Determinants of the intensity of uremic pruritus in patients receiving maintenance hemodialysis: A cross-sectional study

Background Uremic pruritus (UP) is a common and frustrating symptom in patients receiving hemodialysis (HD). The majority of patients have mild to moderate itching of the skin, and a small percentage have severe itching, which seriously affects their quality of life and survival rate. However, little is known about factors that influence the intensity of itching in patients. Methods A cross-sectional study on uremic pruritus in male and female patients receiving HD was conducted in September 2019. This study included 148 eligible patients who received HD at the Blood Purification Center of Xinchang County People’s Hospital, Zhejiang Province, China from March 2019 to June 2019. We collected general data consisted of age, sex, body mass index (BMI), place of residence, educational level, diabetes mellitus status and duration of HD; as well as clinical, biochemical indicators, including serum calcium (Ca), serum phosphorus (P), serum albumin (ALB), haemoglobin (Hb), serum intact parathyroid hormone (iPTH), pre-dialysis serum urea nitrogen (BUN), normalized protein catabolic rate (nPCR), urea nitrogen clearance index (KT/V), ferritin (FER) and pre-dialysis serum creatinine (sCR). We also assayed the inflammatory cytokine serum high sensitivity C-reactive protein (hs-CRP). The Five-Dimensional Itching Scale (5DIS) was used to evaluate the degree of skin itching (none, mild, moderate, or severe). We used multiple logistic regression to analyze influencing factors on the degree of skin itching in patients with UP. Results Of the 148 patients, 60 had uremic pruritus (incidence rate, 40.54%). These included 22 cases of mild skin itching (14.86%), 30 of moderate skin itching (20.27%), and 8 of severe skin itching (5.41%). Compared with uremia patients without skin pruritus, patients with UP had higher levels of iPTH, Hb, BUN, nPCR, and hs-CRP. The composition ratio showed significant differences between urban and rural patients with different degrees of skin itching (P = 0.017); moreover, the difference of iPTH and hs-CRP levels were statistically significant (P = 0.009 and < 0.001, respectively). Using no itching as a reference, multiple logistic regression analysis showed that as hs-CRP level increased, the patient’s risks of mild skin itching (odds ratio [OR] = 1.740; 95% confidence interval [CI], 1.061–2.854; P = 0.028), moderate skin itching (OR = 2.8838 95% CI, 1.744–4.718; P < 0.001), and severe skin itching (OR = 9.440; 95% CI, 3.547–25.124; P < 0.001) all increased as well. Compared with urban residents, rural residents have a higher risk of moderate itching (OR = 3.869; 95% CI, 1.099–13.622; P = 0.035). Conclusion Levels of hs-CRP were associated with the intensity of skin itching in patients with UP. Higher hs-CRP levels were closely related to severe skin itching. The relationship between the intensity of skin itching and the environment in maintenance hemodialysis patients needs further clarification.

