OG / DG
Mean / SD / Mean / SD / p
age / years / 22.3 / ± / 3.4 / 62.4 / ± / 15.1 / <0.01
gender / M/F / 92 / 8 / 41 / 59 / <0.01
BMI / kg/m2 / 32.6 / ± / 2.1 / 26.1 / ± / 5.7 / <0.01
SBP / mmHg / 133.0 / ± / 11.5 / 128.8 / ± / 17.1 / 0.04
DBP / mmHg / 74.5 / ± / 11.3 / 70.2 / ± / 12.0 / 0.01
UpH / 5.9 / ± / 0.5 / 5.9 / ± / 0.8 / 0.74
eGFR / mL/min/1.73m2 / 97.8 / ± / 15.3 / 66.3 / ± / 31.2 / <0.01
AST / IU/L / 29.4 / ± / 17.3 / 23.7 / ± / 10.9 / 0.01
ALT / IU/L / 55.5 / ± / 44.9 / 22.5 / ± / 15.1 / <0.01
PG / mmol/L / 4.9 / ± / 0.8 / 7.8 / ± / 2.5 / <0.01
HbA1c (IFCC) / mmol/mol / - / - / 53.5 / ± / 11.8 / -
TC / mmol/L / 4.9 / ± / 0.9 / 4.6 / ± / 0.8 / 0.04
TG / mmol/L / 1.6 / ± / 0.9 / 1.4 / ± / 0.9 / 0.05
HDL-C / mmol/L / - / - / 1.5 / ± / 0.5 / -
BUN / mmol/L / 4.5 / ± / 1.0 / 7.2 / ± / 5.5 / <0.01
UA / μmol/L / 432.1 / ± / 94.9 / 323.7 / ± / 93.4 / <0.01
UUN / mmol/g Cr / 191.1 / ± / 53.4 / 291.2 / ± / 102.9 / <0.01
UUA / μmol/g Cr / 950.8 / ± / 693.0 / 2590.9 / ± / 1312.9 / <0.01
ACR / mg/g Cr / 24.4 (3.3 – 6710) / -
8-OHdG / ng/g Cr / 3.7 / ± / 1.9 / 8.9 / ± / 3.5 / <0.01
OG: non-diabetic obese group, DG: diabetes mellitus group, SD: standard deviation, BMI: body mass index, SBP: systolic blood pressure, DBP: diastolic blood pressure, UpH: urinary pH, eGFR: estimated glomerular filtration rate, AST: aspartate transaminase, ALT: alanine transaminase, PG: plasma glucose, HbA1c: hemoglobin A1c, IFCC: International Federation of Clinical Chemistry, TC: serum total cholesterol, TG: triglyceride, HDL-C: serum high density lipoprotein cholesterol, BUN: blood urea nitrogen, UA: serum uric acid, UpH: urinary pH, UUN: urinary urea nitrogen excretion, UUA: urinary uric acid excretion, ACR: albumin-to-creatinine ratio (urinary albumin excretion), 8-OHdG: urinary 8-hydroxy-2'-deoxyguanosine excretion.
Compared to the DG, the OG subjects were younger, and had a higher ratio of males, higher BMI, SBP, DBP, eGFR, AST, ALT, TC, TG, and UA, and lower HbA1c, BUN, UUN, UUA, and 8-OHdG. The results may be explained by the drastically different backgrounds between the non-diabetic obese subjects who were recruited during a university freshmen health checkup, and diabetic subjects, who were undergoing outpatient treatment. It appears likely that the OG comprised a large number of subjects with high lipid levels and fatty liver, while the DG comprised a large number of subjects with high blood glucose levels and decreased renal function. The fact that the DG participants were taking numerous prescription drug treatments could have also influenced the results. Oxidative stress was greater in the diabetic subjects than in the non-diabetic obese subjects.
