Hui-Teng Cheng

Notch2 (but not Notch1) is required for proximal fate acquisition in the mammalian nephron

The Notch pathway regulates cell fate determination in numerous developmental processes. Here we report that Notch2 acts non-redundantly to control the processes of nephron segmentation through an Rbp-J-dependent process. Notch1 and Notch2 are detected in the early renal vesicle. Genetic analysis reveals that only Notch2 is required for the differentiation of proximal nephron structures (podocytes and proximal convoluted tubules) despite the presence of activated Notch1 in the nuclei of putative proximal progenitors. The inability of endogenous Notch1 to compensate for Notch2 deficiency may reflect sub-threshold Notch1 levels in the nucleus. In line with this view, forced expression of a γ-secretase-independent form of Notch1 intracellular domain drives the specification of proximal fates where all endogenous, ligand-dependent Notch signaling is blocked by aγ -secretase inhibitor. These results establish distinct (non-redundant), instructive roles for Notch receptors in nephron segmentation.

γ-Secretase activity is dispensable for mesenchyme-to-epithelium transition but required for podocyte and proximal tubule formation in developing mouse kidney

Notch signaling is involved in pronephros development in Xenopus and in glomerulogenesis in mice. However, owing to early lethality in mice deficient for some Notch pathway genes and functional redundancy for others, a role for Notch signaling during early stages of metanephric development has not been defined. Using an antibody specific to the N-terminal end ofγ -secretase-cleaved Notch1, we found evidence for Notch1 activation in the comma and S-shaped bodies of the mouse metanephros. We therefore cultured mouse metanephroi in the presence of a γ-secretase inhibitor, N-S-phenyl-glycine-t-butyl ester (DAPT), to block Notch signaling. We observed slightly reduced ureteric bud branching but normal mesenchymal condensation and expression of markers indicating that mesenchyme induction had occurred. However, fewer renal epithelial structures were observed, with a severe deficiency in proximal tubules and glomerular podocytes, which are derived from cells in which activated Notch1 is normally present. Distal tubules were present but in reduced numbers, and this was accompanied by an increase in intervening, non-epithelial cells. After a transient 3-day exposure to DAPT, proximal tubules expanded, but podocyte differentiation failed to recover after removal of DAPT. These observations suggest that γ-secretase activity, probably through activation of Notch, is required for maintaining a competent progenitor pool as well as for determining the proximal tubule and podocyte fates.

Target Selectivity of Vertebrate Notch Proteins COLLABORATION BETWEEN DISCRETE DOMAINS AND CSL-BINDING SITE ARCHITECTURE DETERMINES ACTIVATION PROBABILITY

All four mammalian Notch proteins interact with a single DNA-binding protein (RBP-jκ), yet they are not equivalent in activating target genes. Parallel assays of three Notch-responsive promoters in several cell lines revealed that relative activation strength is dependent on protein module and promoter context more than the cellular context. Each Notch protein reads binding site orientation and distribution on the promoter differently; Notch1 performs extremely well on paired sites, and Notch3 prefers single sites in conjunction with a proximal zinc finger transcription factor. Although head-head sites can elicit a Notch response on their own, use of CBS (CSL binding site) in tail-tail orientation is context-dependent. Bias for specific DNA elements is achieved by interplay between the N-terminal RAM (RBP-jκ-associated molecule/ankyrin region), which interprets CBS proximity and orientation, and the C-terminal transactivation domain that interacts specifically with the transcription machinery or nearby factors. To confirm the prediction that modular design underscores the evolution of functional divergence between Notch proteins, we generated a synthetic Notch protein (Notch1 ankyrin with Notch3 transactivation domain) that displayed superior signaling strength on the hes5 promoter. Consistent with the prediction that “preferred” targets (Hes1) should respond faster and at lower Notch concentration than other targets, we showed that Hes5-GFP was extinguished fast and recovered slowly, whereas Hes1-GFP was inhibited late and recovered quickly after a pulse of DAPT in metanephroi cultures.

Canonical Notch signaling in the developing lung is required for determination of arterial smooth muscle cells and selection of Clara versus ciliated cell fate.

