Smad4 controls proliferation of interstitial cells in the neonatal kidney.

Expansion of interstitial cells in the adult kidney is a hallmark of chronic disease, whereas their proliferation during fetal development is necessary for organ formation. An intriguing difference between adult and neonatal kidneys is that the neonatal kidney has the capacity to control interstitial cell proliferation when the target number has been reached. In this study, we define the consequences of inactivating the TGFß/Smad response in the mouse interstitial cell lineage. We find that pathway inactivation through loss of Smad4 leads to overproliferation of interstitial cells regionally in the kidney medulla. Analysis of markers for BMP and TGFß pathway activation reveals that loss of Smad4 primarily reduces TGFß signaling in the interstitium. Whereas TGFß signaling is reduced in these cells, marker analysis shows that Wnt/ß-catenin signaling is increased. Our analysis supports a model in which Wnt/ß-catenin-mediated proliferation is attenuated by TGFß/Smad to ensure that proliferation ceases when the target number of interstitial cells has been reached in the neonatal medulla.

Inactivation of MAP3K7 in FOXD1-expressing cells results in loss of mesangial PDGFRΒ and juvenile kidney scarring.

Transforming growth factor-ß (TGFß) plays a central role in renal scarring, controlling extracellular matrix deposition by interstitial cells and mesangial cells. TGFß signals through Smad and mitogen-activated protein kinase (MAPK) pathways. To understand the role of MAPK in interstitial and mesangial cells, we genetically inactivated TGFß-activated kinase-1 ( Map3k7) using Foxd1+/cre. Embryonic kidney development was unperturbed in mutants, but spontaneous scarring of the kidney ensued during the first postnatal week, with retention of embryonic nephrogenic rests and accumulation of collagen IV in the mesangium. MAPK signaling in the mesangium of mutant mice was skewed, with depressed p38 but elevated c-Jun NH2-terminal kinase (JNK) activation at postnatal day 3. Despite normal expression of platelet-derived growth factor receptor-ß (PDGFRß) in the mesangium of mutants at birth, expression was lost concomitantly with the increase in JNK activation, and studies in isolated mesangial cells revealed that JNK negatively regulates Pdgfrß. In summary, we show that MAP3K7 balances MAPK signaling in mesangial cells, suppressing postnatal JNK activation. We propose that the balance of MAPK signaling is essential for appropriate postnatal regulation of mesangial PDGFRß expression.

FOXD1 promotes nephron progenitor differentiation by repressing decorin in the embryonic kidney.

Forkhead transcription factors are essential for diverse processes in early embryonic development and organogenesis. Foxd1 is required during kidney development and its inactivation results in failure of nephron progenitor cell differentiation. Foxd1 is expressed in interstitial cells adjacent to nephron progenitor cells, suggesting an essential role for the progenitor cell niche in nephrogenesis. To better understand how cortical interstitial cells in general, and FOXD1 in particular, influence the progenitor cell niche, we examined the differentiation states of two progenitor cell subtypes in Foxd1(-/-) tissue. We found that although nephron progenitor cells are retained in a primitive CITED1-expressing compartment, cortical interstitial cells prematurely differentiate. To identify pathways regulated by FOXD1, we screened for target genes by comparison of Foxd1 null and wild-type tissues. We found that the gene encoding the small leucine-rich proteoglycan decorin (DCN) is repressed by FOXD1 in cortical interstitial cells, and we show that compound genetic inactivation of Dcn partially rescues the failure of progenitor cell differentiation in the Foxd1 null. We demonstrate that DCN antagonizes BMP/SMAD signaling, which is required for the transition of CITED1-expressing nephron progenitor cells to a state that is primed for WNT-induced epithelial differentiation. On the basis of these studies, we propose a mechanism for progenitor cell retention in the Foxd1 null in which misexpressed DCN produced by prematurely differentiated interstitial cells accumulates in the extracellular matrix, inhibiting BMP7-mediated transition of nephron progenitor cells to a compartment in which they can respond to epithelial induction signals.

Stromal ß-catenin overexpression contributes to the pathogenesis of renal dysplasia.

