International Urology and Nephrology (2024) 56:1651–1661

https://doi.org/10.1007/s11255-023-03843-3

NEPHROLOGY - REVIEW

Evaluation of N‑acetyl‑β‑D‑glucosaminidase as a prognostic marker

for diabetic nephropathy in type 2 diabetics: systematic review
and meta‑analysis
Augusto dos Santos Bitencourt1 · Régis Leães Vargas Filho1 · Gabriele da Silveira Prestes1 ·
Maria Laura Rodrigues Uggioni1 · Fernanda Marçal1 · Tamy Colonetti1 · Maria Inês da Rosa1,2

Received: 25 May 2023 / Accepted: 8 October 2023 / Published online: 29 October 2023
© The Author(s), under exclusive licence to Springer Nature B.V. 2023

Abstract
Objective This review aimed to assess the utility of urinary N-acetyl-β-D-glucosaminidase (uNAG) as a prognostic biomarker
for nephropathy in patients with type 2 diabetes mellitus.
Methods The search for relevant studies was conducted across multiple databases, including PubMed (Medline), EMBASE,
LILACS, CENTRAL, IBECS, and gray literature. We employed a random effects model to calculate the standardized mean
difference and 95% confidence interval. Furthermore, we assessed heterogeneity using Cochrane's Q test and Higgins' I2
statistics.
Results This review included a total of 16 articles involving 1669 patients, with 13 being case–control studies and three
being cohorts. The meta-analysis conducted across all studies revealed significant heterogeneity. However, subgroup analysis
of four studies indicated that an increase in uNAG among normoalbuminuric patients was associated with the development
of macroalbuminuria (DMP = – 1.47; 95% CI = – 1.98 to 0.95; p < 0.00001; I2 = 45%). Conversely, it did not demonstrate
effectiveness in predicting the development of microalbuminuria (DMP = 0.26; 95% CI = – 0.08 to 0.60; p = 0.13; I2 = 17%).
Conclusions Elevated uNAG levels in normoalbuminuric patients may indicate an increased risk for the development of
macroalbuminuria, but not microalbuminuria. However, the high heterogeneity observed among the studies highlights the
necessity for further research to validate these findings.

Keywords N-acetyl-β-D-glucosaminidase · Diabetic nephropathy · Chronic kidney disease · Meta-analysis · Systematic

review

Introduction developing various comorbidities, including systemic arte-

Diabetes mellitus (DM) continues to be a significant global
public health concern, with an estimated 537 million indi-
viduals affected worldwide [1]. Type 2 diabetes mellitus
(T2DM) accounts for 90–95% of these cases. T2DM is a
chronic metabolic disorder characterized by insulin resist-
ance and persistent hyperglycemia. It carries a high risk of
* Maria Inês da Rosa
mir@unesc.net
1 assessing the urinary albumin–creatinine ratio (ACR) and
Laboratory of Translational Biomedicine, University
of Extremo Sul Catarinense, Criciúma, Santa Catarina,
Brazil
2
Rua Cruz e Souza, 510, Bairro Pio Correa, Criciúma,
SC 88811‑550, Brazil
rial hypertension (SAH) and diabetic kidney disease (DKD)
[2–4].
DKD is a microvascular complication of T2DM charac-
terized by a gradual deterioration in kidney function. This
subtle progression affects around 40% of individuals with
T2DM and stands as the leading cause of renal replacement
therapy (RRT). Early detection of DKD enables prompt
intervention and helps to slow down the decline in renal
function. Therefore, it is advisable for all T2DM patients to
undergo annual screening for DKD [5–10].
Screening and monitoring of DKD typically involve
estimating the glomerular filtration rate (GFR) based on
serum creatinine levels. However, the predictability of these
methods is affected by several factors, particularly in the
early stages of the disease. For example, the kidneys can

