Potential Value of Datura stramonium Agglutinin-recognized Glycopatterns in Urinary Protein on Differential Diagnosis of Diabetic Nephropathy and Nondiabetic Renal Disease

Background： Diabetic nephropathy （DN） is the most common and serious microvascular complication of diabetes. To date, the gold standard for identifying DN and nondiabetic renal disease （NDRD） is a renal biopsy; however, there is currently no reliable diagnostic marker to identify DN and NDRD in a noninvasive manner. This study aimed to investigate the different glycopatterns in urine specimens of DN patients and NDRD patients for a differential diagnosis. Methods： In total, 19 DN patients and 18 NDRD patients who underwent renal biopsies between March 2015 and March 2016 at the Chinese People＇s Liberation Army General Hospital were enrolled in this study. A lectin microarray was used to investigate the glycopatterns in the urinary protein of the 37 patients. Ratio analysis and one-way analysis of variance were used to screen altered glycopatterns. Then, the altered glycopatterns between the DN and NDRD groups were verified by a urinary protein microarray among another 32 patients （15 with DN and 17 with NDRD）, and receiver operating characteristic （ROC） curve analysis was used to determine the diagnostic value of the altered glycopatterns in differentiating DN and NDRD. Finally, lectin blotting was used to evaluate the altered glycosylation in protein level. Results： The result of lectin microarrays revealed that the relative abundance of the （13-1,4）-linked N-acetyI-D-glucosamine （GlcNAc） recognized by lectin Datura stramonium agglutinin （DSA） was significantly higher in urinary protein in DN patients than that in NDRD patients （fold change 〉1.50, P 〈 0.001）. Subsequently, the results of urinary protein microarrays were consistent with lectin microarrays （P 〈 0.05）. Furthermore, the ROC curve showed that glycopatterns could effectively distinguish DN from NDRD patients （area under the ROC curve = 0.94, P 〈 0.001）. DSA lectin blotting showed that glycoproteins, with a molecular weight of approximately 50,000, demonstrated a difference in urine samples between DN patients and NDRD patients. Conclusions： The relative abundance of （13-1,4）-linked GIcNAc recognized by/ectin DSA and urinary glycoprotein with a molecular weight of approximately 50,000 are significantly different between DN and NDRD patients, indicating that the glycopatterns could be used as potential biomarkers for a differential diagnosis.

Social dominance-related major urinary proteins and the regulatory mechanism in mice

Major urinary proteins(MUPs)have been proven to be non-volatile male pheromones in mice.Here,we aimed to elucidate the relationship between MUPs and dominance hierarchy,and the underlying molecular mechanisms.Dominance–submission relationship was established by chronic dyadic encountering.We found that at the urinary protein level and hepatic mRNA level,the expression of major MUPs,including Mup20,was enhanced in dominant males compared with subordinate males,indicating that MUPs might signal the social status of male mice.Meanwhile,the mRNA level of hepatic corticotropin releasing hormone receptor 2(CRHR2)was higher in subordinate male mice than in dominant male mice.Castration also enhanced the expression of CRHR2,but suppressed that of MUPs.CRHR2 agonist treatment reduced the expression of MUPs in liver.However,male social status failed to exert significant influence on serum testosterone and corticosterone as well as the mRNA expression of their receptors.These findings reveal that some MUPs,especially Mup20,might constitute potential dominance pheromones and could be downregulated by hepatic CRHR2,which is possibly independent of androgen or corticosterone systems.

Collection and preservation of urinary proteins, using a fluff pulp diaper

Change is the most fundamental property of a biomarker. In contrast to the blood, which is under homeostatic controls, urine reflects changes in the body earlier and is more sensitive, thus making it a better biomarker source. Moreover, drawing blood from infants and toddlers is difficult and not tolerated well. For patients limited by language, communicating their chief complaint is difficult. Thus, monitoring biomarkers in urine can provide valuable clues for the diagnosis of diseases, especially pediatric diseases. Collecting urine from young children and some adult patients is more challenging than collecting it from healthy adults.Here, we propose a method that uses a fluff pulp diaper to collect urine. Urinary proteins are then eluted and adsorbed onto a piece of nitrocellulose membrane, which can be dried and stored in a vacuum bag. SDS-PAGE and LC-MS/MS analysis indicated that this method is reproducible, and similar proteins were identified as those obtained by an acetone precipitation method. With this simple and economical method, it is possible to collect and preserve urine samples from infants, toddlers, and patients with special needs, even for large-scale biomarker studies.

