthesis of ammonia and Nrf2 and glucogenic amino acids.

(Ogawa S, et al: Tohoku J Exp Med 239; 103-110, 2016)

Clin Exp Nephrol. 2017 Mar 22. The relationship between the renal reabsorption of cysteine and the lowered urinary pH in diabetics Ogawa S et al ・100 non-diabetic obese individuals and 100 diabetics ・blood amino acids, urinary amino-acid excretion, and their relationship with the UpH

Urine pH: unrecognized risk factor or marker of metabolic insults

Excessive acid loads

Subclinical acidosis

Inflammation, RO, RAAS

Organ damages

(Goraya N, et al: Kidney Int 86; 1031-1038, 2014)

Error bars (mean±s.e.) of plasma creatinine-calculated estimated GFR (crGFR) (left) and plasma cystatin C–calculated estimated GFR (cysGFR) (right) across four time points including baseline and 3 years’ follow-up.

*P<0.05 vs. Usual Care at 2 year;+P<0.05 vs. Usual Care at 3-year follow-up.

Luminal Alkalinization Attenuates Proteinuria-Induced Oxidative

Damage in Proximal Tubular Cells

Tomokazu Souma,*† Michiaki Abe,*‡ Takashi Moriguchi,† Jun

Takai,† Noriko Yanagisawa-Miyazawa,* Eisuke Shibata,* Yasutoshi

Akiyama,* Takafumi Toyohara,*

Takehiro Suzuki,* Masayuki Tanemoto,* Takaaki Abe,* Hiroshi

Sato,* Masayuki Yamamoto,† and Sadayoshi Ito*

JASN 22:635-48, 2011

Low urinary pH may not only be a marker (or insult) of renal damages but also be a contributor to renal damages

HK-2 Cell

Proximal tubular cells）

Chamber pH：7.0, 6.6, 6.4, 6.0

±OA-Alb15g/L

Am J Physiol Renal Physiol. (2002) 283:F20-8.

O2・- DHE

Ethidium

Influence of pH on OA-Alb-induced ROS generation

HK-2 cells; florescence microscopy

second

Ｅｔｈｉｄｉｕｍ

（Ｕ）

pH6.0

pH6.４

(Souma T, Abe M, et al: JASN 22:635-48, 2011)

Pyk2 connects pH and protein overload

Urine pH: unrecognized risk factor or marker of metabolic insults

Excessive acid loads

Subclinical acidosis

Inflammation, RO, RAAS

Cardio-Renal damages

Low urine pH

Reduced NH 3

Mets