4. The number of patients with severe pruritus is small (n=8) and therefore, meaningful associations with severe pruritus may not be appreciated. Response： In this study, there were a total of 148 patients with uremia and eight patients with severe skin pruritus, accounting for 5.4% of the total patients. 2012-2015 DPPSO study [3] surveyed 7,629 people, divided into five groups (not at all, somewhat, moderately, Very Much, Extremely) according to the degree of itchy skin. Extreme Itching accounted for 7%, which is slightly higher than 5.4% of patients with severe Itching in this study. The reason for the differences may lie in the different criteria used to assess the degree of pruritus in the patient's skin.
This section is detailed on page 14, lines 37-43 Reviewer #2 In this study, the authors studied uremic pruritus using the five-dimensional itch scale in 148 adult patients of end-stage renal disease receiving hemodialysis and report the prevalence of 40.5% and association with parathormone (PTH), hemoglobin and highsensitive C reactive protein (hs-CRP) on bivariate analysis and hs-CRP alone on multiple logistic regression. The study seems to be designed logically, conducted scientifically and analyzed with due statistical rigour. However, the manuscript is lengthy, does not have a smooth flow of information and requires rewriting as per the STROBE checklist. Major observations are described below. Minor observations are inserted in the uploaded version of the manuscript as "sticky notes" at appropriate places. Response: 1)We have rewritten the paper according to the STROBE checklist, details of which can be found in the attached STROBE checklist. 2)Concerning some minor observation, we have followed your comments with deep appreciation.
Major observation: 1. The introduction should briefly summarize the present state of knowledge on uremic pruritus, highlight the gap in understanding the study sought to address and state the study's objective. Comparison with previous studies (measurement of severity of Itching) should be reserved for discussion. Response: The reviewer's suggestions were excellent, and we have corrected them. This section has been moved to page 15, lines 23-29.
2. The stated aims of the study are to "1. explore the prevalence of UP in maintenance HD patients and the demographic and clinical characteristics of pruritus patients. Explore determinants of higher pruritus intensity based on participants' clinical and biochemical parameters and their pruritus assessments by 5DIS at baseline". The second aim is somewhat unclear. Were characteristics of patients with higher pruritus intensity to be compared with those of patients with of lower pruritus intensity or with those of patients without pruritus? Clear statement in the PICO format is suggested. What is the novelty of the research question? This needs to be justified. Response: objectives (1). explore the prevalence of UP in maintenance HD patients and the demographic and clinical characteristics of pruritus patients. (2). analyze the relationship between high-sensitivity C-reactive protein and different degrees of skin itching. This section has been moved to page 2, lines 28-32. 3. The setting of the study may be described in "Methods". Response: This section is detailed on Methods (study setting), page2, lines 38 & page3, lines 1-5. 4.How were biochemical parameters such as serum calcium, phosphorus, creatinine, etc assayed? The detailed methods need not be described but should be referenced. Response: We assayed biochemical indicators such as Ca, P, iPTH, Hb, ALB, sCr, Bun, FER, sp/KTV and hs-CRP. The hospital's central laboratory performed all of the laboratory tests, and auto-analyzers were used to determine biochemical data, iPTH was measured with Roche second-generation assay. The clinical biochemical indicators are based on the data within three months from the survey day. If there is no data within three months, the patient is required to re-test. If the patient is unwilling to cooperate with the test, it is a missing value. spKT/V(single pool KT/V)=-ln(R-0.008t)+(4-3.5R)*(∆BW/BW). Note: K refers to Blood urea clearance rate of dialyzer(L/h), t is the dialysis time (h), and V is the distribution volume of urea (V). R is the ratio of blood urea nitrogen after dialysis to blood urea nitrogen before dialysis; t is single dialysis time in h; ∆BW is the weight change value before and after dialysis, i.e. ultrafiltration, unit L; BW is weight in kg. Blood collection requirements: blood samples before dialysis from the artery end of the vascular path, after dialysis before blood sample collection to stop ultrafiltration, reduce blood flow of 50 ml/min, wait 15 seconds after blood collection from the artery as a blood sample after dialysis.
This section is detailed on page 3, lines 36-41 & page 4, lines 1-7. 5.The study reports no significant association of uremic pruritus with factors such as calcium, phosphorus, creatinine and albumin. However, the sample size calculation was made to estimate the prevalence of uremic pruritus. Could the study have been under powered for the study of these factors? Response: Some studies suggested that UP is related to Ca, P, ALB, and uremic toxins [11,20]. Nevertheless, contradictory data on the impact of Ca and P have been reported [21]. In this study, no statistically significant differences were found in Ca, P, ALB, and creatinine when comparing groups of patients with different degrees of skin pruritus. There may indeed be a lack of test power(Since the data for these factors are nonnormally distributed, the exact distribution is unknown. Based on statistical theory, it is not possible to calculate the true test power with certainty.), which is considered to be related to the small sample size of this study and the unbalanced sample size among the groups. However, single-factor logistic regression analysis also did not find an association between these factors and the degree of skin pruritus. Therefore, these factors were ultimately not included in the multifactor logistic regression analysis. In fact, despite various substances have been discussed as potential pruritogens in chronic renal diseases, a causal relationship could never be established [22]. This section is detailed on page 15, lines 14-22. 6.The following factors influencing uremic pruritus have not been studied: diet, insulin resistance, dialysis adequacy, interleukins 6 & 2, and κ opioid receptor distribution. These need to be acknowledged as limitations. Response: Firstly, this study was a single-center study with a limited number of people on hemodialysis, and therefore the number of people included in the study was limited. Many factors influence skin itching in uremic patients, and it is not practical to include all relevant aspects of the study. Secondly, there is the issue of patient compliance. For example, to study diet factor, we need to distinguish between vegetarian and nonvegan patients, which requires strict control of the patient's diet. For insulin resistance, if the HOMA model is used to assess the resistance of the body to insulin (HOMA-IR), fasting blood glucose and fasting insulin levels need to be measured based on the formula HOMA-IR=FPG×FINS/22.5, therefor, fasting blood needs to be collected before hemodialysis. Some patients are difficult to cooperate, the missing value of the study will be further increased. Thirdly, for dialysis adequacy, we included a urea clearance index (kt/v) to assess this. In conclusion, it is essential to acknowledge the limitations of this study.
This section is detailed on page 13, lines 17-19. to 2015, showed that 26%-48% of patients had at least moderate skin itching, whereas 13%-26% 16 of patients had severe or extreme itching [3]. The higher the degree of skin itching, the higher 17 the mortality rate [4]. Several causes of or factors contributing to uremic pruritus (UP) have been 18 proposed, including increasing systemic inflammation; abnormal levels of serum parathyroid 19 hormone (PTH), serum calcium (Ca), and serum phosphorus (P); opioid receptor imbalance; and 20 neuropathological processes [2]. However, the factors affecting degree of skin itching in patients 21 with uremia are still unclear. We used a five-dimensional itching scale [5] to evaluate skin itching 22 across multiple dimensions in uremic patients undergoing maintenance HD. We also included 23 general patient data such as age, sex, body mass index (BMI), place of residence, educational 24 level, Diabetes mellitus status,and duration of HD; as well as clinical indicators such as Ca, P, 25 serum albumin (ALB), hemoglobin (Hb), PTH, serum creatinine (sCr) ,serum urea nitrogen(BUN), 26 urea nitrogen clearance index (KT/V), ferritin (FER) before dialysis, and hs-CRP to analyze the 27 determinants of itching in these patients.   35 This study is a cross-sectional study on UP in patients receiving HD. This study was approved by the 36 1 XCXRMYY2018-001). 3 we included a total of 148 subjects who received HD at the Blood Purification Center of Xinchang 4

Study setting
County People's Hospital, Zhejiang Province, China from March 2019 to June 2019. each of them 5 signed an informed consent form. Collection of data was by the authors or research personnel, and 6 all personnel received training and guidance from the corresponding author. Data statistics and 7 analysis were from September to December 2019. 8

17
The diagnostic criteria for uremic pruritus adopted internationally are as follows: (1) uremia 18 patients exclude skin pruritus caused by other diseases; (2) itching occurs at least 3 days within 2 19 weeks, and itches several times a day, each time itching It lasts a few minutes and affects the 20 patient's life; (3) Itching in a specific pattern lasts for more than 6 months [6]. We used the Five-21 Dimensional Itching Scale (5DIS) to assess itching based on the five dimensions of degree, duration, 22 direction, disability, and distribution. Total score ranged from 5 (no itching) to 25   is no data within three months, the patient is required to re-test. If the patient is unwilling to cooperate 3 with the test, it is a missing value. spKT/V(single pool KT/V)=-ln( − 0.008 ) + (4 − 3.5 ) * (∆ / ).