13
Vol.:(0123456789)
1652
compensate for nephron loss by increasing the filtration rate,
resulting in a normal GFR despite significant renal damage.
Additionally, DKD encompasses various pathogenic mecha-
nisms, with some causing tubular injury before glomerular
damage and the onset of microalbuminuria [11, 12].
Therefore, it is crucial to identify reliable and specific
biomarkers for DKD to effectively manage diabetic
patients. Biomarkers are measurable substances, structures,
or processes that can predict the incidence or outcome of
a disease. N-acetyl-beta-D-glucosaminidase (NAG) is
one such molecule that exhibits promising potential as a
biomarker for nephropathy [13–17].
NAG is a lysosomal enzyme with a large molecular
weight of 130 kDa. It is primarily found in the proximal
tubules of the kidneys and plays a supportive role in the
hydrolysis of glycoproteins. Under normal conditions, NAG
is present in the urine at low concentrations, which indicates
its physiological excretion by the tubular epithelium.
Due to its high molecular weight, NAG is unable to be
reabsorbed. Therefore, an increase in urinary N-acetyl-
β-D-glucosaminidase (uNAG) levels signifies the loss of
integrity in the proximal tubules. Observational studies
have demonstrated a positive correlation between uNAG
and various forms of kidney damage, including acute kidney
injury (AKI) events and chronic conditions such as DKD
[15, 18–21].
Despite this association, there is still no consensus on the
prognostic value of uNAG in DKD. Therefore, the objective
of this study was to evaluate the prognostic utility of uNAG
as a planned biomarker for nephropathy in patients with type
2 diabetes mellitus.
Methodology
This review was conducted following the guidelines of the
Preferred Reporting Items for Systematic Reviews and Meta-
Analyses (PRISMA) [22]. As it is a systematic review, there
was no requirement to seek evaluation from the Research
Ethics Committee. The study protocol was registered on the
PROSPERO platform (International Prospective Register
of Systematic Reviews) with the registration number
CRD42022338999.
Search strategy
The search was systematically conducted across the major
databases, including Medical Literature Analysis and
Retrieval System Online (MEDLINE) via PubMed, Excerpta
Medical Database (EMBASE), Scopus, Biomed Central,
Web of Science, Index Bibliographic Español en Ciencias de
la Salud (IBECS), Latin American and Caribbean Literature
in Health Sciences (LILACS), and gray literature sources
13
International Urology and Nephrology (2024) 56:1651–1661
(Google Scholar and British Library). The search utilized
the following keywords: "diabetic nephropathy," "diabetic
kidney disease," "type 2 diabetes mellitus," "N-acetyl-β-D-
glucosaminidase," along with their respective synonyms,
combined using the Boolean operators "OR" and "AND".
The search was limited to studies involving human subjects,
with no language restrictions.
Study selection
Evidence-based medicine suggests that clinical dilemmas
that arise in healthcare practice should be deconstructed
and organized using the "PECOS" strategy, which stands
for Patient, Exposure, Comparison, Outcomes, and Study
type [23, 24]. These five components, when delineating the
subject and study approach, form the basis for formulating
the research question:
Population: type 2 diabetics with microalbuminuria or
macroalbuminuria.
Exposure: change in levels of uNAG.
Comparison: type 2 diabetics with normoalbuminuria.
Outcomes: diabetic nephropathy; tubular damage.
Study type: observational (cross-sectional, case–control,
cohort).
Therefore, studies that met the predetermined criteria
in our PECOS search strategy, were included. Studies
that did not meet the defined inclusion criteria or had any
characteristics that could influence the intended evaluation,
such as the use of interventions, were excluded.
Studies were independently identified by two investigators
(ASB and RLVF). The final determination of inclusion or
exclusion of the studies was made based on a set of selection
criteria. In cases of disagreement regarding the inclusion or
exclusion of studies, a third reviewer (MIR) conducted an
assessment to resolve the discrepancies.
Data extraction
Data extraction was conducted by completing a data
collection form, which included information such as author,
year, country, study objectives, number of patients, patient
characteristics, data collection and analysis methods, and
results for each included study. The data extraction process
was carried out independently by two reviewers (ASB and
RLVF), and in cases where necessary, a third reviewer
(MIR) was consulted for resolution.
Methodology quality assessment
The methodological quality assessment of the observational
studies included in the review was conducted using
International Urology and Nephrology (2024) 56:1651–1661
the Newcastle–Ottawa Scale. This tool evaluates eight
items from each study, which are categorized into three
domains: selection of groups, comparability of groups, and
ascertainment of exposure (for case–control studies) or
outcomes (for cohort studies). Each item is assigned a star,
with a maximum of two stars for the comparability domain.
Studies with seven to nine stars were considered to have high
methodological quality and a low risk of bias, while studies
with four to six stars were deemed to have a high risk of
bias, and those with zero to three stars indicated a very high
risk of bias [25].
Data synthesis and statistical analysis
The results were presented through graphs and tables,
including Forest plots to assess the use of uNAG for the
specified outcomes. Data analysis was performed using
RevMan 5.4 software, utilizing the standardized mean
difference (SMD) with a 95% confidence interval (CI).
A descriptive synthesis of the studies was conducted and
presented in tables or text format. Study heterogeneity was
assessed using I2 statistics, with the following interpretation
of values: 0–40%—heterogeneity may not be significant;
30–60%—may indicate moderate heterogeneity; 50–90%—
may indicate substantial heterogeneity; and 75–100%—may
indicate considerable heterogeneity. When heterogeneity
exceeded 40%, the random-effects model was used to
calculate the estimates. In the absence of heterogeneity, the
Mantel–Haenszel fixed-effects model was employed [26,
27].
Results
Study selection
The initial database search yielded 147 records. After remov-
ing duplicates, 125 records remained, which were further
assessed based on their titles and abstracts. Among these,
95 records were excluded as they did not meet the inclusion
criteria, and an additional three studies were excluded due
to unavailability of the full text. The remaining 30 studies
underwent full-text reading, and out of these, 14 studies
were excluded for the following reasons: eight studies used
different renal markers, two did not differentiate between
types of DM, two included a non-diabetic population, and
one study did not have a control group. Ultimately, 16 stud-
ies were included in the systematic review [28–43] (Fig. 1).
Study description
The 16 included studies were published between 1995
and 2021 and involved a total of 1669 patients. Among
1653
these, 13 studies had a cross-sectional design [28–31,
34–42], while three were cohort studies. Among the cross-
sectional studies, nine utilized the first-morning urine
collection method to assess uNAG, while the remaining
studies employed the 24-h urine collection method. The
three cohort studies [32, 33, 43] had an average follow-up
duration of 6 years and 1 month, with two of them analyzing
uNAG levels through the first-morning urine and one
using the 24-h urine collection method. In all studies,
patients were categorized into three groups based on their
albumin-to-creatinine ratio (ACR): normoalbuminuric,
microalbuminuric, and macroalbuminuric. However, the
cutoff points used to define these groups varied among
the studies. Thirteen studies defined concentrations
below 30 mg/g Cr as normoalbuminuria, between 30 and
300 mg/g Cr as microalbuminuria, and above 300 mg/g Cr
as macroalbuminuria.
The three remaining studies utilized different criteria to
define the groups. Banu et al. [34] categorized participants
based on albuminuria levels as follows: less than 20 mg/g
Cr, between 20 and 200 mg/g Cr, and greater than 200 mg/g
Cr. Yashima et al. [30] divided participants into groups with
uNAG levels less than 15 μg/min, between 15 and 200 μg/
min, and greater than 200 μg/min. Lastly, Assal et al. [32]
used different cutoff points to classify albuminuria in
women (< 30 μg/mg Cr, 30–400 μg/mg Cr, and > 400 μg/
mg Cr) and men (< 30 μg/g Cr, between 30 and 300 μg/g
Cr, and > 300 μg/g Cr). Table 1 provides an overview of the
key aspects of the included studies.
Participants description
The 16 included studies encompassed a total of 1699
patients, with 705 classified as normoalbuminuric, 642
as microalbuminuric, and 338 as macroalbuminuric. The
average age of the participants was 62 years, and 54%
of them were female. The average duration of type 2
diabetes mellitus (DM2) was 11 years, and a majority of
the participants exhibited poor glycemic control (mean
HbA1c: 8.1 mmol/L). The average blood pressure (BP) of
the patients was 140/80 mmHg, with consistently higher
readings observed in macroalbuminuric patients. Four
studies (253 patients) did not report blood pressure values;
did not report blood pressure values, while two other studies
excluded patients with hypertension (334 patients). Most
of the patients included in the studies were overweight
or obese, with an average BMI of 31. Table 2 provides a
summary of the key characteristics of the included studies.
13
1654
Fig. 1  Flowchart of study selection
Main outcomes
uNAG analysis in diabetic patients with microalbuminuria
All 16 studies included in the review examined uNAG
concentrations in patients with microalbuminuria.
Among the 12 studies analyzed individually, significant
differences in uNAG concentrations were found between
microalbuminuric and normoalbuminuric patients. This
difference was more pronounced in the more recent
studies. Significant clinical and methodological variations
were observed across the studies, including differences
in urine collection methods and the analytical-laboratory
techniques used to measure uNAG levels. Given the high
13
International Urology and Nephrology (2024) 56:1651–1661
heterogeneity when combining the studies for the overall
evaluation, a sensitivity analysis was conducted with four
studies that could be combined, and these were included in
the meta-analysis.
The meta-analysis revealed a standardized mean differ-
ence (SMD) of – 0.26, with a 95% confidence interval (CI)
of – 0.60 to 0.08 (p = 0.13; I2 = 17%), involving a total of
77 diabetic patients with normoalbuminuria and 146 with
microalbuminuria (Fig. 2). Based on the analysis of these
results, it was observed that there was no statistically sig-
nificant difference in uNAG levels between the evaluated
groups.
International Urology and Nephrology (2024) 56:1651–1661 1655

