Kameswaran Surendran

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.

Wnt-Dependent β-Catenin Signaling Is Activated after Unilateral Ureteral Obstruction, and Recombinant Secreted Frizzled-Related Protein 4 Alters the Progression of Renal Fibrosis

beta-Catenin functions as a transducer of Wnt signals to the nucleus, where it interacts with the T cell factor (TCF) family of DNA binding proteins to regulate gene expression. On the basis of the genes regulated by beta-catenin and TCF in various biologic settings, two predicted functions of beta-catenin/TCF-dependent transcription are to mediate the loss of epithelial polarity and to promote fibroblast activities, such as the increased synthesis of fibronectin during chronic renal disease. These predictions were tested by determination of the expression and function of an inhibitor of Wnt signaling, secreted frizzled-related protein 4 (sFRP4), during renal tubular epithelial injury initiated by unilateral ureteral obstruction (UUO). Despite increased sFRP4 gene expression in perivascular regions of injured kidneys, total sFRP4 protein levels decreased after injury. The decreased sFRP4 protein levels after UUO accompanied increased Wnt-dependent beta-catenin signaling in tubular epithelial and interstitial cells, along with increased expression of markers of fibrosis. Administration of recombinant sFRP4 protein caused a reduction in tubular epithelial beta-catenin signaling and suppressed the progression of renal fibrosis, as evidenced by a partial maintenance of E-cadherin mRNA expression and a reduction in the amount of fibronectin and alpha-smooth muscle actin proteins. Furthermore, recombinant sFRP4 reduced the number of myofibroblasts, a central mediator of fibrosis. It is concluded that beta-catenin signaling is activated in tubular epithelial and interstitial cells after renal injury, and recombinant sFRP4 can interfere with epithelial de-differentiation and with fibroblast differentiation and function during progression of renal fibrosis.

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.

Bone morphogenetic protein 7: a novel treatment for chronic renal and bone disease.

Purpose of reviewWhen last reviewed, bone morphogenetic protein 7 was presented as a potential new renal therapeutic agent, with multiple efficacies in chronic kidney disease. The object of this review is to describe progress from many sources since then in support or denial of the hypothesis. Recent findingsBone morphogenetic protein 7 has been shown to be an effective defence in several forms of chronic kidney disease in animal models, and its mechanisms of action have begun to be elucidated. Bone morphogenetic protein 7 inhibits tubular epithelial cell de-differentiation, mesenchymal transformation and apoptosis stimulated by various renal injuries. Bone morphogenetic protein 7 preserves glomerular integrity and inhibits injury-mediated mesangial matrix accumulation. In renal osteodystrophy, bone morphogenetic protein 7 affects osteoblast morphology and number, eliminates peritrabecular fibrosis, decreases bone resorption, and increases bone formation in secondary hyperparathyroidism. Bone morphogenetic protein 7 restores normal rates of bone formation in the adynamic bone disorder. Bone morphogenetic protein 7 is broadly efficacious in renal osteodystrophy, and importantly increases the skeletal deposition of ingested phosphorus and calcium, improving ion homeostasis in chronic kidney disease. Bone morphogenetic protein 7 was shown to prevent vascular calcification in a model of chronic kidney disease associated with the restoration of osteocalcin expression to normal tissue-restricted sites. SummaryBone morphogenetic protein 7 may be a powerful new therapeutic agent for chronic kidney disease, with the novel attribute of not only treating the kidney disease itself, but also directly inhibiting some of the most important complications of the disease state.

The contribution of Notch1 to nephron segmentation in the developing kidney is revealed in a sensitized Notch2 background and can be augmented by reducing Mint dosage

We previously determined that Notch2, and not Notch1, was required for forming proximal nephron segments. The dominance of Notch2 may be conserved in humans, since Notch2 mutations occur in Alagille syndrome (ALGS) 2 patients, which includes renal complications. To test whether mutations in Notch1 could increase the severity of renal complications in ALGS, we inactivated conditional Notch1 and Notch2 alleles in mice using a Six2-GFP::Cre. This BAC transgene is expressed mosaically in renal epithelial progenitors but uniformly in cells exiting the progenitor pool to undergo mesenchymal-to-epithelial transition. Although delaying Notch2 inactivation had a marginal effect on nephron numbers, it created a sensitized background in which the inactivation of Notch1 severely compromised nephron formation, function, and survival. These and additional observations indicate that Notch1 in concert with Notch2 contributes to the morphogenesis of renal vesicles into S-shaped bodies in a RBP-J-dependent manner. A significant implication is that elevating Notch1 activity could improve renal functions in ALGS2 patients. As proof of principle, we determined that conditional inactivation of Mint, an inhibitor of Notch-RBP-J interaction, resulted in a moderate rescue of Notch2 null kidneys, implying that temporal blockage of Notch signaling inhibitors downstream of receptor activation may have therapeutic benefits for ALGS patients.

Physiological Notch Signaling Maintains Bone Homeostasis via RBPjk and Hey Upstream of NFATc1

Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo.