Supplementary Table2.Major treatment drugs administered to the diabetic group.RASIs / no / 38
mono / 46
combination / 16
CCB / no / 52
mono / 33
combination / 15
Diuretics / no / 73
mono / 22
combination / 5
Other antihypertensive drugs / no / 74
mono / 23
combination / 3
Insulin therapy / no / 35
non- intensive / 14
intensive / 51
GLP-1 analog / no / 92
mono / 8
sulfonylureas / no / 86
mono / 14
glinides / no / 78
mono / 22
α-glucosidase inhibitors / no / 62
mono / 38
biguanides / no / 55
mono / 45
thiazolidine / no / 73
mono / 27
DPP4 inhibitors / no / 66
mono / 34
SGLT2 inhibitors / no / 98
mono / 2
Statins / no / 61
mono / 39
Xithantine oxidase inhibitors / no / 84
mono / 16
Other anti-uric acid drugs / no / 99
mono / 1
RASI: renin-angiotensin system inhibitors, CCB: calcium channel blockers, GLP-1: glucagon-like polypeptide 1, DPP4: dipeptidyl peptidase 4, SGLT2: sodium-glucose linked transporter 2.
Mono: monotherapy, combination: administered more than the two medicines.
Supplementary Table3. Correlation between UpH and clinical examination items in both the diabetic group and the obese group.Group / Diabetic group / Obese group
UpH / UpH
r / p / r / p
age / 0.06 / 0.53 / -0.13 / 0.21
gender / 0.06 / 0.58 / 0.11 / 0.26
BMI / -0.20 / 0.05 / -0.08 / 0.42
SBP / 0.03 / 0.77 / 0.01 / 0.89
DBP / 0.05 / 0.60 / -0.06 / 0.58
AST / 0.13 / 0.18 / 0.17 / 0.09
ALT / 0.08 / 0.43 / 0.09 / 0.39
BUN / -0.19 / 0.07 / 0.07 / 0.47
Cre / -0.02 / 0.82 / 0.01 / 0.92
UA / -0.22 / 0.03 / -0.07 / 0.50
Na/Cl / 0.10 / 0.31
TG / -0.09 / 0.40 / -0.12 / 0.24
TC / -0.17 / 0.08 / 0.02 / 0.88
HDL-C / 0.19 / 0.07
PG / -0.07 / 0.47 / -0.03 / 0.79
HbA1c / 0.10 / 0.35
ACR / -0.13 / 0.19
eGFR / 0.08 / 0.45 / 0.06 / 0.59
8-OHdG / 0.00 / 0.97
UUN / -0.06 / 0.57 / 0.19 / 0.06
UUA / 0.07 / 0.48 / 0.40 / <0.01
WBC / -0.24 / 0.02
Hb / 0.11 / 0.28
Plt / -0.14 / 0.16
UpH: urinary pH, BMI: body mass index, SBP: systolic blood pressure, DBP: diastolic blood pressure, AST: aspartate transaminase, ALT: alanine transaminase, BUN: blood urea nitrogen, Cre: serum creatinine, UA: serum uric acid, Na/Cl: serum sodium/chloride ratio, TG: triglyceride, TC: serum total cholesterol, HDL-C: serum high density lipoprotein cholesterol, PG: plasma glucose, HbA1C: hemoglobin A1c, ACR: albumin-to-creatinine ratio (urinary albumin excretion), eGFR: estimated glomerular filtration rate, 8-OHdG: urinary 8-hydroxy-2'-deoxyguanosine excretion, UUN: urinary urea nitrogen excretion, UUA: urinary uric acid excretion, WBC: white blood cell, Hb: hemoglobin, Plt: platelets.