4
Note: K refers to Blood urea clearance rate of dialyzer(L/h), T is the dialysis time (h), and V is the 5 distribution volume of urea (V). R is the ratio of blood urea nitrogen after dialysis to blood urea nitrogen 6 before dialysis; t is single dialysis time in h; ∆BW is the weight change value before and after dialysis, i.e. 7 ultrafiltration, unit L; BW is weight in kg. Blood collection requirements: blood samples before dialysis from 8 the artery end of the vascular path, after dialysis before blood sample collection to stop ultrafiltration, 9 reduce blood flow of 50 ml/min, wait 15 seconds after blood collection from the artery as a blood sample 10 after dialysis.    Study size 27 Epidemiological data indicated that approximately 40% of patients with end-stage renal disease 28 (ESRD) experience UP [2]. The overall prevalence of uremia and pruritus is 0.4, the tolerance is set to 29 0.1, taking a=0.05, the total number of hemodialysis in our blood purification center is 180 people.  ncss.com/software/pass), the calculated sample size is 121 people, assuming that the dropout rate is 32 20%, and the total sample size is 152 people.

Quantitative variables 34
Quantitative data with a normal distribution is expressed as mean ± standard deviation; intergroup 35 comparisons were made using one-way analysis of variance or t-test; quantitative data with a non-36 normal distribution are expressed as medians, and interquartile ranges, intergroup comparisons of 37 continuous variables were made using a non-parametric test.

Statistical methods
We performed statistical processing using line data from SPSS software version 24.0 (IBM Corp., 1 Armonk, New York, US). Intergroup comparison of classification variables was based on the rank-sum 2 or chi-square test. A multiple logistic regression model analyzed the factors associated with degree of 3 skin itching in patients with uremia. All variables were diagnosed by collinearity; after elimination of 4 the collinearity problem, variables of P<0.05 in single-factor regression analysis and variables with 5 statistical differences between patients with different degrees of skin itching were included in the 6 regression model. Finally, we included four factors including Hb, PTH, hs-CRP, Place of residence in the 7 multiple logistic-regression model. P < 0.05 was considered to be statistically significant. In this study,

21
Descriptive data 22 The primary clinical characteristics of the included patients are shown in Table 1.

5
The composition ratio showed a difference between rural and urban patients with varying degrees of skin itching (P = 0.017). Differences in IPTH and hs-CRP levels of 6 patients with different degrees of skin itching were statistically significant (P = 0.009 and <0.001, respectively), As shown in Table 3. 7 Table 3. Baseline characteristics of patients with varying degrees of skin itching.   Table).    Key results 2 In this study, we found that among 148 eligible patients receiving maintenance HD, 60 had UP 3 (prevalence rate, 40.54%); they included 22 mild cases of skin itching (14.86%), 30 moderate cases 4 (20.27%), and 8 severe cases (5.41%). Compared with patients with no skin pruritus, patients with UP 5 had higher levels of iPTH, Hb, BUN and hs-CRP. The composition ratio of rural to urban patients, levels 6 of IPTH and hs-CRP were statistically significantly different in patients with varying degrees of skin 7 pruritus. Single-factor logistic-regression analysis discovered a link between hs-CRP levels and all three 8 degrees of itching. Moreover, Hemoglobin is associated with moderate and severe Itching, and iPTH is 9 related to severe Itching. Multi-factor logistic regression analysis also indicated that patients with 10 higher hs-CRP levels had higher risks of mild, moderate, and severe skin itching. Interestingly, the 11 multivariate analysis also showed that rural residents have a higher risk of moderate skin itching than 12 urban residents.

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Our study had some limitations. Firstly, it was a single-center, cross-sectional study that did not show a 15 causal relationship between severity of skin itching in UP and hs-CRP, so further validation is needed 16 from multicenter, large-sample, prospective cohort studies. Secondly, this study did not include some 17 of the influences known in the literature such as normalized protein catabolic rate (nPCR), diet, insulin 18 resistance, interleukins 6 and 2, and κ opioid receptor distribution. Thirdly, the number of patients 19 with severe pruritus is small (n = 8), so readers may not understand a meaningful association with 20 severe pruritus. Finally, the sample size of this study was small and uneven between groups.

22
This study showed that median hs-CRP level gradually increased along with degree of skin itching in 23 patients with uremic pruritus (no itching, 0.90; mild skin itching, 1.08; moderate skin itching, 2.15; 24 severe skin itching, 3.93). Furthermore, hs-CRP levels in patients with moderate and severe skin 25 itching were significantly higher than in those without itching. These findings suggested that hs-CRP 26 levels might be associated with severity of skin itching. Jiang et al. [7] compared the clinical effects of 27 high-throughput hemodialysis (HTH) and hemodialysis filtration (HDF) treat patients with end-stage 28 nephropathy and explored possible potential mechanisms. Their results showed that levels of C-29 reactive protein (CRP) and leukocyte interleukin-6 (IL-6) in patients receiving HTH significantly 30 improved, compared with those in patients who received HDF. The study also found that HTH had an 31 exact therapeutic effect in UP that was related to inhibition of microinflammation in dialysis patients.

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Another study also showed that HD patients with UP had significantly higher serum CRP and IL-6 33 levels compared to HD patients without UP, UP seems to be associated with an up-regulation of micro-34 inflammation in uremia [8]. Mahmudpour's team [9] indicated that the average reduction in VAS 35 scores in the montelukast group was greater than that in the placebo group, and average levels of hs-36 CRP in this group showed a downward trend; however, average hs-CRP levels in the placebo group 37 gradually increased, showing that the effect of montelukast treatment in UP might be related to a 38 reduction in an inflammatory response. A factor analysis also shows a close correlation between UP 39 and levels of the inflammatory factors CRP and leukocyte IL-6 [10]. Based on this study and previous 40 studies, inflammatory responses appear to play an important role in UP.

