I want to write 2 part First , write the itnroduction ( 1500 words ) Inculde : 1- infection organisem and infection disease of the kidney which include all this point ( what is the infection of the kidney , what are the organisem that found in the kidney infection with small introduction of all organisem , what are the symptome of infection , how can dignose this organisem . 2- diabetis disease which include ( what is the diabetis disease , how can affect this disease on the kidney , what is the affect of this disease on epithilial cells in urine , what are the symptoms of the disease , what are the type of the disease ) 3- kidney stone which inculde ( what are the kidney stone , type of the kidney stone , symptoms of kidney stone , how the disease occur and how can affect on the kidney ) 4- lupus nephritis disease which include ( what is the lupus nephritis , how can the disease occur , type and stage of this disease , symptoms of this disease ) 5- urine cytology ( what is the urine cytology , ( manual and automated ) , how can to dignosis the kidney disease by this procedure . 2 the second part is liturutrure review about this topic 1- infection ( pathology of the disease , dignosis by the urine cytology ( sedement and smear ) prognosis of disease , change of the epithilial cells in the urine by infection , tretment 2- diabetis ( pathology , diagnosis by urine cytology , change of the epithilial cells in the urine in pateint of diabetis , tretment 3- kidney stone ( pathology , diagnosis by urine cytology , change of the epithilial cells in the urine , tretment ) 4- lupus nephritis ( pathology , diagnosis by urine cytology , change of the epithilial cells in the urine , tretment ) . I need this part 2000 to 3000 words . I have references from which I want the file to be written, and I do not want them to deviate from these references, and I do not want references other than these .
Hindawi
Journal of Diabetes Research
Volume 2019, Article ID 9475637, 7 pages
https://doi.org/10.1155/2019/9475637
Research Article
A Liquid-Based Cytology System, without the Use of
Cytocentrifugation, for Detection of Podocytes in Urine
Samples of Patients with Diabetic Nephropathy
Moritsugu Kimura ,1 Masao Toyoda ,1 Nobumichi Saito,1 Noriko Kaneyama ,1
Han Miyatake ,1 Eitaro Tanaka ,1 Hirotaka Komaba ,1 Masanori Hara,2
and Masafumi Fukagawa 1
1
Division of Nephrology, Endocrinology and Metabolism, Department of Internal Medicine, Tokai University School of Medicine,
Isehara, Japan
2
Iwamuro Health Promotion Center, Niigata, Japan
Correspondence should be addressed to Masao Toyoda; m-toyoda@is.icc.u-tokai.ac.jp
Received 2 June 2018; Revised 3 December 2018; Accepted 25 December 2018; Published 17 February 2019
Academic Editor: Pedro M. Geraldes
Copyright © 2019 Moritsugu Kimura et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Objective. Podocytes have highly differentiated functions and are extremely difficult to grow; thus, damage of podocytes is
associated with glomerular dysfunction. Desquamated podocytes can be detected in urine of patients with severe renal
impairment. Unlike the rapidly progressive glomerular damage in glomerulonephritis, only a few desquamated podocytes are
usually detected in diabetic nephropathy (DN). It is not clear whether the low podocyte count in DN is due to limitation of the
conventional method or true pathological feature. The aim of this study was to compare the conventional method with a newly
modified method in detecting podocytes in morning urine samples of patients with DN. Materials and Methods. The study
subjects were patients with type 2 diabetes. Urine samples from these patients were analyzed by the conventional method
(Cytospin®) and the modified method (SurePath™). We determined the rate of detection of urinary podocytes and the number
of detected cells. Results. The detection rate and podocyte count were significantly higher by the modified method than by the
conventional method. The differences in the detection rates and numbers of podocytes were not significant between patients
with normoalbuminuria and those with macroalbuminuria. However, they were significant in patients with microalbuminuria.
The number of podocytes in the urine correlated significantly with the albumin-to-creatinine ratio, but not with the estimated
glomerular filtration rate. Conclusions. The true number of urinary podocytes, as measured by the modified SurePath™-based
method, in patients with DN is much higher than that estimated by the conventional method.
1. Introduction
Diabetic nephropathy (DN) is currently the leading cause for
initiation of dialysis in Japan. Extensive research has been
carried out to elucidate the etiopathogenesis of DN, though
mesangial cell matrix proliferation and hypertrophy of the
glomerular basement membrane are considered important
factors involved in the development and progression of DN
[1–4]. In addition, impairment of podocytes has also been
considered in recent years to be an important pathomechanism of albuminuria [5–14].