Renal dysplasia, the leading cause of renal failure in children, is characterized by disrupted branching of the collecting ducts and primitive tubules, with an expansion of the stroma, yet a role for the renal stroma in the genesis of renal dysplasia is not known. Here, we demonstrate that expression of ß-catenin, a key transcriptional co-activator in renal development, is markedly increased in the expanded stroma in human dysplastic tissue. To understand its contribution to the genesis of renal dysplasia, we generated a mouse model that overexpresses ß-catenin specifically in stromal progenitors, termed ß-cat(GOF-S) . Histopathological analysis of ß-cat(GOF) (-S) mice revealed a marked expansion of fibroblast cells surrounding primitive ducts and tubules, similar to defects observed in human dysplastic kidneys. Characterization of the renal stroma in ß-cat(GOF) (-S) mice revealed altered stromal cell differentiation in the expanded renal stroma demonstrating that this is not renal stroma but instead a population of stroma-like cells. These cells overexpress ectopic Wnt4 and Bmp4, factors necessary for endothelial cell migration and blood vessel formation. Characterization of the renal vasculature demonstrated disrupted endothelial cell migration, organization, and vascular morphogenesis in ß-cat(GOF) (-S) mice. Analysis of human dysplastic tissue demonstrated a remarkably similar phenotype to that observed in our mouse model, including altered stromal cell differentiation, ectopic Wnt4 expression in the stroma-like cells, and disrupted endothelial cell migration and vessel formation. Our findings demonstrate that the overexpression of ß-catenin in stromal cells is sufficient to cause renal dysplasia. Further, the pathogenesis of renal dysplasia is one of disrupted stromal differentiation and vascular morphogenesis. Taken together, this study demonstrates for the first time the contribution of stromal ß-catenin overexpression to the genesis of renal dysplasia. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Optogenetic activation of UCP1-dependent thermogenesis in brown adipocytes.

Brown adipocytes are unique in that they expend energy and produce heat to maintain euthermia through expression of uncoupling protein-1 (UCP1). Given their propensity to stimulate weight loss and promote resistance to obesity, they are a compelling interventional target for obesity-related disorders. Here, we tested whether an optogenetic approach could be used to activate UCP1-dependent thermogenesis in brown adipocytes. We generated brown adipocytes expressing a bacterial-derived photoactivatable adenylyl cyclase (bPAC) that, upon blue light stimulation, increases UCP1 expression, fuel uptake and thermogenesis. This unique system allows for precise, chemical free, temporal control of UCP1-dependent thermogenesis, which can aid in our understanding of brown adipocyte biology and development of therapies that target obesity-related disorders.

BMP7 promotes proliferation of nephron progenitor cells via a JNK-dependent mechanism.

The iterative formation of nephrons during embryonic development relies on continual replenishment of progenitor cells throughout nephrogenesis. Defining molecular mechanisms that maintain and regulate this progenitor pool is essential to understanding nephrogenesis in developmental and regenerative contexts. Maintenance of nephron progenitors is absolutely dependent on BMP7 signaling, and Bmp7-null mice exhibit rapid loss of progenitors. However, the signal transduction machinery operating downstream of BMP7 as well as the precise target cell remain undefined. Using a novel primary progenitor isolation system, we have investigated signal transduction and biological outcomes elicited by BMP7. We find that BMP7 directly and rapidly activates JNK signaling in nephron progenitors resulting in phosphorylation of Jun and ATF2 transcription factors. This signaling results in the accumulation of cyclin D3 and subsequent proliferation of PAX2(+) progenitors, inversely correlating with the loss of nephron progenitors seen in the Bmp7-null kidney. Activation of Jun and ATF2 is severely diminished in Bmp7-null kidneys, providing an important in vivo correlate. BMP7 thus promotes proliferation directly in nephron progenitors by activating the JNK signaling circuitry.

Follistatin-like 1 regulates renal IL-1ß expression in cisplatin nephrotoxicity.

Follistatin-like 1 (FSTL1) is a secreted protein with homology to both Follistatin and the SPARC/BM40 family of matricellular proteins. In this study, we sought to determine the expression patterns of Fstl1 and its cognate receptor Dip2a in the adult, and to assess the consequences of Fstl1 inactivation on development and homeostasis of the kidney. We find that FSTL1 circulates at high levels in both the human and the mouse and that it is also locally expressed in the loop of Henle in the kidney. To begin to understand the in vivo functions of Fstl1, we generated a mouse mutant using a genetrap approach. The hypomorphic Fstl1 genetrap strain displays a strong reduction in FSTL1 expression at the protein level, but it does not show overt developmental defects. FSTL1 has previously been implicated in diverse disease processes as a regulator of inflammatory cytokine expression, and we therefore evaluated the response of the genetrap strain to cisplatin-mediated acute kidney injury, a disease model with highly cytokine-dependent pathology. We find that although TNF-α and Il6 levels are unchanged relative to wild-type, renal Il-1ß expression is increased in genetrap mice following cisplatin treatment. Furthermore, histopatological analysis, expression of the tissue injury marker Havcr1, and measurement of serum creatinine demonstrate that reduction of Fstl1 expression sensitizes the kidney to acute cisplatin nephrotoxicity, suggesting a role for FSTL1-mediated Il-1ß suppression in protection of the kidney from acute nephrotoxic injury.