1
We analyzed the factors affecting degree of skin itching in UP patients and corrected for patients' 2 levels of Hb and PTH, Place of residence. Multi-factor logistic-regression analysis found that hs-CRP 3 levels were associated with severity of skin itching and were a risk factor. Compared with patients 4 without itching, the OR value for mild skin itching was 1.740, that for moderate skin itching was 2.838, 5 and that for severe skin itching was 9.440, indicating that degree of skin itching increased along with 6 hs-CRP levels. A previous multivariable logistic-regression analysis confirmed that older age and higher 7 CRP levels are associated with severe itching [3]. Still, in our study, neither single-factor nor multi-8 factor logistic-regression analysis showed any differences in skin itching between elderly and non-9 elderly patients. In our study, the OR value for hs-CRP of patients with severe skin itching was 10 significantly higher than that of patients without itching, suggesting that higher hs-CRP levels were 11 closely associated with severe skin itching, which was consistent with the results of an earlier study.

12
Pakfetrat et al. [11] assessed the effect of sertraline on UP. patients were randomly divided into two 13 groups, the trial group receiving sertraline and the control group taking a placebo, and their severity 14 of itching was measured by two scoring systems (visual-simulation score and DUO scoring system 15 [12]). The researchers found that differences in skin itching intensity between the two scoring systems 16 and between the two groups were directly related to CRP level, and they believed that sertraline 17 reduced skin itching, perhaps due to its effect of reducing the number of inflammatory cytokines.

18
Tseng et al. [13]studying a possible association between vegetarians and UP, found that levels of

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In our study, the composition ratio showed a significant difference between rural and urban 24 patients with different degrees of skin itching. Compared with patients without itching, the proportion 25 of rural patients with moderate and severe skin itching was relatively high, however, no statistically 26 significant difference was achieved. Although the single factor analysis did not indicate that the place 27 of residence was related to the intensity of the patient's skin itching, the multi-factor analysis 28 demonstrated that rural residents had a higher risk of moderate skin itching than urban residents. The 29 possible reason is that on the one hand, compared with urban residents, rural residents have lower 30 living habits and lower health awareness; on the other hand, with the development of urbanization, 31 rural areas the air pollution in Xinchang's local rural areas is gradually increasing and the urban 32 environment is slowly improving. UP in patients receiving maintenance HD is associated with air 33 pollutants such as NO2 and CO [14]. A cross-sectional study suggested that UP in patients receiving 34 maintenance HD might be associated with the number of days in which environmental particulate 35 matter ≤2.5 µ in diameter (PM2.5) exceeds standard levels [15]. The above mentioned two studies 36 suggest that itching of the skin might be associated with the patient's living environment. In this study, 37 there were a total of 148 patients with uremia and eight patients with severe skin pruritus, accounting 38 for 5.4% of the total patients.2012-2015 DPPSO study [3] surveyed 7,629 people, divided into five 39 groups (not at all, somewhat, moderately, Very Much, Extremely) according to the degree of itchy 40 skin. Extreme Itching accounted for 7%, which is slightly higher than 5.4% of patients with severe

41
Itching in this study. The reason for the differences may lie in the different criteria used to assess the 42 degree of pruritus in the patient's skin.

43
iPTH levels differed between patients with varying degrees of skin itching. Median iPTH level in 44 patients with severe skin itching was higher than that in patients without itching. Still, there was no 1 statistically significant difference between iPTH levels across groups, and single-factor regression 2 analysis suggested a weak association between higher iPTH levels and severe skin itching. However, 3 after we corrected for hs-CRP and Hb levels, Place of residence, that association disappeared. After 4 parathyroid excision, iPTH level and serum phosphate concentration are significantly reduced, and 5 skin itching disappears [16,17]. A study by Moldovan et al. [18] suggests that the average value of PTH 6 decreased significantly after parathyroidectomy, and the symptoms of bone pain were significantly 7 improved. However, muscle weakness and itching of the skin did not improve. Levy et al. [19] studied 8 the associated symptoms of sHPT in patients receiving maintenance HD. Their results showed that an 9 increase in PTH levels was associated with skin itching worsening over time, and the effect was more

13
Some studies suggested that UP is related to Ca, P, ALB, and uremic toxins [11,20]. Nevertheless, 14 contradictory data on the impact of Ca and P have been reported [21]. In this study, no statistically 15 significant differences were found in Ca, P, ALB, and creatinine when comparing groups of patients 16 with different degrees of skin pruritus. There may indeed be a lack of test power, which is considered 17 to be related to the small sample size of this study and the unbalanced sample size among the groups.

18
However, single-factor logistic regression analysis also did not find an association between these 19 factors and the degree of skin pruritus. Therefore, these factors were ultimately not included in the 20 multifactor logistic regression analysis. In fact, despite various substances have been discussed as 21 potential pruritogens in chronic renal diseases, a causal relationship could never be established [22].

22
At present, only a few studies have explored the factors affecting skin itching intensity in these 23 patients.
Wieczorek et al. [23] documented Intensity of skin itching have a significant negative 24 correlation with expression of κ opioid receptors in patients with HD. Also, hs-CRP levels, male 25 sex, HD duration, insulin resistance, insufficient dialysis adequacy, and hyperphosphatemia are 26 positively correlated with UP intensity [24], increasing the blood flow of the HD machine can 27 reduce the intensity and frequency of skin itching [25]. However, the above preliminary research 28 used visual-analogue scoring (VAS) to evaluate the degree of skin itching. In this study, we used a 29 five-dimensional itching scale to evaluate skin itching across multiple dimensions in uremic 30 patients undergoing maintenance HD. Although VAS is sufficient to assess the severity of 31 symptoms, it does not take into account other aspects of pruritus, such as the relative impact of 32 pruritus on quality of life, changes in pruritus over time, and the location of pruritus. The 5-D 33 score was strongly correlated with the numerical rating scale(NRS)and VAS [5,26], which is a 34 questionnaire with acceptable validity, reliability and sensitivity to change to evaluate pruritus in 35 Thai and Arabic patients [26,27]. The 5-dimensional Pruritus Scale is recommended to assess the 36 severity of pruritus because it is more accurate than VAS and more sensitive to the 37 multidimensional nature of pruritus [28]. 38

Generalizability 39
In conclusion, our study suggested that the intensity of skin itching in patients with UP was associated 40 with hs-CRP and that higher hs-CRP levels were associated with severe skin itching. Thus, this study 41 demonstrates that controlling inflammation can relieve itchy skin in patients with uremia, and is

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The authors received no specific funding for this work.