Supplementary Table 4. Classification of AA based on the increase/decrease of BAA and UAA in the DG when compared with the OG.UAA
DG > OG / DG < OG
BAA
DG > OG / Group A / Group C
taurine / glycine / cystathionine / 1-methyl histidine
serine / citrulline / BAIBA / histidine
asparagine / cysteine / 3-methyl histidine
glutamine / methionine / arginine
DG < OG / Group B / Group D
aspartic acid / AABA / ornithine / tyrosine
threonine / valine / lysine / MEA
glutamate / isoleucine / tryptophan
proline / leucine
alanine / beta-alanine
AA: amino acid, BAA: blood amino acid concentration, UAA: urinary amino acid excretion, DG: diabetes mellitus group, OG: obese group, BAIBA: beta-aminoisobutyrate, AABA: alpha-aminobutyrate, MEA: methanolamine.
Group A: BAA and UAA were DG > OG and DG > OG, respectively; AA whose urinary excretion rate had also increased since blood concentration had increased because of diabetes.
Group B: BAA and UAA were DG < OG and DG > OG, respectively; AA whose blood concentration had decreased since the rate of urinary excretion had increased because of diabetes.
Group C: BAA and UAA were DG > OG and DG < OG, respectively; AA whose blood concentration has increased since the rate of urinary excretion had decreased because of diabetes.
Group D: BAA and UAA were DG < OG and DG < OG; AA whose rate of urinary excretion had decreased since blood concentration had decreased because of diabetes.
Supplementary Table 5A. Correlation between UpH and UAA/BAA in the DG, and correlation between UAA and BAA.
DG
urinary excretion / r / p / blood level / r / p / Correlation
(BAA vs UAA) / r / p
glutamine / 0.29 / <0.01 / taurine / -0.21 / 0.04 / BAIBA / 0.65 / <0.01
cysteine / 0.26 / <0.01 / AABA / -0.20 / 0.04 / 1-methyl histidine / 0.63 / <0.01
alanine / 0.25 / 0.01 / threonine / 0.41 / <0.01
lysine / 0.25 / 0.02 / alanine / 0.38 / <0.01
threonine / 0.23 / 0.02 / serine / 0.33 / 0.01
asparagine / 0.22 / 0.03 / glycine / 0.33 / 0.01
glycine / 0.22 / 0.03 / hydroxy proline / 0.30 / <0.01
arginine / 0.23 / 0.03 / proline / 0.26 / 0.01
histidine / 0.20 / 0.05 / ornithine / 0.25 / 0.02
AABA / 0.22 / 0.03
tyrosine / 0.22 / 0.03
asparagine / 0.21 / 0.04
cysteine / -0.21 / 0.04
Supplementary Table 5B.Correlation between UpH and UAA/BAA in the OG and correlation between UAA and BAA.
OG
urinary excretion / r / p / blood level / r / p / Correlation
BAA vs UAA / r / P
threonine / 0.45 / <0.01 / taurine / -0.26 / 0.01 / BAIBA / 0.86 / <0.01
asparagine / 0.45 / <0.01 / 1-methyl histidine / 0.62 / <0.01
lysine / 0.45 / <0.01 / threonine / 0.41 / <0.01
glutamine / 0.41 / <0.01 / 3-methyl histidine / 0.36 / <0.01
histidine / 0.39 / <0.01 / methionine / 0.31 / <0.01
cysteine / 0.37 / <0.01 / serine / 0.28 / <0.01
serine / 0.35 / <0.01 / glutamine / 0.25 / 0.01
leucine / 0.33 / <0.01 / AABA / 0.25 / 0.01
glycine / 0.32 / <0.01 / glycine / 0.23 / 0.01
AAAA / 0.31 / <0.01 / alanine / 0.24 / 0.02
beta-alanine / 0.30 / <0.01 / asparagine / 0.21 / 0.04
taurine / 0.35 / <0.01
3-methyl histidine / 0.30 / <0.01
hydroxylysine / 0.30 / 0.01
phenylalanine / 0.28 / 0.01
alanine / 0.27 / 0.01
cystathionine / 0.27 / 0.01
arginine / 0.26 / 0.01
isoleucine / 0.24 / 0.02
valine / 0.23 / 0.02
ornithine / 0.23 / 0.02
methanolamine / 0.22 / 0.03
tyrosine / 0.22 / 0.03
citrulline / 0.26 / 0.04
anserine / 0.36 / 0.04
UpH: urinary pH, UAA: urinary amino acid excretion, BAA: blood amino acid concentration, DG: diabetes mellitus group, OG: obese group, AABA: alpha-aminobutyrate, BAIBA: beta-aminoisobutyrate, AAAA: alpha-aminoadipate
In diabetes, the greater the decline in UpH, the greater the decrease in urinary cysteine excretion, and the greater the rise in blood cysteine concentration.
