Regarding the detection of uACR ≥ 30 mg/gCr by a single dipstick measurement, the sensitivity + specificity—1 value was the highest in non-diabetic and diabetic patients with more than trace proteinuria, as shown in Supplemental Table 2. Findings in previous reports have varied, reporting values of “sensitivity 37.1%, specificity 97.3%, positive predictive value (PPV) 71.4%, negative predictive value (NPV) 89.5%” [11]; “sensitivity 69.4%, specificity 86.8%, PPV 27.1%, NPV 97.6%” [12]; and “sensitivity 43.6% specificity 93.6%, PPV 34.6%, NPV 95.5%” [13] for more than trace proteinuria. This difference is believed to be due to the fact that there are many cases of advanced CKD, as the subjects in this study were outpatients with lifestyle-related diseases, and because dipstick evaluations are affected by the urine concentration and test reagents [20]. Although it is necessary to confirm the results when the PPV is low, even if the sensitivity of uACR ≥ 30 mg/gCr is high [12, 13], this study indicated that the PPV was high and suggested the utility of dipstick test, even if a dipstick proteinuria evaluation showed low sensitivity. However, as the NPV was relatively low, a uACR of ≥ 30 mg/gCr cannot be ruled out in cases of negative proteinuria.

When the utility of a single dipstick evaluation was compared with that of the uPCR for detecting of uACR ≥ 30 mg/gCr, the uPCR was shown to be more useful, with a high sensitivity and NPV as well as an almost equivalent specificity and PPV.

It is recommended that microalbuminuria be diagnosed, even when a single uACR measurement is ≥ 30 mg/gCr, if another examination shows uACR ≥ 30 mg/gCr among 2 subsequent early-morning urine tests [3]. Regarding the significance of the three-time dipstick measurement, if the CO of 1 measurement is trace proteinuria, 3 measurements are more useful than 1 measurement, with the CO of 3 measurements indicating sensitivity 55.5% and specificity 79.7% when trace or more proteinuria is shown even once out of 3 times. If the CO of microalbuminuria is set at trace or more proteinuria two out of three times or 1( +) or more proteinuria one out of three times, the sensitivity for detecting microalbuminuria is reduced in non-diabetic patients, and therefore when the dipstick test shows trace levels or higher proteinuria once, it is desirable to confirm the result by measuring either uPCR or uACR at that time.

The sum of three uPCRs in non-diabetic patients was useful for differentiating microalbuminuria and indicated a CO (D, YI) of 0.23 g/gCr, which is approximately 3 times the CO of a single uPCR measurement. However, for qualitative proteinuria measurements, the CO (D, YI) was set at a trace finding being obtained at least one out of three times. The reason for this may be because the median uACRs of trace proteinuria in non-diabetic and diabetic patients were 56 and 73 mg/gCr, respectively, while the first quartiles were at 32.5 and 42 mg/gCr, respectively, which were higher than 30 mg/gCr.

The uPCR was useful for differentiating between uACR < 30 mg/dl and ≥ 30 mg/dl, when we limited our studies to dipstick-negative proteinuria. Furthermore, a single uPCR measurement was useful for predicting microalbuminuria, even when evaluating proteinuria-negative cases via the dipstick test three times in a row, with the sum of three uPCRs also being useful. While proteinuria is generally quantified after detection of proteinuria by a dipstick examination [8], measuring the uPCR, even wnen the result is negative, seems useful for the early detection of microalbuminuria in lifestyle-related diseases.

The CO (YI) of uPCR that differentiates uACR ≥ 30 mg/dl in non-diabetic lifestyle-related diseases remained unchanged from the CO of negative proteinuria, with the values for G1-3a and G3b-4 being 0.07 g/gCr and 0.10 g/gCr, respectively, even when more than trace proteinuria was included. The sensitivity at the same uPCR and AUC increased while the specificity showed a small decrease when more than trace proteinuria was included. This is believed to be due to the fact that the COs is in A1M, while the uACR is mostly ≥ 30 mg/gCr in A1H and A2, along with the fact that the high fraction ratio of uPCR increases with the increase in qualitative proteinuria findings.

Regarding the difference in CO (YI) between G1-3a and 3b-4, the specificity of G1-3a increased more rapidly with the increase in the CO of the uPCR than did that of G3b-4, whereas G3b-4 had a slower decrease in sensitivity than G1-3a. This difference is attributed to the significant difference in the number of cases with uACR < 30 mg/gCr and ≥ 30 mg/gCr by uPCR category between G1-3a and G3b-4, meaning that G1-3a had higher proteinuria selectivity than G3b-4 or tubular protein in the urine may be increased in G3b-4.