Table 1  Characteristics of the included studies

Author Year Country Study type Mean age BMI HbA1c Time of DM2 Participants
Normo Micro Macro Total

Al-Hazmi et al. [41] 2020 Saudi Arabia Cross-sectional 53 years 31 8.6 8 years 26 30 30 86
(47–64)
Asare-Anane et al. [40] 2016 Gana Cross-sectional 55.3 years 28.6 – 8.2 years 39 26 – 65
(43–67)
Assal et al. [32] 2013 Egypt Cohort 51.9 years 23.5 8.6 8.1 years 20 25 25 70
(45–59)
Banu et al. [34] 1995 Japan Cross-sectional 62 years 22.3 8.0 16 years 85 54 33 172
(35–86)
Bouvet et al. [31] 2014 Argentina Cross-sectional 66 years 27 7.4 13.5 years 19 17 – 36
(52–80)
Fu et al. [38] 2011 China Cross-sectional 53.9 years – 8.2 2.7 years 61 24 16 101
(50–67)
Fufaa et al. [43] 2015 USA Cohort 42.5 years 35.3 9.8 11 years 138 72 50 260
(32–53)
Fujita et al. [29] 2006 Japan Cross-sectional 61 years 24 9.4 16 years 19 18 16 43
(52–68)
Ito et al. [36] 2001 Japan Cross-sectional 62 years 23 7.0 – 15 14 12 41
(53–68)
Miyauchi et al. [33] 1995 Japan Cohort 59 years 22 7.6 7.9 years 16 15 7 38
(49–70)
Morii et al. [28] 2003 Japan Cross-sectional 60 years 24 8.3 – 29 25 18 72
(51–68)
Petrica et al. [42] 2021 Romania Cross-sectional 60 years 32.5 7.3 8.5 years 48 45 43 136
(53.5–67)
Piwowar et al. [37] 1999 Poland Cross-sectional 67 years – 7.6 8 years 5 21 11 41
(42–78)
Piwowar et al. [35] 2006 Poland Cross-sectional 62 years – 8.94 11.5 years 14 89 27 130
(32–81)
Yashima et al. [30] 1999 Japan Cross-sectional 58 years – 7.6 8.1 years 91 56 50 197
(49–67)
Zhang et al. [39] 2011 China Cross-sectional 55.3 years 27 9.1 14 years 100 111 – 211
(45–65)

uNAG analysis in diabetic patients with macroalbuminuria
The evaluation of uNAG levels in macroalbuminuric
diabetics was conducted in ten cross-sectional studies and
three cohort studies. Among the ten cross-sectional studies,
nine demonstrated statistically significant differences in
uNAG concentrations between macroalbuminuric patients
and normoalbuminuric patients. Additionally, seven studies
showed significant differences in uNAG concentrations
between macroalbuminuric and microalbuminuric patients.
The cohort studies yielded similar results, with significant
differences observed in uNAG concentrations between
macroalbuminuric patients and both normoalbuminuric
and microalbuminuric patients. Similar to the analysis of
microalbuminuric patients, the combined evaluation of
macroalbuminuric patients exhibited high heterogeneity.
Therefore, a sensitivity analysis was performed,
incorporating four studies into the meta-analysis.
The meta-analysis revealed a standardized mean dif-
ference (SMD) of – 1.47, with a 95% confidence interval
(CI) ranging from – 1.98 to – 0.95 (p < 0.00001; I2 = 45%),
involving a total of 77 diabetic patients with normoalbu-
minuria and 73 diabetic patients with macroalbuminuria
(Fig. 3). The analysis of the results indicates a statistically
significant difference in uNAG levels between macroalbumi-
nuric and normoalbuminuric patients, suggesting that uNAG
dosage could serve as a potential prognostic marker for DKD
in these individuals.
Risk of bias
Eight cross-sectional studies achieved the highest score on
the Newcastle–Ottawa Scale, indicating high methodological
quality. Additionally, four other cross-sectional studies,
although not reaching the maximum score, demonstrated
high methodological quality and a low risk of bias (Table 3).

