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Detection of renal brush border membrane enzymes for evaluation of renal injury in neonatal scleredema.

Byline: Qing Ren Yongjun Zhang Jinying Yang Lixia Wei Lili Zhao and Qiaozhi Yang

ABSTRACT

Objective: To evaluate renal brush border membrane enzymes in urine as an indicator for renal injury in neonatal scleredema(NS).

Methods: Sixty nine NS patients in our hospital were enrolled and divided into mild group and moderate/ severe group. Patients were further randomly divided into therapy and control subgroups for 7 days ligustrazine administration. Urine samples were collected to detect renal brush border membrane enzymes (RBBME) by ELISA and AY2-microglobulin (AY -MG) by radioimmunoassay (RIA). The results were compared with those of 30 normal neonates. Data were statistically analyzed using SPSS13.0 software.

Results: Both RBBME and AY -MG were found to be higher in urine in NS patients than normal controls (P less than 0.01). Level of RBBME increased with the severity of NS (P less than 0.05) while urinary AY -MG did not (P greater than 0.05). After being treated with ligustrazine a medicine for renal function recovery both RBBME and AY -MG were similarly significantly decreased comparing to untreated groups (P less than 0.05). 79.7% of NS patients showed abnormal RBBME while only 52.2% had an abnormal urinary AY -MG (2=11.65 P less than 0.01).

Conclusion: RBBME was more sensitive than AY -MG in reflecting the renal injury in NS. Examination of

RBBME effectively reflected the recovery of renal injury after treatment with ligustrazine.

KEY WORDS: AY2-microglobulin Ligustrazine Neonatal scleredema Renal brush border membrane enzyme Renal injury.

INTRODUCTION

Neonatal Scleredema (NS) is also called neonatal cold injury syndrome which is characterized by diffuse hardening of the subcutaneous tissue low body temperature and edema with minimal inflammation.12 NS often affect preterm neonates in the first week of life.3 Serious NS may cause multiple organ dysfunctions. One of the complications of NS is impaired renal function; mainly refer to proximal tubule lesions with clinical symptoms including oliguria anuria proteinuria acute tubular necrosis and even kidney failure. Diagnosis on renal dysfunction was traditionally based on the elevated blood urea nitrogen (BUN) and creatinine (Cr) as well as decreased urine volume. However it is difficult to achieve signs for early neonatal renal damage with these tests.

Clinically it has proven that the proximal tubule of the kidney is especially susceptible to ischemic inflammatory or toxic events.

The enzymes that bound to the brush border of microvillous membrane including alkaline phosphatase (ALP) leucineaminopeptidase (LAP) -glutamyltransferase (-GT) are collectively called renal brush boarder membrane enzyme (RBBME). The shedding of the tubular epithelial membrane (and consequently the RBBME) might occur before the histopathological damage and this enzymuria could be a useful early marker of renal damage. In the present study we evaluated clinical significance of the RBBME assay in renal injury diagnosis in NS patients. We used AY2-microglobulin(AY2-MG) as a parallel proof for the renal injury and ligustrazine treatment as a secondary proof to observe RBBME changes while renal injury was treated.

METHODS

Patients: The study was approved by the Ethic Committee of Liaocheng People's Hospital and the written informed consent was obtained from each patient's parents. Patients were eligible for enrollment if NS was diagnosed and urine test showed higher RBBME above the normal range obtained from normal control (normal new born infants was enrolled as control). NS was diagnosed with typical skin harden swelling and lower body temperature. Severity of the patients was classified as shown in Table-I. Patients who needed treatment with dopamine phentolamine anisodamine or other vasoactive drugs were excluded from the study.

Sample collection: Urine and blood samples from normal control group were obtained for one time at the clinic. Blood and urine samples from NS patients were collected at the time of hospitalization and before and after ligustrazine treatment.

Detection of RBBME AY2-MG BUN and creatinine: Urine samples for RBBME detection were treated with preservative solution at 9:1 ratio and tested immediately or stored at -80C freezer. RBBME were detected using the detection kit provided by Dr. Jingti Deng of Shandong University School of Medicine with ELISA method described earlier.8

Tests results were considered abnormal when the value was equal or higher than the mean + 2SD

Type###Body temperature###Involved area

###T Anus TAxil TAnus (% Color)

I (mild)###35C###positive###less than 20 pale

II (moderate) less than 35C###0 or positive 20-50 dark red

III(severe)###less than 30C###negative###greater than 50 cyanotic

(standard deviation) of the normal control group. AY2-MG was measured by radioimmunoassay that was routinely operated in clinical lab. Blood urea nitrogen (BUN) and creatinine (Cr) test results were also obtained from clinical labs.