Chordin-like 1 and twisted gastrulation 1 regulate BMP signaling following kidney injury.

Stimulation of the bone morphogenetic protein (BMP) pathway protects the kidney from acute and chronic injury. Numerous regulators in the kidney control BMP signaling, offering many targets for therapeutic manipulation. Here, we screened for modulators of BMP signaling in the ischemia-sensitive S3 segment and found that Chordin-like 1 is expressed in this segment of both the mouse and human nephron. Chordin-like 1 specifically antagonizes BMP7, which is expressed in the neighboring distal nephron, and this depends on the presence of the protein Twisted gastrulation. Upon ischemia-induced degeneration of the S3 segment, we observed a reduction in Chordin-like 1 expression coincident with intense BMP signaling in tubules of the recovering kidney. Restored expression accompanied proximal tubule epithelia redifferentiation, again coincident with decreased BMP signaling. We propose that Chordin-like 1 reduces BMP7 signaling in healthy proximal tubules, and the loss of this activity upon sloughing of injured epithelia promotes BMP7 signaling in repopulating, dedifferentiated epithelia. As regenerating epithelia differentiate, Chordin-like 1 is again expressed, antagonizing BMP7. These data suggest a mechanism for dynamic regulation of renoprotective BMP7 signaling in the S3 segment of the proximal tubule.

Kinetics of transmission, infectivity, and genome stability of two novel mouse norovirus isolates in breeding mice.

Murine noroviruses are a recently discovered group of viruses found within mouse research colonies in many animal facilities worldwide. In this study, we used 2 novel mouse norovirus (MNV) wildtype isolates to examine the kinetics of transmission and tissue distribution in breeding units of NOD.CB17-Prkdc(scid)/J and backcrossed NOD.CB17-Prkdc(scid)/J x NOD/ShiLtJ (N1) mice. Viral shedding in feces and dissemination to tissues of infected offspring mice were monitored by RT-PCR over a 6-wk period postpartum. Histologic sections of tissues from mice exposed to MNV were examined for lesions and their sera monitored for the presence of antibodies to MNV. Viruses shed in feces of parental and offspring mice were compared for sequence homology of the Orf2 gene. Studies showed that the wildtype viruses MNV5 and MNV6 behaved differently in terms of the kinetics of transmission and distribution to tissues of offspring mice. For MNV5, virus transmission from parents to offspring was not seen before 3 wk after birth, and neither isolate was transmitted between cages of infected and control mice. Susceptibility to infection was statistically different between the 2 mouse strains used in the study. Both immunodeficient NOD.CB17-Prkdc(scid)/J mice and NOD. CB17-Prkdc(scid)/J x NOD/ShiLtJ offspring capable of mounting an immune response shed virus in their feces throughout the 6-wk study period, but no gross or histologic lesions were present in infected tissues. Progeny viruses isolated from the feces of infected offspring showed numerous mutations in the Orf2 gene for MNV5 but not MNV6. These results confirm previous studies demonstrating that the biology of MNV in mice varies substantially with each virus isolate and mouse strain infected.

Control of kidney, eye and limb expression of Bmp7 by an enhancer element highly conserved between species.