Introduction
Background Itchy skin is a common and distressing symptom in patients with chronic kidney disease. Uremic pruritus mainly manifests as skin itching of varying degrees, either systemically or locally, of which the back, extremities, chest, and head are common. Itching appears as paroxysmal episodes of varying duration. Those with milder symptoms can have intermittent attacks, lasting several minutes each time, while those with more severe symptoms last longer, and the symptoms are usually most obvious at night [1]. Epidemiological data indicated that approximately 40% of patients with end-stage renal disease (ESRD) experience moderate to severe itching [2]. The Dialysis Outcomes Practice Patterns Study (DOPPS), conducted from 2012 to 2015, showed that 26%-48% of patients had at least moderate skin itching, whereas 13%-26% of patients had severe or extreme itching [3]. The higher the degree of skin itching, the higher the mortality rate [4]. Several causes of or factors contributing to uremic pruritus (UP) have been proposed, including increasing systemic inflammation; abnormal levels of serum parathyroid hormone (PTH), serum calcium (Ca), and serum phosphorus (P); opioid receptor imbalance; and neuropathological processes [2]. However, the factors affecting degree of skin itching in patients with uremia are still unclear. Wwe used a five-dimensional itching scale [59] to evaluate skin itching across multiple dimensions in uremic patients undergoing maintenance HD. We also included general patient data such as age, sex, body mass index (BMI), place of residence, educational level, Diabetes mellitus status,and duration of HD; as well as clinical indicators such as Ca, P, serum albumin (ALB), hemoglobin (Hb), PTH, serum creatinine (sCr) ,serum urea nitrogen(BUN), urea nitrogen clearance index (KT/V), ferritin (FER) before dialysis, and hs-CRP to analyze the determinants of itching in these patients. The aims of this study are to: objectives 1. explore the prevalence of UP in maintenance HD patients and the demographic and clinical characteristics of pruritus patients.

Study design
This study is a cross-sectional study on UP in patients receiving HD. This study was approved by the Ethics Committee of Xinchang County People's Hospital, Zhejiang Province, China (Approval No.

Study setting
we included a total of 148 subjects who received HD at the Blood Purification Center of Xinchang County People's Hospital, Zhejiang Province, China from March 2019 to June 2019. each of them signed an informed consent form. Collection of data was by the authors or research personnel, and all personnel received training and guidance from the corresponding author. Data statistics and analysis were from September to December 2019.

Pparticipants and data collection
Inclusion criteria were as follows: (1) ≥1 month of HD in dialysis units and (2) age 20-90 years.
Exclusion criteria were as follows: (1) primary skin diseases (e.g., eczema, psoriasis, neurodermatitis, allergic dermatitis, and drug rash), (2) use of right-cyclic saccharin, analgesics, antibiotics, or lipidlowering drugs, (3) peripheral neuropathy, thyroid disease, leukemia, lymphoma, bile siltation, liver lesions, or pregnancy, (4) patients with hearing/communication impairments who could not complete the study, and (5) patients with infections, malignant tumors, cardiovascular disease, or tissue damage.Thesedamage. These criteria were applied to each patient. Collection of data was by the authors or research personnel and all personnel received training and guidance from the first author. Patient data included sex, age, BMI, place of residence (rural or urban), educational level (illiterate or semi-literate, primary, junior high school, high school, or university or above), and duration of maintenance HD (months), which were obtained by the investigator interviewing the patients. We assayed biochemical indicators such as Ca, P, PTH, Hb, ALB, sCr, and hs-CRP. All of the laboratory tests were performed by the hospital's central laboratory, and auto-analyzers were used to determine biochemical data. The clinical biochemical indicators are based on the data within 3 months from the survey day. If there is no data within 3 months, the patient is required to re-test. If the patient is unwilling to cooperate with the test, it is a missing value. BMI (kg/m 2 ) was calculated using the following formula: weight (kg) / height (m) 2 . Lean patients were defined as those with BMI ≦18.4 kg/m 2 , normal patients with BMI 18.5-23.9 kg/m 2 , heavy patients with BMI 24.0-27.9 kg/m 2 , and obese patients with BMI ≧8.0 kg/m 2 . According to World Health Organization (WHO) standards, elderly patients were defined as those age ≧65, and non-elderly patients as those age <65.

Determining UP
The diagnostic criteria for uremic pruritus adopted internationally are as follows: (1) uremia . We used the Five-Dimensional Itching Scale (5DIS) to assess itching based on the five dimensions of degree, duration, direction, disability, and distribution. Total score ranged from 5 (no itching) to 25 (most severe itching). Patients with scores <5 points were defined as having no itching; those with scores 6-25 points were considered itch patients. Score of 6-10 points indicated mild itching, 11-20 points indicated moderate itching, and 21-25 points indicated severe Itching. Diagnosis of uremia pruritus meets the following conditions: 1. The total score of pruritus according to 5DIS is between 6-25 points. 2. Meet the international diagnostic criteria for uremic pruritus (2)