13
1656 International Urology and Nephrology (2024) 56:1651–1661

Table 2  Measured outcomes

Author Year Diagnostic Sample Cut-off point uNAG
method
Normo Micro Macro

Al-Hazmi et al. 2020 ELISA First morning < 30 mg/g Cr 26.7 ± 7.3 41.1 ± 11.7 48.8 ± 11.3
[41] urine 30–300 mg/g Cr (ng/mL) (ng/mL) (ng/mL)
> 300 mg/g Cr
Asare-Anane 2016 – First morning < 30 mg/g Cr 11.83 ± 6.76 26.7 ± 17.2 –
et al. [40] urine 30–300 mg/g Cr (U/g Cr) (U/g Cr)
Assal et al. [32] 2013 Spectrophotometry First morning < 30 μg/mg Cr 11.2 ± 4.5 18.5 ± 3.7 22.7 ± 5.2
urine 30–300 μg/mg Cr (ng/mL) (ng/mL) (ng/mL)
30–400 μg/mg Cr
> 300 μg/mg Cr
> 400 μg/mg Cr
Banu et al. [34] 1995 Spectrophotometry First morning < 20 mg/g Cr 5.8 ± 0.4 9.4 ± 1.0 14.4 ± 1.4
urine 20–200 mg/g Cr (U/g Cr) (U/g Cr) (U/g Cr)
> 200 mg/g Cr
Bouvet et al. [31] 2014 Spectrophotometry First morning < 30 mg/g Cr 4.4 (1.5- 9.2) 17.0 (5.9–23.3) –
urine 30–300 mg/g Cr (U/g Cr) (U/g Cr)
> 300 mg/g Cr
Fu et al. [38] 2011 Spectrophotometry First morning < 30 mg/g Cr 10.5 (8.2–16.2) 15.5.5 (12.6– 17.8 (14.6–31.8)
urine 30–300 mg/g Cr (U/g Cr) 20.5) (U/g Cr)
> 300 mg/g Cr (U/g Cr)
Fufaa et al. [43] 2015 Spectrophotometry First morning < 30 mg/g Cr 0.768 (0.452– 6.92 (2.26–15.2) 9.81 (3.22–25.0)
urine 30–300 mg/g Cr 1.22) (mU/mmol Cr) (mU/mmol Cr)
> 300 mg/g Cr (mU/mmol Cr)
Fujita et al. [29] 2006 Spectrophotometry First morning < 30 mg/g Cr 4.5 (2.8–7.8) 5.2 (2.3–10.5) 10.5 (4.2–18.3)
urine 30–300 mg/g Cr (U/g Cr) (U/g Cr) (U/g Cr)
> 300 mg/g Cr
Ito et al. [36] 2001 Spectrophotometry First morning < 30 mg/g Cr 6.64(3.03–14.39) 6.86(4.50–12.97) 15.66 (9.19–36.02)
urine 30–300 mg/g Cr (U/g Cr) (U/g Cr) (U/g Cr)
> 300 mg/g Cr
Miyauchi et al. 1995 Spectrophotometry 24 h urine < 30 mg/g Cr 3.1 (1.9–19.1) 6.6 (4.2–11.6) 12.1 (6.4–24.6)
[33] 30–300 mg/g Cr (U/dia) (U/dia) (U/dia)
> 300 mg/g Cr
Morii et al. [28] 2003 Spectrophotometry First morning < 30 mg/g Cr 4.28 (2.84–16.82) 5.18 (2.32–14.20) 10.51 (4.21–18.28)
urine 30–300 mg/g Cr (U/g Cr) (U/g Cr) (U/g Cr)
> 300 mg/g Cr
Petrica et al. [42] 2021 Molecular First morning < 30 mg/g Cr 3.17 (1.88–5.28) 10.5 (9.92–11.96) 16.74 (16.05–18.0)
Fluorescence urine 30–300 mg/g Cr (U/g Cr) (U/g Cr) (U/g Cr)
> 300 mg/g Cr
Piwowar et al. 1999 Molecular 24 h urine < 30 mg/g Cr 1.40 (0.58–5.76) 4.51 (0.53–20.44) 10.96 (2.6–48.8)
[37] Fluorescence 30–300 mg/g Cr (U/g Cr) (U/g Cr) (U/g Cr)
> 300 mg/g Cr
Piwowar et al. 2006 Molecular First morning < 30 mg/g Cr 1.06 (0.60— 2.29 (1.38–22.40) 9.26 (2.2–30.6)
[35] Fluorescence urine 30–300 mg/g Cr 11.30) (U/g Cr) (U/g Cr)
> 300 mg/g Cr (U/g Cr)
Yashima et al. 1999 Spectrophotometry 24 h urine < 15 μg/min Cr 4.4 ± 3.5 8.1 ± 5.0 10.0 ± 4.6
[30] 15–200 μg/min (U/dia) (U/dia) (U/dia)
Cr
> 200 μg/min Cr
Zhang et al. [39] 2011 Spectrophotometry 24 h urine < 30 mg/g Cr 11.7 ± 6.62 13.9 ± 16.2 –
30–300 mg/g Cr (U/L) (U/L)
> 300 mg/g Cr

Among them is the study by Morii et al. [28], which did uncertainty and potential biases into the study. Similarly, the
not specify the origin and selection criteria for patients in study by Fujita et al. [29], did not provide details on how
the control group. The lack of information regarding the patients in the control group were recruited, and it employed
source and selection process for the control group introduces different assessment methods for cases and controls. The

13
International Urology and Nephrology (2024) 56:1651–1661 1657

Fig. 2  Forest plot of the meta-analysis evaluating uNAG in diabetic patients with microalbuminuria

Fig. 3  Forest plot of the meta-analysis evaluating in diabetic patients with macroalbuminuria

Table 3  Assessment of the Author, year Selection Comparability Exposure/ Total

quality of the included studies Outcome
using the Newcastle–Ottawa
scale 1 2 3 4 1 2 3