Superoxide Destabilization of β-Catenin Augments Apoptosis of High-Glucose-Stressed Mesangial Cells

Intense mesangial cell apoptosis contributes to the pathogenesis of diabetic nephropathy. Although reactive oxygen radicals and Wnt signaling components are potent regulators that modulate renal tissue remodeling and morphogenesis, cross-talk between oxidative stress and Wnt/beta-catenin signaling in controlling high-glucose-impaired mesangial cell survival and renal function have not been tested. In this study, high glucose induced Ras and Rac1 activation, superoxide burst, and Wnt5a/beta-catenin destabilization and subsequently promoted caspase-3 and poly (ADP-ribose) polymerase cleavage and apoptosis in mesangial cell cultures. The pharmacological and genetic suppression of superoxide synthesis by superoxide dismutase and diphenyloniodium, dominant-negative Ras (S17N), and dominant-negative Rac1 (T17N) abrogated high-glucose-induced glycogen synthase kinase (GSK-3beta) activation and caspase-3 and poly (ADP-ribose) polymerase degradation. Inactivation of Ras and Racl also reversed Wnt/beta-catenin expression and survival of mesangial cells. Stabilization of beta-catenin by the transfection of stable beta-catenin (Delta45) and kinase-inactive GSK-3beta attenuated high-glucose-mediated mesangial cell apoptosis. Exogenous superoxide dismutase administration attenuated urinary protein secretion in diabetic rats and abrogated diabetes-mediated reactive oxygen radical synthesis in renal glomeruli. Immunohistological observation revealed that superoxide dismutase treatment abrogated diabetes-induced caspase-3 cleavage and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) and increased Wnt5a/beta-catenin expression in renal glomeruli. Taken together, high glucose induced oxidative stress and apoptosis in mesangial cells. The Ras and Rac1 regulation of superoxide appeared to raise apoptotic activity by activating GSK-3beta and inhibiting Wnt5a/beta-catenin signaling. Controlling oxidative stress and Wnt/beta-catenin signaling has potential for protecting renal tissue against the deleterious effect of high glucose.

Reduced Notch Signaling Leads to Renal Cysts and Papillary Microadenomas

The formation of proximal nephron segments requires canonical Notch2 signaling, but other functions of Notch signaling during renal development are incompletely understood. Here, we report that proximal tubules forming with reduced Notch signaling, resulting from delayed conditional inactivation of Notch1 and/or Notch2, are prone to cyst formation and tubular epithelial stratification. Conditional inactivation of the DNA binding factor RBP-J, which mediates Notch signaling, also resulted in multiple congenital cysts arising from the proximal tubule. Moreover, a few stratified foci/microadenomas containing hyperproliferative cells, resembling precursors of papillary renal cell carcinoma, formed in these proximal tubules. Epithelial stratification correlated neither with reduced expression of the transcriptional regulator of ciliary proteins TCF2/HNF1beta nor with loss of apical-basal polarity. Instead, Notch signaling helped to restrict the orientation of epithelial mitotic spindles to a plane parallel to the basement membrane during nephron elongation. In the absence of Notch, random spindle orientation may explain the epithelial stratification and cyst formation. Furthermore, post hoc analysis of human class 1 papillary renal cell carcinoma revealed reduced Notch activity in these tumors, resulting from abundant expression of a potent inhibitor of canonical Notch signaling, KyoT3/FHL1B. In summary, these data suggest that canonical Notch signaling maintains the alignment of cell division in the proximal tubules during nephrogenesis and that perturbations in Notch signaling may lead to cystic renal disease and tumorigenesis.

The Role of Notch Signaling in Kidney Development and Disease

The kidney is the bodys filter, responsible for the removal of metabolic waste and the excretion or reabsorption of electrolytes to control blood composition and pH balance. The functional unit of this filter is the nephron, whose segmented architecture has been largely conserved in form and function throughout eukaryotic evolution. Not surprisingly, the core developmental pathways that regulate the formation of the nephron have also been conserved. In particular, the Notch signaling pathway functions in both primitive and advanced nephrons to pattern domains required for the kidneys diverse functions. In this chapter, we will discuss the role that Notch plays in directing cell fate decisions during embryonic development of the pronephros and metanephros. We will go on to discuss the later role of Notch signaling as a cyst-suppressor and the consequences of aberrant or absent Notch activity in disease and cancer. The work discussed here highlights the fundamental importance of Notch during development and homeostasis of the kidney and underlies the need for mechanistic understanding of its role towards the treatment of human disease.

Chromatin-based Mechanisms of Renal Epithelial Differentiation

Successful regenerative renal medicine depends on understanding the molecular mechanisms by which diverse phenotypes of epithelial cells differentiate from metanephric mesenchyme to populate nephrons. Whereas many genes are maintained in a poised state within the population of pluripotent progenitors, specialized epithelial functions reflect the selective expression of a subset of genes and the repression of all others. Here we highlight some common mechanisms of cell differentiation and epigenetic regulation to discuss their implications for renal epithelial development, repair, and disease.