In the OG, a positive correlation was seen between UpH and the urinary excretion of threonine, asparagine, lysine, glutamine, histidine, cysteine, serine, leucine, glycine, alpha-aminoadipate, beta-alanine, taurine, 3-methyl histidine, hydroxylysine, phenylalanine, alanine, cystathionine, arginine, isoleucine, valine, ornithine, methanolamine, tyrosine, citrulline, and anserine, whereas a negative correlation was seen between UpH and blood taurine concentration.
A multiple regression analysis was conducted that used UpH as the dependent variable, and used the amount of urinary excretion of threonine, asparagine, lysine, glutamine, histidine, cysteine, serine, leucine, glycine, and alpha-aminoadipate (10factors, starting with those whose p is the smallest) as the independent variables.Only alpha-aminoadipate was extracted as anindependent variable (β = 0.0093034, p = 0.04, 95%CI = 0.0003991–0.0182077).
In the OG, moreover, a positive correlation was seen between BAA and UAA in terms of BAIBA, 1-methyl histidine, threonine, 3-methyl histidine, methionine, serine, glutamine, AABA, glycine, alanine, and asparagine.
Supplementary Table 6A. Reabsorption rate (%) of amino acid whose significant correlation with urinary pH (UpH) was seen in the diabetic group (DG) and the non-diabetic obese group (OG).DG / OG
Reabsorption rate / UpH / Reabsorption rate / UpH
r / p / r / p
glutamine / -0.23 / 0.02 / lysine / -0.48 / <0.01
1-methyl histidine / -0.21 / 0.04 / threonine / -0.48 / <0.01
3-methyl histidine / -0.20 / <0.05 / leucine / -0.45 / <0.01
lysine / -0.20 / <0.05 / asparagine / -0.45 / <0.01
cystathionine / -0.81 / <0.05 / taurine / -0.44 / <0.01
glutamine / -0.43 / <0.01
serine / -0.43 / <0.01
histidine / -0.41 / <0.01
valine / -0.40 / <0.01
isoleucine / -0.40 / <0.01
bBeta- alanine / -0.39 / <0.01
glycine / -0.36 / <0.01
phenylalanine / -0.36 / <0.01
alanine / -0.35 / <0.01
1- methyl histidine / -0.34 / <0.01
ornithine / -0.31 / <0.01
3-methyl histidine / -0.28 / 0.01
tryptophan / -0.28 / 0.01
tyrosine / -0.26 / 0.01
cysteine / -0.74 / 0.01
methionine / -0.24 / 0.02
In the OG, UpH correlated negatively with the reabsorption rates (%) of lysine, threonine, leucine, asparagine, taurine, glutamine, serine, histidine, valine, isoleucine, beta-alanine, glycine, phenylalanine, alanine, 1-methyl histidine, ornithine, 3-methyl histidine, tryptophan, tyrosine, and methionine (cysteine was eliminated because n = 10).