Cohort studies
Miyauchi et al., 1995 [33] * * – * * * * * 7
Fufaa et al., 2015 [43] * * * * ** * * * 9
Assal et al., 2013 [32] * – * * ** * * * 8
Cross-sectional studies
Piwowar et al., 2006 [37] * * – * ** * * * 8
Petrica et al., 2021 [42] * * – * ** * * * 8
Morii et al., 2003 [28] * * * * ** * * * 9
Ito etal., 2001 [36] * * – * ** * * * 8
Fujita et al., 2006 [29] * * * * ** * * * 9
Bouvet et al., 2014 [31] * * – * * * * * 7
Fu et al., 2011 [38] * – – * * * * * 6
Banu et al., 1995 [34] * * * * * * * * 8
Al-Hazmi et al., 2020 [41] * * * * ** * * * 9
Zhang et al., 2011 * * * * ** * * * 9
Yashima et al., 1999 [30] * * * * * * * * 8
Asare-Anane et al., 2016 [40] * * * * ** * * * 9
Piwowar et al., 1999 [35] * – * * * * * * 7

The quality assessment of the included studies was performed using the Newcastle–Ottawa scale (NOS).
Scales were used to assess cohort studies (3 studies) and cross-sectional studies (thirteen studies). Cohort
Study: Selection: (1) Representativeness of the exposed cohort; (2) Selection of the unexposed cohort; (3)
Exposure check; (4) Demonstration that the outcome of interest was not present at the beginning of the
study; Comparability: (1) Comparability of cohorts based on design or analysis; Result: (1) Evaluation of
the result; (2) The follow-up was long enough for the results to occur; (3) Adequacy of cohort follow-up.
Cross-sectional study: Selection: (1) Sample representativeness; (2) Sample size; (3) Non-respondents;
(4) Verification of exposure (risk factor); Comparability: (1) Subjects in different outcome groups are
comparable, based on study design; Result: (1) Evaluation of the result; (2) Statistical test