The predictive probability of uACR ≥ 30 mg/gCr based on a logistic model using uPCR and the uSG or uCr in both non-diabetic and diabetic patients with negative proteinuria was significantly better than the uPCR alone for differentiating of uACR ≥ 30 mg/gCr. This appears to be due to the fact that the uSG, uCr and uACR exhibit a negative correlation at the same uPCR value of ≤ 0.10 g/gCr, suggesting that the uACR may be increased in diluted urine and decreased in concentrated urine. While the reason for this is unclear, the reabsorption of filtered albumin in the renal proximal tubules [21] may be increased in concentrated urine compared with nonalbumin protein in the urine.

The KDIGO CKD guideline [3] recommended that even if a uACR ≥ 30 mg/gCr is noted in a single spot urine measurement, the results should be confirmed by measuring the uACR using early-morning urine in order to exclude the possibility of postural proteinuria. Another reason for using the early morning urine based on the findings of this study is that the uACR of the specimen may have been reduced, as early-morning urine is expected to be more concentrated than spot urine. When predicting the uACR from the uPCR, it may also be desirable to use early-morning urine, although the uPCR itself is a major predictive factor of microalbuminuria.

The uACR estimates the daily albuminuria excretion under the assumption that the daily urinary creatinine excretion is 1 g, with a good correlation reportedly having been shown in patients with a normal renal function [22]. It appears that the significance thereof in G3b and G4 is unclear. The urinary Cr excretion per unit time is expressed as (serum Cr value × GFR + amount of creatinine secreted by renal tubules). As renal dysfunction progresses, the tubular secretion of creatinine per unit nephron increases, while the number of functional nephrons is expected to decrease. While serum creatinine increases as eGFR decreases, this study found that the median age at stages G1, 2, 3a, 3b and 4 indicated 46.4, 60.6, 71.0, 77.3 and 79.5 years old for non-diabetic patients, respectively, and 55.0, 65.7, 75.0, 76.1 and 76.0 years old for diabetic patients, respectively, with significantly more elderly patients showed a decreased renal function than younger patients (Kruskal–Wallis test P < 0.001, respectively). In addition to aging, a study on CKD patients with minimal dietary interventions reported that patients with lower creatinine clearance had a spontaneous decrease in dietary protein intake, reduced 24-h urine Cr excretion [23], and prevalence of sarcopenia is increased as CKD progressed [24]. Judging from the above, it is predicted that muscle mass will decrease in patients with a decreased renal function, with serum Cr value × GFR also potentially decreasing. With respect to improving the limitations of the daily albuminuria excretion prediction by uACR, it has been reported that the estimated albumin excretion rate, calculated by multiplying the spot uACR value by the estimated urinary creatinine excretion rate (g/24 h), improved the prediction of the measured 24-h albumin excretion [25]. Nonalbuminuric renal dysfunction have been reported in type 2 diabetic patients and the general population [26, 27], so it is necessary to investigate the estimated albumin excretion rate and the estimated protein excretion rate for predicting it in cases of a decreased renal function.

The BMI and abdominal circumference were higher in diabetic patients than in non-diabetic patients, and while significant positive correlations between the BMI and uPCR and the BMI and uACR were noted in non-diabetic patients, no such correlations were noted in diabetic patients. The lack of correlations in diabetic patients may be due to a small number of this study. It is reported that high waist-to-hip ratio and BMI are independently positively associated with albuminuria due to intraglomerular haemodynamics resulting from excess adiposity [28]. uACR and uPCR were higher in hypertensive cases than in non-hypertensive cases among non-diabetic patients in this study. There is the possibility that hypertension and obesity correlate with the severity of global nephrosclerosis in non-diabetic nephrosclerosis [29], and it is speculated that systemic and intraglomerular hypertension cause glomerular sclerosis, resulting in high uACR and uPCR values in non-diabetic hypertensive or obese patients. In addition, glomerular lesions, which are typically found in diabetic nephropathy (diabetic glomerulopathy) [29], may be involved in the increase in uACR and uPCR in diabetic patients.