Supplementary Table 6B. Relationship between the reabsorption rates(%) of each amino acid, and eGFR and ACR in the diabetic group.Reabsorption rate / eGFR / Reabsorption rate / ACR
r / p / r / p
beta-alanine / 0.71 / <0.01 / beta alanine / -0.50 / <0.01
proline / 0.57 / <0.01 / proline / -0.36 / <0.01
alpha-amino butyrate / 0.51 / <0.01 / phenylalanine / -0.34 / <0.01
taurine / 0.46 / <0.01 / methanolamine / -0.29 / <0.01
glutamate / 0.41 / <0.01 / alpha-amino butyrate / -0.28 / 0.01
alanine / 0.40 / <0.01 / asparagine / -0.27 / 0.01
hydroxy proline / 0.38 / <0.01 / hydroxy proline / -0.27 / 0.01
serine / 0.36 / <0.01 / valine / -0.26 / 0.01
phenylalanine / 0.36 / <0.01 / alanine / -0.25 / 0.01
cysteine / -0.32 / <0.01 / glutamate / -0.25 / 0.01
tryptophan / 0.32 / <0.01 / taurine / -0.22 / 0.03
methanolamine / 0.31 / <0.01 / threonine / -0.22 / 0.03
1 methyl histidine / -0.31 / <0.01 / aspartic acid / -0.21 / 0.03
asparagine / 0.28 / 0.01 / tyrosine / -0.20 / 0.04
isoleucine / -0.27 / 0.01
valine / 0.27 / 0.01
cystathionine / 0.89 / 0.02
arginine / 0.23 / 0.02
glycine / 0.20 / <0.05
eGFR: estimated glomerular filtration rate, ACR: urinary albumin-to-creatinine ratio.
In amultiple regression analysis that used the top 10most correlated factors as the independent variables, the reabsorption rates of the following amino acidswere shown to be factors independently-related to eGFR: beta-alanine (β = 2.561682, p < 0.01, 95%CI = 1.173709-3.949656), proline (β = –16.1163, p 0.01, 95%CI = –23.5935 to –8.6392), alpha-aminobutyrate (β = 8.039175, p < 0.01, 95%CI = 4.564425-11.51392), and cysteine (β = –0.82147, p < 0.01, 95%CI = –1.16225 to –0.48069).
Moreover, in the DG, ACR correlated negatively with the reabsorption rates of beta-alanine, proline, phenylalanine, methanolamine, alpha-aminobutyrate, asparagine, hydroxyproline, valine, alanine, glutamate, taurine, threonine, aspartate, and tyrosine.In the multiple regression analysis that used the top 10most correlated factors as the independent variables, anindependent factor for the ACR could not be identified.
Supplementary Figure 1
The relationshipbetween urinary pH and cysteine use in the renal proximal tubules under diabetic conditions.
Under diabetic conditions, oxidative stress (OS) increases in the renal proximal tubules.Nuclear factor-E2 p45-related factor (Nrf) 2 reactivates to counteract the increased OS. Nrf2 promotes the synthesis of glutathione from glutamate and cysteine. A greater increase in OS is associated with a greater amount of glutamate consumed to synthesize glutathione.
Ammonia synthesis involves the production of α-ketoglutarate from glutamate. As a result of Nrf2 reactivation, glutamate is used for glutathione synthesis, which decreases ammonia production and, in turn, lowers the urinary pH.
The glutamate reabsorption rate is already at 100%, thus it is not possible to increase it any further. However, as the cysteine reabsorption rate is usually about 50% it can be increased. Under diabetic conditions, glutathione production is solely dependent on the amount of cysteine reabsorption (i.e. not glutamate), so the relationship between urinary pH and urinary cysteine excretion becomes extremely strong.
Supplementary Figure 2
Cysteine originally has a lower reabsorption rate [70.53% in the non-diabetic obese group {OG} (A)] than that of glutamate [99.93% (B)], so it is possible to increase the reabsorption rate per residual nephron [increased to about 88% in the diabetic group {DG} (A)].This increase in the reabsorption rate occurs if the number of nephrons decreases because of increased OS (A, B).
