Biliary atresia is a progressive infantile cholangiopathy of complex pathogenesis. Although early diagnosis and surgery are the best predictors of treatment response, current diagnostic approaches are imprecise and time consuming. We used large-scale, quantitative serum proteomics at the time of diagnosis of biliary atresia and other cholestatic syndromes (serving as disease controls) to identify biomarkers of disease. In a discovery cohort of 70 subjects, the lead biomarker was matrix metalloproteinase-7 (MMP-7), which retained high distinguishing features for biliary atresia in two validation cohorts. Notably, the diagnostic performance reached 95% when MMP-7 was combined with gamma-glutamyltranspeptidase (GGT), a marker of cholestasis. Using human tissue and experimental model of biliary atresia, we found that MMP-7 is primarily expressed by cholangiocytes, is released upon epithelial injury, and promotes the experimental disease phenotype. Thus, we propose that serum MMP-7 (alone or in combination with GGT) is a diagnostic biomarker for biliary atresia and may serve as a therapeutic target.
Angiotensin converting enzyme 2 (ACE2) and neprilysin (NEP) are metalloproteases that are highly expressed in the renal proximal tubules. ACE2 and NEP generate renoprotective angiotensin (1-7) from angiotensin II and angiotensin I, respectively, and therefore could have a major role in chronic kidney disease (CKD). Recent data demonstrated increased urinary ACE2 in patients with diabetes with CKD and kidney transplants. We tested the hypothesis that urinary ACE2, NEP, and a disintegrin and metalloproteinase 17 (ADAM17) are increased and could be risk predictors of CKD in patients with diabetes. ACE2, NEP, and ADAM17 were investigated in 20 nondiabetics (ND) and 40 patients with diabetes with normoalbuminuria (Dnormo), microalbuminuria (Dmicro), and macroalbuminuria (Dmacro) using ELISA, Western blot, and fluorogenic and mass spectrometric-based enzyme assays. Logistic regression model was applied to predict the risk prediction. Receiver operating characteristic curves were drawn, and prediction accuracies were calculated to explore the effectiveness of ACE2 and NEP in predicting diabetes and CKD. Results demonstrated that there is no evidence of urinary ACE2 and ADAM17 in ND subjects, but both enzymes were increased in patients with diabetes, including Dnormo. Although there was no detectable plasma ACE2 activity, there was evidence of urinary and plasma NEP in all the subjects, and urinary NEP was significantly increased in Dmicro patients. NEP and ACE2 showed significant correlations with metabolic and renal characteristics. In summary, urinary ACE2, NEP, and ADAM17 are increased in patients with diabetes and could be used as early biomarkers to predict the incidence or progression of CKD at early stages among individuals with type 2 diabetes.
Biliary atresia is a severe inflammatory and obstructive disease of bile ducts occurring in infancy. Although the cause is unknown, activation of the innate and adaptive immune systems injures the bile duct epithelium. In this study we found that patients' livers had increased expression of inflammasome genes. Using mice engineered to inactivate individual inflammasome genes, the epithelial injury and bile duct obstruction were prevented by the loss of Il1r1 or Nlrp3, with a decreased activation of natural killer cells and expression of cytokines and chemokines. In contrast, the loss of Casp1 did not change the disease phenotype. Combined, the findings point to a differential role of inflammasome gene products in the pathogenic mechanisms of biliary atresia.
Diabetes and its associated chronic kidney disease (CKD) is a major health burden and there is an urgent need for new sensitive biomarkers to detect and monitor the progression of CKD. Albuminuria is still the gold standard for the evaluation of kidney function. However, its sensitivity and reliability have recently been questioned. ACE2 is highly expressed in renal tubules and has been shown to be shed in the urine of diabetic patients with CKD. The aim of the study was to investigate whether urinary ACE2 is increased in diabetic patients with CKD before the onset of microalbuminuria. Participants were recruited from Dayton VA Medical Center (Dayton, OH, USA). Baseline urinary albumin creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) were determined three months before initiation of the study in non-diabetic patients (UACR <30 mg/g, eGFR=97.40±16 ml/min/1.73 m
2
), and in diabetic patients with normoalbuminuria (UACR <30 mg/g, eGFR=83.08±17 ml/min/1.73 m
2
), microalbuminuria (UACR = 30-300 mg/g, eGFR=47.13±23 ml/min/1.73 m
2
), and macroalbuminuria (UACR >300 mg/g, eGFR=39.68±20 ml/min/1.73 m
2
). Using fluorogenic and mass spectrometry-based enzyme assays, we measured urinary and plasma ACE2 activity in patients. Urinary ACE2 activity was significantly increased in diabetic patients with normoalbuminuria (0.58±0.2 nmol/hr/mg creatinine), microalbuminuria (1.19 ±0.5 nmol/hr/mg creatinine), and macroalbuminuria (2.265±0.4 nmol/hr/mg creatinine) compared with non-diabetic controls (0.06 ± 0.02 nmols/hr/mg creatinine) (p<0.0001). These results were confirmed by detecting the ACE2 product Ang-(1-7) (
m/z
899) in incubations of urine samples with the natural substrate Ang II (
m/z
1046) using mass spectrometry-based enzyme assays. In addition, urinary ACE2 expression was significantly increased in diabetic patients as determined by western blot analysis (p<0.05). Plasma ACE2 activity was not detectable in control and diabetic patients. In conclusion, urinary ACE2 is increased in diabetic patients with CKD which suggests that urinary ACE2 could be used as an early, noninvasive biomarker for diabetic nephropathy before the onset of microalbuminuria.
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