Data source/measurement
Patients data included Sex, Age, BMI, Place of residence (rural or urban), educational level (illiterate or semi-literate, primary, junior high school, high school, or university or above), diabetes mellitus (Yes or No) and duration of maintenance HD (Months), which were obtained by the investigator interviewing the patients. We assayed biochemical indicators such as Ca, P, iPTH, Hb, ALB, sCr, Bun, FER, sp/KTV and hs-CRP. The hospital's central laboratory performed all of the laboratory tests, and auto-analyzers were used to determine biochemical data, iPTH was measured with Roche second-generation assay. The clinical biochemical indicators were are based on the data within three months from the survey day. If there is no data within three months, the patient is required to re-test. If the patient is unwilling to cooperate with the test, it is a missing value. spKT/V(single pool KT/V)=-ln( − 0.008 ) + (4 − 3.5 ) * (∆ / ). Note: K refers to Blood urea clearance rate of dialyzer(L/h), T is the dialysis time (h), and V is the distribution volume of urea (V). R is the ratio of blood urea nitrogen after dialysis to blood urea nitrogen before dialysis; t is single dialysis time in h; ∆BW is the weight change value before and after dialysis, i.e. ultrafiltration, unit L; BW is weight in kg. Blood collection requirements: blood samples before dialysis from the artery end of the vascular path, after dialysis before blood sample collection to stop ultrafiltration, reduce blood flow of 50 ml/min, wait 15 seconds after blood collection from the artery as a blood sample after dialysis.

1.Non-response bias
This study recruited 160 patients, 3 of whom were non-responders, and these non-responders may have differed from responders in some critical clinical features or exposures; however, the proportion (1.87%) was small.

2.Subject bias and researcher bias
During participation in the questionnaire of this study, some patients may be limited by their cultural level or subjective perceptions, which may lead to discrimination in the individual skin itch scale scores, thus affecting the accuracy of the assessment of the itchiness of the patient's skin. For the study participants who took part in the questionnaire, we trained two research staff, and the final results were taken as the mean of the two-pruritus skin itch scale scores.

selective bias
Of the 60 patients with itchy skin, 22 had mild skin pruritus, 30 had moderate skin pruritus, and 8 had severe skin pruritus. The proportion of patients with severe skin pruritus was small (13.33%), resulting in an uneven balance of patients between the groups.

Study size
Epidemiological data indicated that approximately 40% of patients with end-stage renal disease (ESRD) experience UP [2]. The overall prevalence of uremia and pruritus is 0.4, the tolerance is set to 0.1, taking a=0.05, the total number of hemodialysis in our blood purification center is 180 people.

Ethics
Quantitative data with a normal distribution is expressed as mean ± standard deviation; intergroup comparisons were made using one-way analysis of variance or t-test; quantitative data with a nonnormal distribution are expressed as medians, and interquartile ranges, intergroup comparisons of continuous variables were made using a non-parametric test.

Statistical methodsanalysis
We performed statistical processing using line data from SPSS software version 24.0 (IBM Corp., Armonk, New York, US).We performed statistical processing using line data from SPSS software

Descriptive data
The primary clinical characteristics of the included patients are shown in Table 1.

Comparison of patients with and without skin pruritus
Compared with uremia patients without skin pruritus, patients with UP had higher levels of PTH, Hb, and hs-CRP. No statistically significant differences were found in demographic characteristics such as sex, age, educational level, BMI, or residence; in levels of Ca, P, ALB, or sCr; or in HD duration ( Table 2).

Outcome data
Compared with uremia patients without skin pruritus, patients with UP had higher levels of IPTH, Hb, BUN and hs-CRP. No statistically significant differences were found in demographic characteristics such as Sex, Age, educational level, BMI, Diabetes mellitus status or Place of residence; in levels of Ca, P, ALB, FER, KT/V, or sCr; or in HD duration ( Table 2).

Comparison of patients with different degrees of skin itching
The composition ratio showed a difference between rural and urban patients with varying degrees of skin itching (P = 0.017). Differences in PTH and hs-CRP levels of patients with varying degrees of skin itching were statistically significant (P = 0.015 and <0.001, respectively), As shown in Table 3. Further comparison between the two groups found that compared with mild skin itching, the proportion of rural residents with moderate skin itching was significantly higher than that of urban residents (P = 0.004). Levels of hs-CRP in patients with moderate and severe skin itching were statistically significantly higher than in patients without itching (all P <0.001); the level of hs-CRP in patients with severe itching was higher than in those with mild skin itching, to a statistically significant degree (P = 0.007). There was no statistically significant difference in PTH levels in patients with severe itching (all P > 0.05), As shown in S1 Fig. The composition ratio showed a difference between rural and urban patients with varying degrees of skin itching (P = 0.017). Differences in IPTH and hs-CRP levels of patients with different degrees of skin itching were statistically significant (P = 0.009 and <0.001, respectively), As shown in Table 3. and Place of residence was associated with severity of skin itching, as shown in (Fig 2, S1 Table).  P=0.035). as shown in (Fig 3, S2 Table).

Other analyses
Levels of hs-CRP in patients with moderate and severe skin itching were statistically significantly higher than in patients without Itching (all P <0.001); the level of iPTH in patients with mild Itching was higher than in those with no skin itching, to a statistically significant degree (P <0.05). There was no statistically significant difference in Place of residence in patients with mild, moderate and severe Itching compared with no skin itching (all P > 0.05), As shown in

Key results
In this study, we found that among 148 eligible patients receiving maintenance HD, 60 had UP pruritus. The proportion of rural residents with severe skin itching was significantly higher than that of urban residents, and there were statistically significant differences in levels of PTH and hs-CRP in patients with different degrees of skin itching. Single-factor logistic-regression analysis discovered a link between hs-CRP levels and all three degrees of itching. Moreover, Hemoglobin is associated with moderate and severe Itching, and iPTH is related to severe Itching. Multi-factor logistic regression analysis also indicated that patients with higher hs-CRP levels had higher risks of mild, moderate, and severe skin itching. Interestingly, the multivariate analysis also showed that rural residents have a higher risk of moderate skin itching than urban residents. Therefore, hs-CRP levels were associated with the intensity of skin itching in patients receiving maintenance HD, and higher hs-CRP levels were associated with severe itching.