Lung development is the result of complex interactions between four tissues: epithelium, mesenchyme, mesothelium and endothelium. We marked the lineages experiencing Notch1 activation in these four cellular compartments during lung development and complemented this analysis by comparing the cell fate choices made in the absence of RBPjκ, the essential DNA binding partner of all Notch receptors. In the mesenchyme, RBPjκ was required for the recruitment and specification of arterial vascular smooth muscle cells (vSMC) and for regulating mesothelial epithelial-mesenchymal transition (EMT), but no adverse affects were observed in mice lacking mesenchymal RBPjκ. We provide indirect evidence that this is due to vSMC rescue by endothelial-mesenchymal transition (EnMT). In the epithelium, we show that Notch1 activation was most probably induced by Foxj1-expressing cells, which suggests that Notch1-mediated lateral inhibition regulates the selection of Clara cells at the expense of ciliated cells. Unexpectedly, and in contrast to Pofut1-null epithelium, Hes1 expression was only marginally reduced in RBPjκ-null epithelium, with a corresponding minimal effect on pulmonary neuroendocrine cell fate selection. Collectively, the primary roles for canonical Notch signaling in lung development are in selection of Clara cell fate and in vSMC recruitment. These analyses suggest that the impact of γ-secretase inhibitors on branching in vitro reflect a non-cell autonomous contribution from endothelial or vSMC-derived signals.

Molecular insights into segmentation along the proximal-distal axis of the nephron.

The structure of a mammalian kidney is parsed into large collections of polarized nephrons, and each segment is home to a diverse community of cells that specialize in renal endocrine and excretory functions. Early developmental lengthening and diversification of nephron segments along a proximal--distal axis initiate all subsequent facets of tubular growth and function. Morphogenic cues and biochemical interactions that are critical to this process are starting to emerge. The underlying principles of regional cell signaling and transcriptional control organizing early segmentation are the subject of this review.

Metabolic Syndrome and Insulin Resistance as Risk Factors for Development of Chronic Kidney Disease and Rapid Decline in Renal Function in Elderly

CONTEXT Studies addressing the association of metabolic syndrome and insulin resistance with the risks of incident chronic kidney disease (CKD) and the progression of renal function were either lacking or inconclusive. OBJECTIVE The aim of this study was to define the effect of metabolic syndrome and insulin resistance on the development of new CKD and the decline in renal function. DESIGN AND SETTING A prospective cohort study was conducted at a tertiary university-based hospital in Taiwan. PATIENTS AND OTHER PARTICIPANTS We studied a total of 1456 Asians 65 or older who were followed for an average of 3.15 yr. Within the cohort, we measured insulin resistance using the homeostasis model assessment formula in 652 nondiabetic participants. INTERVENTIONS There were no interventions. MAIN OUTCOME MEASURES We measured the prevalence and incidence of CKD and the annual decline of the estimated glomerular filtration rate. RESULTS We found that the adjusted odds ratio for prevalent CKD in association with metabolic syndrome was 1.778 (95% confidence interval, 1.188 to 2.465), the hazard ratio for rapid decline in renal function was 1.042 (0.802-1.355), and the hazard ratio for incident CKD was 1.931 (1.175-3.174). With each one-unit increment of insulin resistance, the odds ratio of prevalent CKD and proteinuria were raised 1.312-fold (1.114 to 1.545) and 1.278-fold (1.098 to 1.488), respectively. Insulin resistance was not associated with incident CKD. Increment of insulin resistance per unit was associated with 1.16-fold (1.06 to 1.26) elevation in the hazard ratios of the decline in renal function. CONCLUSIONS Metabolic syndrome predicts the risks of prevalent and incident CKD, whereas insulin resistance is associated with prevalent CKD and rapid decline in renal function in elderly individuals.

Endoplasmic reticulum stress implicated in the development of renal fibrosis.

Endoplasmic reticulum (ER) stress-associated apoptosis plays a role in organ remodeling after insult. The effect of ER stress on renal tubular damage and fibrosis remains controversial. This study aims to investigate whether ER stress is involved in tubular destruction and interstitial fibrosis in vivo. Renal cell apoptosis was proven by terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) stain and poly-ADP ribose polymerase expression in the unilateral ureteral obstruction (UUO) kidney. ER stress was evoked and confirmed by the upregulation of glucose-regulated protein 78 (GRP78) and the common Lys-Asp-Glu-Leu (KDEL) motif of ER retention proteins after UUO. ER stress-associated proapoptotic signals, including B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2-associated × protein (BAX) expression, caspase-12 and c-Jun N-terminal kinase (JNK) phosphorylation, were activated in the UUO kidney. Prolonged ER stress attenuated both unsplicing and splicing X-box binding protein 1 (XBP-1) protein expression, but continued to activate inositol-requiring 1α (IRE1α)-JNK phosphorylation, protein kinase RNA-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2α subunit (eIF2α), activating transcription factor (ATF)-4, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) and cleavage activating transcription factor 6 (cATF6)-CHOP signals, which induce ER stress-related apoptosis but attenuate adaptive unfolded protein responses in UUO kidneys. However, renal apoptosis and fibrosis were attenuated in candesartan-treated UUO kidney. Candesartan was associated with maintenance of XBP-1 expression and attenuated ATF4, cATF6 and CHOP protein expression. Taken together, results show that overwhelming ER stress leads to renal cell apoptosis and subsequent fibrosis; and candesartan, at least in part, restores renal integrity by blocking ER stress-related apoptosis. Reducing ER stress may present a way to attenuate renal fibrosis.