Compared with cysteine, it is difficult to increase the reabsorption rate of glutamate any further because the original reabsorption rate of glutamate is about 100%.Instead, the reabsorption rate of glutamate decreases in diabetic patients, because of the decrease in nephrons [99.93 ± 0.04, 99.42 ± 0.78, p < 0.01 (B)].
Even if the number of nephrons decreasesdue to increased OS in diabetic patients, the cysteine reabsorption rate per single nephron in the renal proximal tubules (RPTs) increases [70.53 ± 11.60, 88.09 ± 16.97, p < 0.01, (A)].Therefore, the increase in the amount of urinary cysteine excretion [49.56 ± 24.62, 109.11 ±131.41, p < 0.01, increased approximately 2.20-fold (C)] becomes smaller than what can be anticipated from a rise in the blood concentration [1.34 ± 0.48: 16.92 ± 15.59, p < 0.01, increased approximately 12.63-fold (D)] (Table 1). In other words, the amount of urinary cysteine excretion increased only roughly 2.20-fold, contrary to the 12.63-fold increase that was anticipated from the rise in blood concentration.
Supplementary documents-1
Measurements
Urinary pH (UpH)
Since UpH is liable to fluctuate, we used the urine taken in the early morning on an empty stomach after having fasted for 12 hours, instead of spot urine.
UpH was measured, using an AX-4030 (ARKRAY, Inc., Kyoto, Japan), a fully automated urine analyzer with a preset test-paper. The first morning urine was collected after a fasting period of more than 12 hours.
The plasma amino acids concentration and urinary amino acids excretion were measured using the following method.
After protein had been removed from the urine samples using 6% sulfosalicylic acid, the specimen was subjected to derivatization, using a MassTrak AAA reagent kit (a substance in which 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and AQC had been dissolved in acetonitrile, and mixed with a sample with added borate buffer), we measured the amino acid concentration using an UltraPerformance Liquid Chromatography (MassTrak™Amino Acid Analysis, column 2.1 x 150 mm, ACQUITY UPLC® system, and Empower™ 2 software) (UV 260 nm).
Supplementary document-2
UpH and urinary 8-OHdG excretion showed a negative correlation (r = 0.41, p < 0.01). This, however, was deemed to have been not only because oxidative stress had specified a reduction in UpH, but also because a reduction in UpH had increased the oxidative stress. UpH and cysteine’s reabsorption rate showed a tendency to correlate (r = -0.19, p = 0.06). The reason the correlation was not significant was believed to have been because cysteine’s reabsorption volume was specified by both a rise in serum concentration and an increase in reabsorption rates, and was not being specified by the reabsorption rate alone. UpH did not correlate with glutamate(r = -0.08, p = 0.45) but correlated negatively with glutamine (r = -0.23, p = 0.02). This was believed to have been because the shortage of glutamate was being compensated for by the supply of glutamine. It is estimated that UpH does not drop if the shortage of glutamine can be replenished by this increased supply of glutamine (elevation of serum concentration and increase in RR).
Urinary 8-OHdG excretion showed no correlation with cysteine-RR, glutamate-RR or glutamine-RR. (They were r = 0.05, p = 0.65, r = -0.02, p = 0.85, and r = 0.16, p = 0.12, respectively). This was believed to have been due to the fact that excretion of urinary 8-OHdG does not necessarily represent oxidative stress in RPT only, that glutathione is not the only system that eliminates oxidative stress, and that cysteine and glutamate elevate not only reabsorption rates but also serum concentration to increase the reabsorption amount.
UpH correlated negatively with serum uric acid (UA) concentration (an anti-oxidant) (r = -0.22, p = 0.03). Cysteine RR, moreover, showed a positive correlation with UA (r = 0.40, p < 0.01), but UA did not correlate with the RR of glutamate or glutamine (r = 0.03, p = 0.79, and r = 0.05, p = 0.61, respectively). This may have been because both a rise in UA and an increase in cysteine RR were related to a decline in eGFR. Another reason may be that UA is an acid, and is one of the supply sources of H+.