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

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 β2-microglobulin (β2-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 β2-MG were found to be higher in urine in NS patients than normal controls (P < 0.01). Level of RBBME increased with the severity of NS (P <0.05), while urinary β2-MG did not (P >0.05). After being treated with ligustrazine, a medicine for renal function recovery, both RBBME and β2-MG were similarly significantly decreased comparing to untreated groups (P < 0.05). 79.7% of NS patients showed abnormal RBBME while only 52.2% had an abnormal urinary β2-MG (χ2=11.65，P < 0.01). Conclusion: RBBME was more sensitive than β2-MG in reflecting the renal injury in NS. Examination of RBBME effectively reflected the recovery of renal injury after treatment with ligustrazine.


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. 1,2 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. [4][5][6][7] The enzymes that bound to the brush border of microvillous membrane, including alkaline phosphatase (ALP), leucineaminopeptidase (LAP), γ-glutamyltransferase (γ-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 β2-microglobulin(β 2 -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.

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, β2-MG, BUN and creatinine: Urine samples for RBBME detection were treated with preservative solution at 9:1 ratio and tested immediately or stored at -80°C 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 (standard deviation) of the normal control group. β2-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 mean±SD. 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 twosided 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.    Table-II. No significant differences were found in sex, age, and birth weight among these groups ( Table-II).

General characteristics of patients:
Correlation of β2-MG and RBBME with the severity of NS: All test values from enrolled patients were showed in Table-III. Both RBBME andβ2-MG values were significantly higher in NS patients than the control group (P<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 (p<0.05). And by linear correlation analysis, RBBME and β2-MG had a significant positive correlation (r = 0.560, p <0.01). ROC curves of both RBBME and β2-MG were generated as shown in Fig.1. The area under the curve (AUC) for RBBME and β2-MG were 0.939 and 0.834 respectively, indicating higher diagnostic accuracy of RBBME for NS kidney damage. Youden index 10 was calculated to determine the cutting points for RBBME to be 36.75U/L and β2-MG 3.85 mg/L, at which that RBBME exhibited a sensitivity of 88.2%, specificity of 81.5%, while the corresponding sensitivity of β2-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 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. 11,12 Ligustrazine was administered at 6mg/kg in 30ml 5% glucose solution via iv infusion once daily for 5 consecutive days. Both RBBME and β 2 -MG decreased significantly in both mild and mod/sev groups after application of ligustrazine(p<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 A novel marker for renal injury in NS   early renal damage, which was further proven in this study. β2-MG is a widely used indicator in clinical detection for early renal tubular dysfunction. β2-MG is filtered through the glomerulus and almost completelyre-absorbed and lysed by the proximal tubular cells. 13 Impairment of β2-MG tubular uptake results in a raised intact β2-MG urinary excretion. Therefore, urinary β2-MG is a sensitive indicator reflecting of renal tubular dysfunction. However, multiple factors can influence the result, including β2-MG production, filtration function of glomerulus, and presence of proteinuria. 14-18 RBBME can be a more direct indicator of the tubular function that is less affected by other factors. 19 Shedding of RBBME reflects the acute tubular injury that can be detected before any other symptoms has been developed. 20 Assay of RBBME has been used in evaluation of drug-induced nephrotoxicity, 21 post transplantation kidney function surveillance, 22 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 antiplatelet aggregation and radical scavenging effect. 23 Ligustrazine has shown protective effect on early renal injury induced by various factors, [24][25][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 β2-MG were found to be significantly declined after application of ligustrazine compared with the untreated group, suggesting that both RBBME and β2-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 β 2 -MG in the mild group were significantly higher than control group (p<0.01). With the increase of severity of NS, the RBBME was found to be elevated significantly (p<0.05), whereas β 2 -MG only showed minor increase which had no statistical significance (p>0.05), suggesting that RBBME was a better indicator representing for the severity of NS than β 2 -MG. By analysis in ROC curve, RBBME exhibited to be a better marker with higher sensitivity for renal damage in NS than β 2 -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 β 2 -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 β 2 -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 β 2 -MG for early detection and better accuracy.

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