Compared to other cell components of the glomerulus,
podocytes have specific biological properties, such as peculiar
morphology, highly differentiated functions, and poor
growth, and thus, disturbance of podocyte function is usually
associated with marked glomerular dysfunction [12–17].
Therefore, clinical assessment of podocyte impairment is
important in the diagnosis and treatment of glomerular
diseases, including DN.
Various urinary biomarkers have been used in recent
years for clinical assessment of renal function [18]. Most of
them are biomarkers for interstitial lesions and renal tubular
2
Journal of Diabetes Research
Table 1: Clinical parameters of patients of the conventional method group, modified method group, and normal subjects.
p value∗
Normal subjects (n = 20)
Conventional method (n = 41)
Modified method (n = 41)
Age (years)
12/8
40 6 ± 8 5
31/10
62 5 ± 10
27/14
63 9 ± 11 0
0.4008
BMI (kg/m2)
22 0 ± 1 9
25 6 ± 3 8
25 8 ± 3 8
0.9372
HbA1c (%)
5 6±0 4
7 4±0 9
7 4±1 7
0.3985
SBP (mmHg)
119 ± 12
133 ± 9 8
131 ± 11 9
0.4521
Gender (M/F)
DBP (mmHg)
2
eGFR (ml/min./1.73m )
UACR (mg/g Cr)
0.3316
69 ± 6 0
76 ± 11 3
74 ± 10 8
0.3830
84 1 ± 9 0
59 4 ± 15 3
55 8 ± 22 2
0.4142
ND
721 ± 1344 9
702 ± 1301 2
0.4305
∗
Statistical comparison of the two groups was performed only between the conventional method and the modified method. Data are mean ± SD. ND: not
determined, SBP: systolic blood pressure, DBP: diastolic blood pressure.
function, where such urinary tests likely reflect areas of
damage. Only a few biomarkers are currently available to
assess glomerular lesions. Importantly, podocytes located
on the external side of the glomerular basement membrane
(GBM), namely, on the urinary space side, are different from
the damaged endothelial cells located inside mesangial cells,
such that damage of the podocytes is reflected directly in
the urine and that injury-related desquamated and excreted
podocytes can be detected in the urine. In their attempt to
detect damaged podocytes in the urine, Nakamura et al.
[19] immunostained urine smeared on glass slides after
Cytospin® centrifugation using a podocalyxin monoclonal
antibody and designed a method for quantification of urinary
podocytes (direct calculation of the number of podocytes).
Using this method, they reported the presence of desquamated and excreted damaged podocytes in urine [19]. Their
results confirmed the presence of numerous podocytes in
the urine of patients with inflammatory glomerular diseases,
who develop classical symptoms of acute inflammation of the
glomeruli, particularly with the formation of acute extratubular lesions. Furthermore, their findings confirmed that the
presence of podocytes in the urine reflected the acute phase
of the disease, and their detection was useful for the selection
of appropriate treatment [10, 11]. At this stage, however,
there is little or no information on the presence or absence
of podocytes in the urine of subjects with normal renal function and in patients with chronic and mild inflammatory glomerular disease, such as DN.
The presence of podocytes in urine samples of patients
with DN is controversial. On the one hand, some investigators using histopathological examination demonstrated the
presence of a low number of podocytes in patients with DN
and confirmed the clinical importance of this finding, while
others reported that the low urinary podocyte count was
unrelated to the type of diabetes [6, 17, 20, 21]. Therefore,
measurement and quantification of urinary podocytes seem
helpful to determine and predict not only the severity of
DN and prognosis but also the selection of treatment for DN.
Compared to glomerulonephritis, which is characterized
by rapid progression, DN progresses slowly over a long
period of time, and the number of desquamated podocytes
excreted in the urine is markedly low, although the latter is
probably due to complexity of the podocyte detection
method [22]. Therefore, improving the rate of detection of
podocytes in the urine requires modification and simplification of the conventional method to allow universal application and clinical use. Modification of the method could be
useful especially if the modified method is simple and convenient as well as if it imposes little burden on patients.
In the present study, we describe a newly modified simple
method of liquid-based cytology system for the detection of
podocytes in the urine using SurePath™, which does not
require the use of Cytospin® cytocentrifugation but rather
employs the use of a separating reagent. Here, we describe
and assess the utility of the modified method.