13
1658
absence of clear information on the recruitment of patients
in the control group and the use of inconsistent assessment
methods raise concerns about the comparability between the
study groups and the validity of the results. In the study by
Fu et al. [38], there was a limitation in terms of case repre-
sentation. By including only patients with diabetes for less
than 5 years, the study may not capture the full spectrum
of individuals with diabetes and nephropathy, resulting in
a limited representation of cases and potential biases in the
findings. The studies by Yashima et al. [30] and Bouvet et al.
[31] demonstrated moderate methodological quality and a
moderate risk of bias. These concerns arise from potential
selection biases associated with the inclusion of hospitalized
patients, the lack of description regarding patient recruit-
ment in the control group, and the absence of presentation
of additional risk factors beyond type 2 diabetes. These
limitations and potential biases should be considered when
interpreting the results and assessing the overall quality and
reliability of these studies.
The three cohor t studies demonstrated high
methodological quality and low risk of bias. The study by
Assal et al. [32] obtained the highest score, while the study
by Miyauchi et al. [33] did not specify the origin of the
patients recruited for the control group.
Discussion
This systematic review aimed to evaluate the use of urinary
N-acetyl-β-D-glucosaminidase (uNAG) as a biomarker of
nephropathy in patients with type 2 diabetes mellitus. The
uNAG values were compared with albuminuria stages. The
results obtained indicate that microalbuminuric patients did
not show significant changes in uNAG levels compared to
normoalbuminuric patients. However, macroalbuminuric
patients exhibited significant differences in uNAG levels
when compared to normoalbuminuric patients. Cianci and
colleagues (2023) conducted a case series study of patients
treated with percutaneous transluminal renal angioplasty
for renovascular hypertension and ischemic nephropathy,
measuring bilateral renal resistance indices, circulating renal
stem cells, and neutrophil Gelatinase Associated Lipocalin
(NGAL) before and after the procedure, with the absence
of NGAL reduction after treatment being associated with
worsening renal function [44]. These findings suggest that
uNAG can be considered a prognostic biomarker for patients
with type 2 diabetes mellitus who are at risk of developing
diabetic kidney disease (DKD).
Given that glomerular involvement alone may not fully
indicate kidney damage and can be inaccurate in certain
cases, there is a need for additional methods to complement
the prognostic evaluation of patients with diabetic kidney
disease (DKD). Tubulointerstitial damage has been identified
13
International Urology and Nephrology (2024) 56:1651–1661
as a significant risk factor for end-stage kidney disease
(ESKD) [32, 45–47], but its assessment has traditionally
required invasive renal biopsies. The emergence of urinary
tubular markers has provided a less invasive and cost-
effective alternative for evaluating tubulointerstitial damage.
In light of this, the present review aimed to investigate the
prognostic potential of uNAG, a tubular biomarker well-
known in the context of DKD.
Thus, after analyzing the sensitivity of four primary
studies, it was found that the measurement of uNAG in
normoalbuminuric patients was not capable of indicating
progression to microalbuminuria. A similar result was
observed in the review by Driza et al. [48], which evaluated
the diagnostic and prognostic power of uNAG in type 1
and type 2 diabetics. In their study, the uNAG levels of
normoalbuminuric and microalbuminuric patients showed
no statistically significant difference (AUC = 0.69; 95%
CI = 0.65–0.73; p = 0.66).
Our findings contrast with those observed in the
case–control study by Kern et al. [49]. This study involved
322 individuals with type 1 diabetes, aged 13–39 years,
who were free of micro or macrovascular complications of
diabetes mellitus (DM). The aim of the study was to verify
the ability of uNAG to predict the development of micro
and/or macroalbuminuria. After adjusting for confounders,
baseline uNAG excretion remained independently associated
with future microalbuminuria (adjusted OR = 1.86; 95%
CI = 1.41–2.46; p = 0.001). Furthermore, the risk of
microalbuminuria increased by 80% for each 50% increase
in baseline uNAG excretion.
The study by Sheira et al. [50] demonstrated a
statistically significant increase in the uNAG/Cr index in
diabetics with microalbuminuria compared to those with
normoalbuminuria. Additionally, all subgroups of diabetic
patients, regardless of ACR or GFR, exhibited elevated
levels of uNAG compared to the control group. Furthermore,
a proportional increase in uNAG excretion was observed
with the deterioration of DRD, indicating a potential role
for uNAG in quantifying the extent of damage.
These findings align with those observed in the individual
analysis of the studies included in this review. It was
discovered that the majority of type 2 diabetics exhibit a
higher degree of tubular damage at baseline compared
to the non-diseased population. Furthermore, it was
observed that in most studies, uNAG values above a certain
threshold (RV < 4.0 U/g Cr) preceded the development of
microalbuminuria. However, cohort studies with larger and
more representative samples are needed to elucidate the
temporal behavior of these markers in the early stages of
DRD. Recognizing early alterations in albuminuria would be
crucial for optimizing preventive measures in these patients.
Furthermore, it was demonstrated that the extent of uNAG
elevation at baseline was more pronounced in individuals
International Urology and Nephrology (2024) 56:1651–1661
with poor glycemic control. This finding is in line with the
results from the cohort study conducted by Yamanouchi
et al. [19]. Over a three-year period, 1062 pre-diabetic
individuals and 112 newly diagnosed type 2 diabetics
underwent assessments of RAC and uNAG levels. It was
observed that uNAG levels were correlated with fasting
blood glucose (r = 0.512, p < 0.0001) and HbA1c (r = 0.351,
p = 0.001).
The present review, after conducting a meta-analysis of
four primary studies, has found that elevated levels of uNAG
in normoalbuminuric individuals with type 2 diabetes are a
risk factor for the development of macroalbuminuria. This
finding is consistent with the results from the aforementioned
study by Kern et al. [49], in which baseline uNAG values
were consistently higher in macroalbuminuric patients
compared to those with normal or microalbuminuria (15.6
vs 10.7 U/g Cr; p < 0.005). Furthermore, when adjusting
for various variables, both initial NAG excretion (adjusted