Treatment: Conventional treatments were applied for all patients to ensure proper management including restoration of body temperature energy supply and fluid infusion correction of acidosis and electrolyte imbalance symptomatic treatment for organ malfunctioning and if necessary oxygen or antibiotics therapy. Patients were further randomly separated into two groups based on their enrollment number (odd number was ligustrazine group and even number was un-treated group). Ligustrazine(Shanghai Modern Hasen Pharmaceutical Co China) was administered at 6mg/kg in 30ml 5% glucose solution i.v. infusion once daily for 5 consecutive days.

Statistical analysis: Data were statistically analyzed using SPSS13.0 software and expressed in meanSD. Median was used to represent data of none normal distribution. Data comparison between groups was performed with student's t test. Pearson correlation or Chi square test was analyzed and two-tailed probability at 0.05 was taken as significant level. The sample size of 60 patients for the study was estimated by using a two- sided t-test at the 5% significance level (a= 0.05) and 80% power (b= 0.2). Adjusting by 10% to account for ineligibility resulted in a final targeted sample size of 66 patients.

RESULTS

General characteristics of patients: Sixty nine infants with NS were enrolled from June 2009 - March 2013 in our hospital including 40 males and 29 females with an age of 8 hour - 28 day at the time of enrollment (2.81.2 d) birth weight ranging from 1.21 - 3.99 kg (2.68 0.8 kg) gestational age from 32 to 43 wks (37.8 2.6 wks). Based on the grading standards published in Practical Neonatology (Version 4)939 cases were diagnosed as mild NS and 30 as moderate to severe (Mod/Sev)NS.

Another 30 normal infants were enrolled as control

Table-II: Demographic characteristics of patients.

Group No.###Age###Gestational Weight

(M/F)###(median###Term###(meanSD

###day) (meanSD wks)###kg)

Mild###39 (22/17)###3.6###38.12.5###2.910.72

Mod/Sev30 (18/12)###2.4###35.82.7###2.790.69

Control 30 (17/13)###3.8###37.52.4###2.530.83

Table-III: Comparison of RBBME and 2-MG between Mild and Mod/Sev groups.

###Group###Number###RBBME (U/L)###2-MG (mg/L)###BUN(mmol/L)###Cr(mol/L)

###Mild###39###38.576.70 a

###4.451.18 a

###5.401.80###72.4819.03

###Mod/Sev###30###42.067.59ab###4.911.49 a c###6.301.78###80.7719.13

###Control###30###23.195.62###2.490.77###5.051.14###69.0011.97

including 17 males and 13 females 33 to 42 weeks of gestational age birth weight 1.51 ~ 4.00Kg. Demographic data for groups of the study are listed in Table-II. No significant differences were found in sex age and birth weight among these groups (Table-II).

Correlation of AY2-MG and RBBME with the severity of NS: All test values from enrolled patients were showed in Table-III. Both RBBME andAY2-MG values were significantly higher in NS patients than the control group (Pless than 0.01) while both BUN and Cr tests showed normal results. RBBME level in NS group was correlated with the severity of the disease. Significantly higher RBBME was found in Mod/Sev group than that of the mild group (pless than 0.05). And by linear correlation analysis RBBME and AY2-MG had a significant positive correlation (r = 0.560 p less than 0.01). ROC curves of both RBBME and AY2-MG were generated as shown in Fig.1. The area under the curve (AUC) for RBBME and AY2-MG were 0.939 and 0.834 respectively indicating higher diagnostic accuracy of RBBME for NS kidney damage. Youden index10 was calculated to determine the cutting points for RBBME to be 36.75U/L and AY2- MG 3.85 mg/L at which that RBBME exhibited a sensitivity of 88.2% specificity of 81.5%

while the corresponding sensitivity of AY2-MG was 82.4% specificity 80.0% for the diagnosis of renal injury of NS.

RBBME as an indicator for the efficacy of ligustrazine treatment: In order to evaluate the capability of RBBME test for reflection of renal function recovery NS patients in each level were

Table-IV: Comparison of RBBME and AY2-MG as indicators for efficacy of ligustrazine treatment.