Limitations
Our study had some limitations. Firstly, it was a single-centre, cross-sectional study that did not show a causal relationship between severity of skin itching in UP and hs-CRP, so further validation is needed from multicentre, large-sample, prospective cohort studies. Secondly, this study did not include some of the influences known in the literature such as normalized protein catabolic rate (nPCR), diet, insulin resistance, interleukins 6 and 2, and κ opioid receptor distribution. Thirdly, the number of patients with severe pruritus is small (n = 8), so readers may not understand a meaningful association with severe pruritus. Finally, the sample size of this study was small and uneven between groups.

Interpretation
This study showed that median hs-CRP level gradually increased along with degree of skin itching in patients with uremic pruritus (no itching, 0.90; mild skin itching, 1.08; moderate skin itching, 2.15; severe skin itching, 3.93). Furthermore, hs-CRP levels in patients with moderate and severe skin itching were significantly higher than in those without itching. These findings suggested that hs-CRP levels might be associated with severity of skin itching. Jiang et al.
[711] compared the clinical effects of high-throughput hemodialysis (HTH) and hemodialysis filtration (HDF) treat patients with end-stage nephropathy and explored possible potential mechanisms. Their results showed that levels of Creactive protein (CRP) and leukocyte interleukin-6 (IL-6) in patients receiving HTH significantly improved, compared with those in patients who received HDF. The study also found that HTH had an exact therapeutic effect in UP that was related to inhibition of microinflammation in dialysis patients.
Another study also showed that HD patients with UP had significantly higher serum CRP and IL-6 levels compared to HD patients without UP, UP seems to be associated with an up-regulation of micro- We analyzed the factors affecting degree of skin itching in UP patients and then corrected for patients' levels of Hb and PTH, Place of residence, P. Multi-factor logistic-regression analysis found that hs-CRP levels were associated with severity of skin itching and were a risk factor. Compared with patients without itching, the OR value for mild skin itching was 1.74020, that for moderate skin itching was 2.83884, and that for severe skin itching was 9.4408.929, indicating that degree of skin itching increased along with hs-CRP levels. A previous multivariable logistic-regression analysis confirmed that older age and higher CRP levels are associated with severe itching [3]., Still,but in our study, neither single-factor nor multi-factor logistic-regression analysis showed any differences in skin itching between elderly and non-elderly patients. In our study, the OR value for hs-CRP of patients with severe skin itching was significantly higher than that of patients without itching, suggesting that higher hs-CRP levels were closely associated with severe skin itching, which was consistent with the results of an earlier study. Pakfetrat et al. [1114] assessed the effect of sertraline on UP. patients were randomly divided into two groups, the trial group receiving sertraline and the control group taking a placebo, and their severity of itching was measured by two scoring systems (visual-simulation score and DUO scoring system [1215]). The researchers found that differences in skin itching intensity between the two scoring systems and between the two groups were directly related to CRP level, and they believed that sertraline reduced skin itching, perhaps due to its effect of reducing the number of inflammatory cytokines. Tseng et al.
[1316]studying a possible association between vegetarians and UP, found that levels of inflammatory factors such as leukocyte IL-2 and hs-CRP, visual-simulation score (Analog Score and VAS), and itching score were also lower in vegetarian patients than in nonvegetarian ones; this demonstrated that a vegetarian diet might be associated with amelioration of uremic-pruritus severity in hemodialysis patients.
In our study, the composition ratio showed a significant difference between rural and urban patients with different degrees of skin itching. Compared with patients without itching, the proportion of rural patients with moderate and severe skin itching was relatively high, however, no statistically significant difference was achieved. Although the single factor analysis did not indicate that the place of residence was related to the intensity of the patient's skin itching, the multi-factor analysis demonstrated that rural residents had a higher risk of moderate skin itching than urban residents. SomeOther studies suggested that UP is related to Ca, P, ALB, and uremic toxins [11,2014,23].
Nevertheless, contradictory data on the impact of Ca and P have been reported [21]. In this study, no statistically significant differences were found in Ca, P, ALB, and creatinine when comparing groups of patients with different degrees of skin pruritus. There may indeed be a lack of test power, which is considered to be related to the small sample size of this study and the unbalanced sample size among the groups. However, single-factor logistic regression analysis also did not find an association between these factors and the degree of skin pruritus. Therefore, these factors were ultimately not included in the multifactor logistic regression analysis. In fact, despite various substances have been discussed as potential pruritogens in chronic renal diseases, a causal relationship could never be established [22].We did not find an association between sCR, BUN, ALB and uremic pruritus because there were no statistical differences in comparisons between groups of patients with different degrees of skin pruritus. Besides, a single factor analysis did not reveal an association between these factors and the degree of pruritus. In fact, despite various substances have been discussed as potential pruritogens in chronic renal diseases, a causal relationship could never be established [22]. Our At present, only a few studies have explored the factors affecting skin itching intensity in these patients. Wieczorek et al. [23] documented Intensity of skin itching have a significant negative correlation with expression of κ opioid receptors in patients with HD. Also, hs-CRP levels, male sex, HD duration, insulin resistance, insufficient dialysis adequacy, and hyperphosphatemia are positively correlated with UP intensity [24], increasing the blood flow of the HD machine can reduce the intensity and frequency of skin itching [25]. However, the above preliminary research used visual-analogue scoring (VAS) to evaluate the degree of skin itching. In this study, we used a five-dimensional itching scale to evaluate skin itching across multiple dimensions in uremic patients undergoing maintenance HD. Although VAS is sufficient to assess the severity of symptoms, it does not take into account other aspects of pruritus, such as the relative impact of pruritus on quality of life, changes in pruritus over time, and the location of pruritus. The 5-D score was strongly correlated with the numerical rating scale(NRS)and VAS [5,26], which is a questionnaire with acceptable validity, reliability and sensitivity to change to evaluate pruritus in Thai and Arabic patients [26,27]. The 5-dimensional Pruritus Scale is recommended to assess the severity of pruritus because it is more accurate than VAS and more sensitive to the multidimensional nature of pruritus [28].