OR, 2.26; 95% CI, 1.41–3.60; p < 0.001) and changes in
NAG levels relative to baseline (adjusted OR, 1.09; 95%
CI, 1.03–1.14; p < 0.002) were predictive of progression to
macroalbuminuria.
This systematic review has limitations, including the
inclusion of observational studies that may still have residual
differences between groups despite efforts to minimize bias.
There is a lack of clinical standardization among participants
due to incomplete understanding of factors influencing
uNAG concentrations, leading to a lack of uniformity in
participant characteristics. The included studies show high
clinical heterogeneity with variations in urine collection
methods, storage conditions, and laboratory techniques
used. Most studies were conducted in Asia and Eastern
Europe, limiting generalizability. Categorizing studies
based on ethnicity or geography is flawed due to complex
interactions of dietary, environmental, and genetic factors,
contributing to significant heterogeneity. Despite limitations,
the findings provide valuable insights for evaluating patients
with DKD, indicating that uNAG measurement enhances the
assessment of progression risk. Further studies with larger
populations, different tolerances, and stricter protocols are
needed, particularly cohort studies exploring the relationship
between DKD, markers, and primary outcomes.
Conclusion
In this systematic review and meta-analysis, the analysis
of uNAG values in macroalbuminuric patients revealed
a significant difference compared to normoalbuminuric
patients. However, no significant difference was
observed between patients with normoalbuminuria and
1659
microalbuminuria. Additionally, it was observed that the
majority of type 2 diabetics without nephropathy exhibited
uNAG values above a certain threshold. Conversely,
diabetics with nephropathy showed an increase in uNAG
levels corresponding to the severity of the disease. Thus,
uNAG appears to be a promising prognostic marker for
DKD, although further robust evidence is needed for its
validation and wider acceptance.
Funding The authors declare that the research was conducted without
any commercial or financial relationships.
Declarations
Conflict of interest The authors declare that they have no conflicts of
interest.
References
1. International Diabetes Federation (2021) International Diabetes
Federation. Diabetes Research and Clinical Practice
2. DeFronzo RA, Ferrannini E, Groop L, Henry RR, Herman WH,
Holst JJ, Hu FB, Kahn CR, Raz I, Shulman GI, Simonson DC,
Testa MA, Weiss R (2015) Type 2 diabetes mellitus. Nat Rev Dis
Prim 1:1–23 (PMID: 27189025)
3. Amorim RG, Guedes GS, Vasconcelos SML, Santos JCF (2019)
Kidney disease in diabetes mellitus: cross-linking between hyper-
glycemia, redox imbalance and inflammation. Arq Bras Cardiol
112(5):577–587 (PMID: 31188964)
4. International K (2020) KDIGO 2020 clinical practice guideline
for diabetes management in chronic kidney disease. Kidney Int
98(4):S1–S115 (PMID: 32998798)
5. Li L, Astor BC, Lewis J, Hu B, Appel LJ, Lipkowitz MS, Toto
RD, Wang X, Wright JT, Greene TH (2012) Longitudinal progres-
sion trajectory of GFR among patients with CKD. Am J Kidney
Dis 59(4):504–512 (PMID: 22284441)
6. American Association of Diabetes (2021) ADA standards of dia-
betes care 2021. Diabetes Care. pp S21–S226
7. Oshima M, Shimizu M, Yamanouchi M, Toyama T, Hara A,
Furuichi K, Wada T (2021) Trajectories of kidney function
in diabetes: a clinicopathological update. Nat Rev Nephrol
17(11):740–750
8. Levey AS, Grams ME, Inker LA (2022) Uses of GFR and albu-
minuria level in acute and chronic kidney disease. N Engl J Med
386(22):2120–2128 (PMID: 35648704)
9. Weldegiorgis M, de Zeeuw D, Li L, Parving HH, Hou FF,
Remuzzi G, Greene T, Heerspink HJL (2018) Longitudinal esti-
mated GFR trajectories in patients with and without type 2 dia-
betes and nephropathy. Am J Kidney Dis 71(1):91–101
10. Vallon V, Thomson SC (2020) The tubular hypothesis of
nephron filtration and diabetic kidney disease. Nat Rev Nephrol
16(6):317–336
11. Thomson SC, Vallon V, Blantz RC (2004) Kidney function in
early diabetes: the tubular hypothesis of glomerular filtration. Am
J Physiol Ren Physiol 286(1):F8–F15 (PMID: 14656757)
12. Ix JH, Shlipak MG (2021) The promise of tubule biomarkers
in kidney disease: a review. Am J Kidney Dis 78(5):719–727
(PMID: 34051308)
13. Lobato GR, Lobato MR, Thomé FS, Veronese FV
(2017) Performance of urinary kidney injury molecule-1,
13
1660
neutrophil gelatinase-associated lipocalin, and N-acetyl-β-D-
glucosaminidase to predict chronic kidney disease progression
and adverse outcomes. Braz J Med Biol Res. https://​doi.​org/​10.​
1590/​1414-​431X2​01761​06. (PMID: 28380198)
14. Liu Q, Zong R, Li H, Yin X, Fu M, Yao L, Sun J, Yang F (2021)
Distribution of urinary N-acetyl-beta-D-glucosaminidase and the
establishment of reference intervals in healthy adults. J Clin Lab
Anal 35(5):e23748
15. Nauta FL, Boertien WE, Bakker SJL, Van Goor H, Van Oeveren
W, De Jong PE, Bilo H, Gansevoort RT (2011) Glomerular and
tubular damage markers are elevated in patients with diabetes.
Diabetes Care 34(4):975–981 (PMID: 21307379)
16. Kalansooriya A, Jennings P, Haddad F, Holbrook I, Whiting PH
(2007) Urinary enzyme measurements as early indicators of renal
insult in type 2 diabetes. Br J Biomed Sci 64(4):153–156 (PMID:
18236735)
17. Omozee EB, Okaka EI, Edo AE, Obika LF (2019) Urinary
N-acetyl-beta-d-glucosaminidase levels in diabetic adults. J Lab
Physicians 11(1):001–004
18. Hiratsuka N, Shiba K, Nishida K, Iizima S, Kimura M, Kobayashi
S (1998) Analysis of urinary albumin, transferrin, N-Acetyl-b-D-
Glucosaminidase and b 2-microglobulin in patients with impaired
glucose tolerance. J Clin Lab Anal 12(6):351–355
19. Yamanouchi T, Kawasaki T, Yoshimura T, Koshibu E, Ogata N,
Funato H (1998) Relationship between Serum 1, 5-anhydrogluci-
tol and urinary excretion of N-acetylglucosaminidase and albumin
determined at onset of NIDDM with 3-year follow-up. Diabetes
Care 21(4):619–624
20. Siddiqui K, Al-Malki B, George TP, Nawaz SS, Al Rubeaan K
(2019) Urinary N-acetyl-beta-d-glucosaminidase (NAG) with
neutrophil gelatinase-associated lipocalin (NGAL) improves the
diagnostic value for proximal tubule damage in diabetic kidney
disease. 3 Biotech 9:66
21. Mohammadi-Karakani A, Asgharzadeh-Haghighi S, Ghazi-Khan-
sari M, Hosseini R (2007) Determination of urinary enzymes as a
marker of early renal damage in diabetic patients. J Clin Lab Anal
21(6):413–417 (PMID: 18022929)
22. Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC,
Mulrow CD, Shamseer L, Tetzlaff JM, Akl EA, Brennan SE, Chou
R, Glanville J, Grimshaw JM, Hróbjartsson A, Lalu MM, Li T,
Loder EW, Mayo-Wilson E, McDonald S, McGuinness LA, Stew-
art LA, Thomas J, Tricco AC, Welch VA, Whiting P, Moher D