In this study, uPCR was useful for determining microalbuminuria in both non-diabetic and diabetic patients, even when testing negative using a dipstick test, and the CO of uPCR for microalbuminuria in cases with three consecutive negative proteinuria findings for both groups was 0.06 g/gCr. In Japan, the measurement of albuminuria is covered by insurance only when incipient diabetic nephropathy is supposed. Obesity [30] and metabolic syndrome [31] are risk factors for diabetes. uACR and uPCR were high in obese and hypertensive cases of non-diabetic patients in this study. Therefore, in the group at high-risk for diabetes, even if the patient is found to be a proteinuria-negative case based on the findings of a dipstick test, it is desirable to measure uPCR, and by maintaining uPCR at a level of ≤ 0.05 g/gCr, it is possible for the albuminuria to remain in the normal range, even if diabetes has already developed. Should microalbuminuria be suspected, then encouraging the patient to make changes in their lifestyle may reduce albuminuria, decrease the risk of a progression to diabetes, and also prevent the development of nephrosclerotic lesions in cases that have developed diabetes.

Albuminuria is a risk factor for renal dysfuction and cardiovascular disease in diabetic patients [4], and this study targeted patients with normo- and microalbuminuria. Exacerbation of albuminuria indicates an increasing risk of renal dysfunction and cardiovascular disease, while amelioration from macroalbuminuria to microalbuminuria or microalbuminuria to normoalbuminuria indicates a reduced risk following the onset of diabetes [4, 6, 32]. This study indicated that changes in uACR and uPCR exhibited a significant positive correlation even if urinary protein was negative three times via the dipstick test, with changes in uACR appearing to be predictable from changes in uPCR. When the dipstick proteinuria was negative three times, the increase/decrease in uACR per uPCR 0.01 g/gCr was approximately 3 mg/gCr, which tended to be lower than approximately 5–6 mg/gCr which included trace or more urinary protein qualitative findings. This is probably because uPCR values in cases of positive dipstick protein findings are higher than the uPCR values in cases of negative proteinuria, and the ratio of albumin in total proteinuria increases as uPCR increases. In Japan, uACR measurement is covered by insurance only once in three months for diabetic patients, so uPCR may be able to evaluate the therapeutic effect and detect deterioration at an early stage during that period.

It has been reported that the use of RAAi (such as ACEi, ARB, and MRB) [10, 33, 34] and SGLT2i [10, 35] reduces albuminuria and the accompanying renal dysfunction and cardiovascular disease in patients with chronic kidney disease with or without diabetic patients. Although the use of ACEi and ARB has been reported to be associated with high uACR/uPCR levels [36], the effects of SGLT2i are unclear. This study indicated that the CO of uPCR, which distinguishes uACR 30 mg/gCr or more in cases using SGLT2i, tended to be higher than that in non-use cases (Supplemental Table 14), so the effect of taking SGLT2i on uACR/uPCR and the CO of microalbuminuria shall be investigated going forward.

The inconsistency between the CO (D) and CO (YI) was recognized in differentiating uACR ≥ 30 mg/gCr in proteinuria-negative cases among non-diabetic patients with G3b-4 and microalbuminuria in triple proteinuria-negative cases of G1-3a. The CO (YI) is reportedly preferable because it maximizes the overall rate of correct classification when the criteria do not agree [19]. However, D2 = min [-2(sensitivity + specificity -1) + (sensitivity2 + specificity2)], and (sensitivity2 + specificity2) is smallest at the CO where the sensitivity and specificity are equal and when (sensitivity + specificity) is constant. In cases of lifestyle-related diseases, the risk of cardiovascular disease increases starting at uACR < 30 mg/gCr [5], so there is little disadvantage in intervening in patients with normoalbuminuria. As high sensitivity for detecting microalbuminuria is considered　necessary for early prevention of cardiovascular disease, the CO (D) seems useful in that it takes into consideration both the accuracy and the balance between sensitivity and specificity.

One limitation associated with this study was its single-center setting, with different results potentially being obtained depending on the target patient, dipstick test, proteinuria, albumin quantification measurement conditions, and measurement methods [20]. In addition, the number of patients was relatively small, but there have been no reports evaluating the factors for microalbuminuria based on urine dipstick, uPCR, and uACR measurements in the same urine sample three times. The median time between the three uACR measurements was about four to five months, which was relatively long, so the results may have been affected by the treatment of lifestyle-related diseases during this period, although the KDIGO guideline recommends a further two measurements of the uACR within two months after the first measurement [3].

The main results of this study were summarized in Table 5.