2. Materials and Methods
2.1. Subjects. The study subjects were type 2 diabetes patients,
with renal function ranging from normoalbuminuria to predialysis chronic renal failure, who received outpatient treatment at Tokai University Hospital and provided written
consent to participate in the study. The following exclusion
criteria were applied: patients on dialysis and patients with
clinical suspicion of complications of kidney diseases other
than DN, based on clinical data (absence of diabetic retinopathy, predominant hematuria compared to proteinuria, overt
proteinuria occurring within 5 years after the onset of diabetes (urinary protein: 1 g/g creatinine or higher), rapid renal
dysfunction, and rapid aggravation of proteinuria within a
short period of detection [23]).
A total of 150 patients who satisfied the above criteria
were enrolled. They included 50 patients in whom urinary
samples had already been obtained and subjected to analysis
of podocytes using the conventional method (the conventional method group) and 100 patients whose urine samples
were analyzed by the modified method (the modified method
group). Data of the two groups were compared, including
age, sex, body mass index (BMI), blood pressure, HbA1c, disease duration, estimated glomerular filtration rate (eGFR),
and urine albumin-to-creatinine ratio (UACR) in morning
urine samples (Table 1).
The study also included 20 healthy controls whose blood
and urine laboratory tests were within normal values. These
subjects were recruited to assess the difference between the
rate of detection of urinary podocytes and the podocyte
Journal of Diabetes Research
number between healthy subjects and diabetic individuals.
The characteristics of these subjects are listed in Table 1.
Ethical approval for this study was granted by the Tokai
University Institutional Review Board for Clinical Research,
and all participants provided written informed consent.
2.2. Urinary Podocyte Detection Method. The presence of
podocytes was checked using the conventional and modified
methods. The study participants were asked to provide urine
voided in the morning, which was stored at −70°C within 2 h
of collection.
2.2.1. Conventional Method
(1) Thirty-milliliter samples of the morning urine were
collected and centrifuged at 1500 revolutions per
minute (700 ×g) for 5 minutes
(2) The supernatant was pipetted out, and the urinary sediment was washed with 0.01 mol/l
phosphate-buffered saline (PBS, pH 7.2)
(3) The urinary sediment was resuspended in 10 ml PBS,
and autosmears were prepared by Cytospin®
cytocentrifugation
(4) The samples were air-dried for 30 minutes and
washed with PBS
(5) Then, the slides were incubated in anti-podocalyxin
primary antibody (22A4, mouse monoclonal antibody [10]; dilution, 1 : 80) for 1 hour at room
temperature
(6) The slides were washed in PBS before incubation in
FITC-labeled rabbit anti-mouse IgG secondary antibody (Dako) for 1 hour at room temperature
(7) After washing the slides in PBS, the cell nuclei were
stained using DAPI (Sigma, St. Louis, MO)
(8) The number of podocytes in each slide was calculated
using a fluorescence microscope
Since each sample was prepared from 30 ml urine, the
number was divided by 30 to calculate the number of podocytes per 1 ml of urine.
2.2.2. Modified Method (Figure 1). To enhance podocyte cell
adhesion to the glass slide prior to the process of immunostaining, we modified the cell attachment technique from
the autosmear system (Pap smear cytology) to liquid-based
cytology system. The use of a dedicated precoated glass slide
promotes urinary podocyte adherence. Thus, apart from the
use of SurePath™, the remaining steps of our method are
similar to those followed in the conventional method:
(1) Similar to step 1 of the conventional method
(2) Similar to step 2 of the conventional method
(3) The glass slide centrifugation mentioned in step 3
above was omitted
3
(4) The slides were treated with SurePath™
This product enhances gravitational sedimentation and
electrical adherence of the tissue to the glass slide.
(5) Similar as in step 5 of the conventional method
(6) Similar to step 6 of the conventional method
(7) Similar to step 7 of the conventional method
(8) Similar to step 8 of the conventional method
As described above, since the samples were prepared
from 30 ml urine, the number of podocytes per 1 ml of urine
was calculated by dividing the number by 30.
2.2.3. Urinary Podocyte Count and Urinary Podocyte
Detection Rate. The number of desquamated podocytes in
the urine sample was counted in both methods using a fluorescence microscope; DAPI-positive cells were considered to
be nucleated, and the number of podocytes was measured by
counting all DAPI- and podocalyxin-positive cells (Figure 2).
The number of podocytes in 1 ml urine was considered as
the urinary podocyte count, and the “number of participants with podocytes detected in the urine” divided by
the “total number of participants” represented the urinary
podocyte detection rate. The urinary podocyte count and
urinary podocyte detection rate were compared in patients
with normoalbuminuria (UACR < 30 mg/g creatinine),
microalbuminuria (UACR ≥ 30 to
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