Lean Body Mass Predicts Long-Term Survival in Chinese Patients on Peritoneal Dialysis

Background Reduced lean body mass (LBM) is one of the main indicators in malnutrition inflammation syndrome among patients on dialysis. However, the influence of LBM on peritoneal dialysis (PD) patients’ outcomes and the factors related to increasing LBM are seldom reported. Methods We enrolled 103 incident PD patients between 2002 and 2003, and followed them until December 2011. Clinical characteristics, PD-associated parameters, residual renal function, and serum chemistry profiles of each patient were collected at 1 month and 1 year after initiating PD. LBM was estimated using creatinine index corrected with body weight. Multiple linear regression analysis, Kaplan–Meier survival analysis, and Cox regression proportional hazard analysis were used to define independent variables and compare survival between groups. Results Using the median LBM value (70% for men and 64% for women), patients were divided into group 1 (n = 52; low LBM) and group 2 (n = 51; high LBM). Group 1 patients had higher rates of peritonitis (1.6 vs. 1.1/100 patient months; p<0.05) and hospitalization (14.6 vs. 9.7/100 patient months; p<0.05). Group 1 patients also had shorter overall survival and technique survival (p<0.01). Each percentage point increase in LBM reduced the hazard ratio for mortality by 8% after adjustment for diabetes, age, sex, and body mass index (BMI). Changes in residual renal function and protein catabolic rate were independently associated with changes in LBM in the first year of PD. Conclusions LBM serves as a good parameter in addition to BMI to predict the survival of patients on PD. Preserving residual renal function and increasing protein intake can increase LBM.

Glycosylated Hemoglobin and Albumin-Corrected Fructosamine Are Good Indicators for Glycemic Control in Peritoneal Dialysis Patients

Purpose Diabetes mellitus (DM) is the most common cause of end-stage renal disease and is an important risk factor for morbidity and mortality after dialysis. However, glycemic control among such patients is difficult to assess. The present study examined glycemic control parameters and observed glucose variation after refilling different kinds of fresh dialysate in peritoneal dialysis (PD) patients. Methods A total of 25 DM PD patients were recruited, and continuous glucose monitoring system (CGMS) was applied to measure interstitial fluid (ISF) glucose levels at 5-min intervals for 3 days. Patients filled out diet and PD fluid exchange diaries. The records measured with CGMS were analyzed and correlated with other glycemic control parameters such as fructosamine, albumin-corrected fructosamine (AlbF), glycosylated hemoglobin (HbA1c), and glycated albumin levels. Results There were significant correlations between mean ISF glucose and fructosamine (r = 0.45, P<0.05), AlbF (r = 0.54, P<0.01), and HbA1c (r = 0.51, P<0.01). The ISF glucose levels in glucose-containing dialysate increased from approximately 7–8 mg/dL within 1 hour of exchange in contrast to icodextrin dialysate which kept ISF glucose levels unchanged. Conclusion HbA1c and AlbF significantly correlated with the mean ISF glucose levels, indicating that they are reliable indices of glycemic control in DM PD patients. Icodextrin dialysate seems to have a favorable glycemic control effect when compared to the other glucose-containing dialysates.

AlGaN/GaN high electron mobility transistors for protein–peptide binding affinity study

Abstract Antibody-immobilized AlGaN/GaN high electron mobility transistors (HEMTs) were used to detect a short peptide consisting of 20 amino acids. One-binding-site model and two-binding-site model were used for the analysis of the electrical signals, revealing the number of binding sites on an antibody and the dissociation constants between the antibody and the short peptide. In the binding-site models, the surface coverage ratio of the short peptide on the sensor surface is relevant to the electrical signals resulted from the peptide–antibody binding on the HEMTs. Two binding sites on an antibody were observed and two dissociation constants, 4.404×10−11 M and 1.596×10−9 M, were extracted from the binding-site model through the analysis of the surface coverage ratio of the short peptide on the sensor surface. We have also shown that the conventional method to extract the dissociation constant from the linear regression of curve-fitting with Langmuir isotherm equation may lead to an incorrect information if the receptor has more than one binding site for the ligand. The limit of detection (LOD) of the sensor observed in the experimental result (∼10pM of the short peptide) is very close to the LOD (around 2.7–3.4pM) predicted from the value of the smallest dissociation constants. The sensitivity of the sensor is not only dependent on the transistors, but also highly relies on the affinity of the ligand-receptor pair. The results demonstrate that the AlGaN/GaN HEMTs cannot only be used for biosensors, but also for the biological affinity study.