Group No.###RBBME###2-MG

###(U/L)###(mg/L)

Mild###Treated###24###26.866.00###2.900.90

###Untreated###15###31.115.72###3.470.82

Mod/Sev###Treated###11###29.806.58###3.190.69

###Untreated###19###35.206.33###3.700.66

further divided into two groups randomly and applied ligustrazine to one group. The other group received no treatment. Ligustrazine is a Chinese herb extracts that has known function to restore normal renal function.1112 Ligustrazine was administered at 6mg/kg in 30ml 5% glucose solution via iv infusion once daily for 5 consecutive days. Both RBBME and AY2-MG decreased significantly in both mild and mod/sev groups after application of ligustrazine(pless than 0.01) as shown in Table-IV.

DISCUSSION

NS is a common disease in the northern territory of China while not many reports are seen from western countries. Its clinical manifestation is very similar to the sclerema neonatorum whereas basic treatment method is about the same. Organ dysfunction including renal dysfunction is a severe complication in NS which was found in 20% of patients (Our unpublished observation). BUN and Cr are routine clinical tests for renal function. However these indicators were not able to reflect early renal damage which was further proven in this study. AY2-MG is a widely used indicator in clinical detection for early renal tubular dysfunction. AY2- MG is filtered through the glomerulus and almost completelyre-absorbed and lysed by the proximal tubular cells.13 Impairment of AY2-MG tubular uptake results in a raised intact AY2-MG urinary excretion. Therefore urinary AY2-MG is a sensitive indicator reflecting of renal tubular dysfunction.

However multiple factors can influence the result including AY2-MG production filtration function of glomerulus and presence of proteinuria.14-18RBBME can be a more direct indicator of the tubular function that is less affected by other factors.19Shedding of RBBME reflects the acute tubular injury that can be detected before any other symptoms has been developed.20Assay of RBBME has been used in evaluation of drug-induced nephrotoxicity21post transplantation kidney function surveillance22 etc.

Ligustrazine a purified and chemical identified component of a Chinese herbal remedy has been used clinically widely in treating cardiovascular disease and improve microcirculation. It has strong effects on scavenging cytotoxic oxygen free radicals and promoting blood flow. It also has anti- platelet aggregation and radical scavenging effect.23

Ligustrazine has shown protective effect on early renal injury induced by various factors24-26 and is able to improve microcirculation reduce glomerular lipid peroxidation injury delay glomerulosclerosis process and regulate arachidonic acid metabolism etc.27 Our previous study has demonstrated that ligustrazine could reduce renal dysfunction associated with attenuating lipid peroxidation (LPO) apoptosis and ICAM-1 expression.28 In this study ligustrazine was administered to patients with low dosages for two purposes. One was as a treatment measurement for renal injury and second was to further verify the RBBME assay as an early marker of renal injury. A reverse of RBBME level in patients after being treated with ligustrazine would further indicate the effectiveness of the measurement. Both RBBME and AY2-MG were found to be significantly declined after application of ligustrazine compared with the untreated group

suggesting that both RBBME and AY2-MG were effective indicators for renal function recovery. Application of ligustrazine in this study couldn't prove its direct effect on renal damage but still justified the clinical the use of the medicine for NS patients.

In this study we found both RBBME and AY2- MG in the mild group were significantly higher than control group (pless than 0.01). With the increase of severity of NS the RBBME was found to be elevated significantly (pless than 0.05) whereas AY2-MG only showed minor increase which had no statistical significance (pgreater than 0.05) suggesting that RBBME was a better indicator representing for the severity of NS than AY2-MG. By analysis in ROC curve RBBME exhibited to be a better marker with higher sensitivity for renal damage in NS than AY2-MG.

Detection of RBBME has been evolved greatly to the current methodology. Deng et al. has developed a reliable detection methodology using specific antibodies against RBBME. The measurement is rapid reproducible highly specific sensitive and can simultaneously measuring a large number of specimens.29

Theologically the shedding of RBBME from brush border of microvillous membrane would be an early sign of renal function defect. We have found that both RBBME and AY2-MG exhibited positive signs for renal damage while BUN and Cr were normal. This suggests that RBBME can be used as early detection of renal damage in NS patients. Since the value of RBBME has a positive correlation with the severity of NS we propose that RBBME may be a more effective indicator for renal damage than AY2-MG in NS patients.

In summary detection of urine RBBME was useful indicator for renal dysfunction as well as treatment efficacy. With the improvement of the methodology RBBME assay is likely to replace AY2- MG for early detection and better accuracy.

ACKNOWLEDGEMENT

This study was sponsored by institutional funding of Liaocheng People's Hospital 2009.

Conflict of Interests: The authors declare that they have no conflict of interest.

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