Generalizability
In conclusion, our study suggested that the intensity of skin itching in patients with UP was associated with hs-CRP and that higher hs-CRP levels were associated with severe skin itching. Thus, this study demonstrates that controlling inflammation can relieve itchy skin in patients with uremia, and is particularly clinically relevant in patients with severe itchy skin. Besides, the environment may be related to the degree of itchy skin in uremia patients. Overall, our study suggested that the intensity of skin itching in patients with UP was associated with hs-CRP and that higher hs-CRP levels were associated with severe skin itching. The limitation of this study is that it was a single-center, crosssectional study that did not show a causal relationship between severity of skin itching in UP and hs-CRP, so further validation is needed from multi-center, large-sample, prospective cohort studies. intergroup comparison of the composition ratio of rural to urban residents. ****P < 0.0001, **P < 0.01. Error line represents minimum and maximum values. S1 Table. Single-factor logistic-regression analysis of degree of skin itching in patients with uremic pruritus. S2 Table. Multiple logistic-regression analysis of factors influencing skin itching in patients with uremic pruritus, using hs-CRP as a variable.

Response to the PLOS ONE's editor
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Response to Reviewer
Thank the reviewers for these lovely comments concerning our manuscript entitled "Determinants of the intensity of uremic pruritus in patients receiving maintenance hemodialysis: A cross-sectional study". These comments are all valuable and very helpful for revising and improving our paper, as well as the essential guiding significance to our researches. We have studied comments carefully and have made corrections which we hope to meet with approval. The response to the reviewer's comments are as follows:

Reviewer #1
The authors performed a cross-sectional analysis of 148 hemodialysis patients to identify factors associated with uremic pruritus. They found that higher levels of PTH, hemoglobin, and hs-CRP, as well as the rural residence of patients were associated with worse pruritus.
There are major concerns with the lack of dialysis and other pertinent laboratory data.
Overall, the results do not add new data to the existing literature.
1. There is no data provided for dialysis adequacy, which has been shown to be associated with pruritus in prior studies. Likewise, BUN levels, nPCR, ferritin levels, and diabetic status should be included in the analysis.

Response:
The revision adds these factors, such as urea nitrogen, ferritin level, diabetes, and single pool KT/V(spKT/V), to the list of factors related to hemodialysis. Normalized protein catabolic rate (nPCR) is a nutritional index for hemodialysis patients. Its measurement requires multiple blood collections, which can be challenging to obtain, especially with the inclusion of serum albumin, which is a nutritional index. The spKT/V index reflects the adequacy of single hemodialysis.
2. The authors used the Five-Dimensional Itching Scale (5DIS) to assess the degree of pruritus in patients, noting that this scale would "produce more objective and comprehensive results" compared to the visual-analogue scoring scale. The authors should provide data to justify this statement.

Response:
In this study, we used a five-dimensional itching scale to evaluate skin itching across multiple dimensions in uremic patients undergoing maintenance HD. Although VAS is sufficient to assess the severity of symptoms, it does not take into account other aspects of pruritus, such as the relative impact of pruritus on quality of life, changes in pruritus over time, and the location of pruritus. The 5-D score was strongly correlated with the numerical rating scale(NRS)and VAS [5,26], which is a questionnaire with acceptable validity, reliability and sensitivity to change 4.How were biochemical parameters such as serum calcium, phosphorus, creatinine, etc assayed? The detailed methods need not be described but should be referenced.

Response:
We assayed biochemical indicators such as Ca, P, iPTH, Hb, ALB, sCr, Bun, FER, sp/KTV and hs-CRP. The hospital's central laboratory performed all of the laboratory tests, and auto-analyzers were used to determine biochemical data, iPTH was measured with Roche second-generation assay. The clinical biochemical indicators are based on the data within three months from the survey day. If there is no data within three months, the patient is required to re-test. If the patient is unwilling to cooperate with the test, it is a missing value. spKT/V (single pool KT/V) =-ln( − 0.008 ) + (4 − 3.5 ) * (∆ / ). Note: K refers to Blood urea clearance rate of dialyzer(L/h), t is the dialysis time (h), and V is the distribution volume of urea (V). R is the ratio of blood urea nitrogen after dialysis to blood urea nitrogen before dialysis; t is single dialysis time in h; ∆BW is the weight change value before and after dialysis, i.e. ultrafiltration, unit L; BW is weight in kg. Blood collection requirements: blood samples before dialysis from the artery end of the vascular path, after dialysis before blood sample collection to stop ultrafiltration, reduce blood flow of 50 ml/min, wait 15 seconds after blood collection from the artery as a blood sample after dialysis.
5.The study reports no significant association of uremic pruritus with factors such as calcium, phosphorus, creatinine and albumin. However, the sample size calculation was made to estimate the prevalence of uremic pruritus. Could the study have been under powered for the study of these factors?

Response:
Some studies suggested that UP is related to Ca, P, ALB, and uremic toxins [11,20].
Nevertheless, contradictory data on the impact of Ca and P have been reported [21]. In this study, no statistically significant differences were found in Ca, P, ALB, and creatinine when comparing groups of patients with different degrees of skin pruritus. There may indeed be a lack of test power(Since the data for these factors are non-normally distributed, the exact distribution is unknown. Based on statistical theory, it is not possible to calculate the true test power with certainty.), which is considered to be related to the small sample size of this study and the unbalanced sample size among the groups. However, single-factor logistic regression analysis also did not find an association between these factors and the degree of skin pruritus. Therefore, these factors were ultimately not included in the multifactor logistic regression analysis. In fact, despite various substances have been discussed as potential pruritogens in chronic renal diseases, a causal relationship could never be established [22].
This section is detailed on page 15, lines 14-22.