(2021) The PRISMA 2020 statement: an updated guideline for
reporting systematic reviews. BMJ 372:n71 (PMID: 33782057)
23. Akobeng AK (2005) Principles of evidence-based medicine. Arch
Dis Child 90(8):837–840 (PMID: 16040884)
24. Fontenelles MJ, Simões MG, Farias SH, Fontenelles RGS (2009)
Metodologia da pesquisa científica: diretrizes para elaboração de
um protocolo de pesquisa. Rev Para Med 23(3):1–8
25. Wells GA, Shea B, O’Connell D, Peterson J, Welch V, Losos M,
Tugwell P (2000) The Newcastle-Ottawa Scale (NOS) for assess-
ing the quality of nonrandomised studies in meta-analyses
26. Deeks J, Higgins J, Altman D (2022) Cochrane handbook for sys-
tematic reviews of interventions version 6.3
27. Jaljuli I, Benjamini Y, Shenhav L, Panagiotou O, Heller R (2023)
Quantifying replicability and consistency in systematic reviews.
Stat Biopharm Res 15(2):372–385
28. Morii T, Fujita H, Narita T, Shimotomai T, Fujishima H, Yosh-
ioka N, Imai H, Kakei M, Ito S (2003) Association of monocyte
chemoattractant protein-1 with renal tubular damage in diabetic
nephropathy. J Diabetes Complicat 17(1):11–15
29. Fujita H, Morii T, Koshimura J, Kato M, Miura T, Sasaki H,
Narita T, Ito S, Kakei M (2006) Possible relationship between adi-
ponectin and renal tubular injury in diabetic nephropathy. Endocr
J 53(6):745–752
13
International Urology and Nephrology (2024) 56:1651–1661
30. Yashima I, Hirayama T, Shiiki H, Kanauchi M, Dohi K (1999)
Diagnostic significance of urinary immunoglobulin G in diabetic
nephropathy. Nihon Jinzo Gakkai Shi 41(8):787–796
31. Bouvet BR, Paparella CV, Arriaga SMM, Monje AL, Amarilla
AM, Almará AM (2014) Avaliação da N-acetil-beta-D-glu-
cosaminidase urinária como marcador de dano renal precoce
em pacientes com diabetes melito tipo 2. Arq Bras Endocrinol
Metabol 58(8):798–801 (PMID: 25465599)
32. Assal HS, Tawfeek S, Rasheed EA, El-Lebedy D, Thabet EH
(2013) Serum cystatin C and tubular urinary enzymes as bio-
markers of renal dysfunction in type 2 diabetes mellitus. Clin Med
Insights Endocrinol Diabetes 6:7–13
33. Miyauchi E, Hosojima H, Morimoto S (1995) Urinary angioten-
sin-converting enzyme activity in type 2 diabetes mellitus: its
relationship to diabetic nephropathy. Acta Diabetol 32(3):193–197
34. Banu N, Hara H, Okamura M, Egusa G, Yamakido M (1995)
Urinary excretion of type IV collagen and laminin in the evalu-
ation of nephropathy in NIDDM: comparison with urinary
albumin and markers of tubular dysfunction and/or damage.
Diabetes Res Clin Pract 29(1):57–67
35. Piwowar A, Knapik-Kordecka M, Buczynska H, Warwas M
(1999) Plasma cystatin C concentration in non-insulin-depend-
ent diabetes mellitus: relation with nephropathy. Arch Immunol
Ther Exp-Engl Ed 47:327–331
36. Ito S, Fujita H, Narita T, Yaginuma T, Kawarada Y, Kawagoe M,
Sugiyama T (2001) Urinary copper excretion in type 2 diabetic
patients with nephropathy. Nephron 88(4):307–312
37. Piwowar A, Knapik-Kordecka M, Fus I, Warwas M (2006) Uri-
nary activities of cathepsin B, N-acetyl-beta-D-glucosamini-
dase, and albuminuria in patients with type 2 diabetes mellitus.
Med Sci Monit: Int Med J Exp Clin Res 12(5):CR210–CR214
38. Fu WJ, Li BL, Wang SB, Chen ML, Deng RT, Ye CQ, Liu L,
Fang AJ, Xiong SL, Wen S, Tang HH, Chen ZX, Huang ZH,
Peng LF, Zheng L, Wang Q (2012) Changes of the tubular mark-
ers in type 2 diabetes mellitus with glomerular hyperfiltration.
Diabetes Res Clin Pract 95(1):105–109 (PMID: 22015481)
39. Zhang Y, Yang J, Zheng M, Wang Y, Ren H, Xu Y, Chang B
(2015) Clinical characteristics and predictive factors of sub-
clinical diabetic nephropathy. Exp Clin Endocrinol Diabetes
123(2):132–138
40. Asare-Anane H, Twum F, Kwaku Ofori E, Torgbor EL, Aman-
quah SD, Osafo C (2016) urinary lysosomal enzyme activities
and albuminuria in Ghanaian patients with type 2 diabetes mel-
litus. Dis Markers. https://​doi.​org/​10.​1155/​2016/​28106​39
41. Al-Hazmi SF, Gad HG, Alamoudi AA, Eldakhakhny BM,
Binmahfooz SK, Alhozali AM (2020) Evaluation of early bio-
markers of renal dysfunction in diabetic patients. Saudi Med J
41(7):690
42. Petrica L, Hogea E, Gadalean F, Vlad A, Vlad M, Dumitrascu
V, Popescu R (2021) Long noncoding RNAs may impact podo-
cytes and proximal tubule function through modulating miRNAs
expression in early diabetic kidney disease of type 2 diabetes mel-
litus patients. Int J Med Sci 18(10):2093
43. Fufaa GD, Weil EJ, Nelson RG, Hanson RL, Bonventre JV, Sabbi-
setti V, Waikar SS, Mifflin TE, Zhang X, Xie D, Hsu CY, Feldman
HI, Coresh J, Vasan RS, Kimmel PL, Liu KD (2015) Associa-
tion of urinary KIM-1, L-FABP, NAG and NGAL with incident
end-stage renal disease and mortality in American Indians with
type 2 diabetes mellitus. Diabetologia 58(1):188–198 (PMID:
25316431)
44. Cianci R, Simeoni M, Gigante A, Marco Perrotta A, Ronchey S,
Mangialardi N, Ferri C (2023) Renal stem cells, renal resistive
index, and neutrophil gelatinase associated lipocalin changes after
revascularization in patients with renovascular hypertension and
ischemic nephropathy. Curr Pharm Des 29(2):133–138
International Urology and Nephrology (2024) 56:1651–1661
45. Ruggenenti P, Cravedi P, Remuzzi G (2010) The RAAS in the
pathogenesis and treatment of diabetic nephropathy. Nat Rev
Nephrol 6(6):319–330
46. Alicic RZ, Rooney MT, Tuttle KR (2017) Diabetic kidney disease:
challenges, progress, and possibilities. Clin J Am Soc Nephrol
12(12):2032–2045 (PMID: 28522654)
47. Zhang Y, Yang J, Zheng M, Wang Y, Ren H, Xu Y, Yang Y,
Cheng J, Han F, Yang X, Chen L, Shan C, Chang B (2015) Clini-
cal characteristics and predictive factors of subclinical diabetic
nephropathy. Exp Clin Endocrinol Diabetes 123(2):132–138
(PMID: 25607340)
48. Driza AR, Kapoula GV, Bagos PG (2021) Urinary N-Acetyl-β-
d-glucosaminidase (uNAG) as an indicative biomarker of early
diabetic nephropathy in patients with diabetes mellitus (T1DM,
T2DM): a systematic review and meta-analysis. Diabetology
2(4):272–285
49. Kern EF, Erhard P, Sun W, Genuth S, Weiss MF (2010) Patho-
genesis and treatment of kidney disease early urinary markers
1661
of diabetic kidney disease: a nested case-control study from
the diabetes control and complications trial (DCCT). YAJKD
55:824–834
50. Sheira G, Noreldin N, Tamer A, Saad M (2015) Urinary biomarker
N-acetyl-β-D-glucosaminidase can predict severity of renal dam-
age in diabetic nephropathy. J Diabetes Metab Disord. https://​doi.​
org/​10.​1186/​s40200-​015-​0133-6
Publisher's Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Springer Nature or its licensor (e.g. a society or other partner) holds
exclusive rights to this article under a publishing agreement with the
author(s) or other rightsholder(s); author self-archiving of the accepted
manuscript version of this article is solely governed by the terms of
such publishing agreement and applicable law.
13