Association of NFkB1 Gene Polymorphism with Inflammatory Markers in Patients of Type 2 Diabetes Mellitus with or without Renal Involvement in Eastern India

Aims: To evaluate the association of Nuclear factor kappa B1(NFkB1) gene polymorphism with inflammatory markers Urinary Monocyte Chemoattractant Protein 1 (UMCP1) and Tumor Necrosis Factor alfa (TNF alfa) in Patients of diabetes mellitus with or without renal involvement in Eastern India. Material and Methods: Consecutive Patients of Type 2 Diabetes Mellitus (DM) with or without microalbuminuria attending SCB MEDICAL COLLEGE and HOSPITAL Medical OPDs in between September 2018 to September 2019 were recruited in this study. Patients were subjected to blood and urine investigations. DNA extraction and Restriction fragment Length Polymorphism (RFLP) was done in Department of Biochemistry. Controls were unrelated healthy attendants with no history of Diabetes Mellitus, HTN, Chronic Kidney Disease (CKD). Results: Mean Systolic BP, Fasting Blood Glucose, Post Prandial Blood Glucose, HBA1c, Total Cholesterol were significantly higher in diabetes mellitus and diabetic nephropathy groups than control group. Estimated Glomerular Filtration Rate was significantly lower in diabetic nephropathy (p value < 0.001). UMCP1, Urinary Albumin Creatinine Ratio, TNF alfa were higher in diabetes mellitus and nephropathy with p value (<0.001, 0.006 < 0.001) respectively. In between DM and Diabetic Nephropathy groups nfkb1 gene expression, umcp1 and tnf alfa levels were significantly increased in Diabetic nephropathy with p value 0.019, <0.01, 0.001 respectively. Insertion/insertion NFkB1 gene polymorphisms were more in diabetic nephropathy group and were positively correlated with inflammatory markers UMCP1 (r = 0.517, p < 0.01) and TNF alfa (r = 0.172, p = 0.19). Conclusion: insertion/insertion NFkB1 gene polymorphism increases the risk of nephropathy by 2.52 times (OR = 2.52, 95% CI: 0.04 - 0.63, p value = 0.019) in diabetes patients in eastern India.

Male pheromones and their reception by females are co-adapted to affect mating success in two subspecies of brown rats

Pheromonal communication plays a key role in the sociosexual behavior of rodents.The coadaptation between pheromones and chemosensory systems has been well illustrated in insects but poorly investigated in rodents and other mammals.We aimed to investigate whether coadaptation between male pheromones and female reception might have occurred in brown rats Rattus norve-gicus.We recently reported that major urinary protein(MUP)pheromones are associated with male mating success in a brown rat subspecies,R.n.humiliatus(Rnh).Here,we discovered that MUPs were less polymorphic and occurred at much lower concentrations in males of a parapatric subspecies,R.n.caraco(Rnc),than in Rnh males,and found no association between pheromones and paternity success.Moreover,the observation of Rnc males that experienced chronic dyadic encounters and established dominance-submission relationships revealed that the dominant males achieved greater mating success than the subordinate males,but their MUP levels did not differ by social status.These findings suggest that male mating success in Rnc rats is related to social rank rather than to pheromone levels and that low concentration of MUPs might not be a reliable signal for mate choice in Rnc rats,which is different from the findings obtained in Rnh rats.In addition,compared with Rnh females,Rnc females exhibited reduced expression of pheromone receptor genes,and a lower number of vomeronasal receptor neurons were activated by MUP pheromones,which imply that the female chemosensory reception of pheromones might be structurally and functionally coadapted with male pheromone signals in brown rats.