Bmp7 is expressed in numerous tissues throughout development and is required for morphogenesis of the eye, hindlimb and kidney. In this study we show that the majority if not all of the cis-regulatory sequence governing expression at these anatomical sites during development is present in approximately 20 kb surrounding exon 1. In eye, limb and kidney, multiple distinct enhancer elements drive Bmp7 expression within each organ. In the eye, the elements driving expression in the pigmented epithelium and iris are spatially separated. In the kidney, Bmp7 expression in collecting ducts and nephron progenitors is driven by separate enhancer elements. Similarly, limb mesenchyme and apical ectodermal ridge expression are governed by separate elements. Although enhancers for pigmented epithelium, nephrogenic mesenchyme and apical ectodermal ridge are distributed across the approximately 20 kb region, an element of approximately 480 base pairs within intron 1 governs expression within the developing iris, collecting duct system of the kidney and limb mesenchyme. This element is remarkably conserved both in sequence and position in the Bmp7 locus between different vertebrates, ranging from Xenopus tropicalis to Homo sapiens, demonstrating that there is strong selective pressure for Bmp7 expression at these tissue sites. Furthermore, we show that the frog enhancer functions appropriately in transgenic mice. Interestingly, the intron 1 element cannot be found in the Bmp7 genes of vertebrates such as Danio rerio and Takifugu rubripes indicating that this modification of the Bmp7 gene might have arisen during the adaptation from aquatic to terrestrial life. Mutational analysis demonstrates that the enhancer activity of the intron 1 element is entirely dependent on the presence of a 10 base pair site within the intron 1 enhancer containing a predicted binding site for the FOXD3 transcription factor.

An in vivo reporter of BMP signaling in organogenesis reveals targets in the developing kidney.

BACKGROUND: Bone morphogenetic proteins (BMPs) regulate essential processes during organogenesis, and a functional understanding of these secreted proteins depends on identification of their target cells. In this study, we generate a transgenic reporter for organogenesis studies that we use to define BMP pathway activation in the developing kidney. RESULTS: Mouse strains reporting on BMP pathway activation were generated by transgenically expressing beta-galactosidase under the control of BMP responsive elements from Id1. Reporter expression corresponds well with immunoassays for pathway activation in all organs studied, validating the model. Using these reporters we have generated a detailed map of cellular targets of BMP signaling in the developing kidney. We find that SMAD dependent BMP signaling is active in collecting duct trunks, but not tips. Furthermore, glomerular endothelial cells, and proximal nephron tubules from the renal vesicle stage onward show pathway activation. Surprisingly, little activation is detected in the nephrogenic zone of the kidney, and in organ culture BMP treatment fails to activate SMAD dependent BMP signaling in nephron progenitor cells. In contrast, signaling is efficiently induced in collecting duct tips. CONCLUSION: Transgenic reporters driven by control elements from BMP responsive genes such as Id1 offer significant advantages in sensitivity and consistency over immunostaining for studies of BMP pathway activation. They also provide opportunities for analysis of BMP signaling in organ and primary cell cultures subjected to experimental manipulation. Using such a reporter, we made the surprising finding that SMAD dependent BMP signaling is inactive in nephron progenitors, and that these cells are refractory to activation by applied growth factors. Furthermore, we find that the BMP pathway is not normally active in collecting duct tips, but that it can be ectopically activated by BMP treatment, offering a possible explanation for the inhibitory effects of BMP treatment on collecting duct growth and branching.

Nephron progenitor cell death elicits a limited compensatory response associated with interstitial expansion in the neonatal kidney.

The final nephron number in an adult kidney is regulated by nephron progenitor cell availability and collecting duct branching in the fetal period. Fetal environmental perturbations that cause reductions in cell numbers in these two compartments result in low nephron endowment. Previous work has shown that maternal dietary factors influence nephron progenitor cell availability, with both caloric restriction and protein deprivation leading to reduced cell numbers through apoptosis. In this study, we evaluate the consequences of inducing nephron progenitor cell death on progenitor niche dynamics and on nephron endowment. Depletion of approximately 40% of nephron progenitor cells by expression of diphtheria toxin A at embryonic day 15 in the mouse results in 10-20% nephron reduction in the neonatal period. Analysis of cell numbers within the progenitor cell pool following induction of apoptosis reveals a compensatory response in which surviving progenitor cells increase their proliferation and replenish the niche. The proliferative response is temporally associated with infiltration of macrophages into the nephrogenic zone. Colony stimulating factor 1 (CSF1) has a mitogenic effect on nephron progenitor cells, providing a potential explanation for the compensatory proliferation. However, CSF1 also promotes interstitial cell proliferation, and the compensatory response is associated with interstitial expansion in recovering kidneys which can be pharmacologically inhibited by treatment with clodronate liposomes. Our findings suggest that the fetal kidney employs a macrophage-dependent compensatory regenerative mechanism to respond to acute injury caused by death of nephron progenitor cells, but that this regenerative response is associated with neonatal interstitial expansion.

Integrated ß-catenin, BMP, PTEN, and Notch signalling patterns the nephron.

The different segments of the nephron and glomerulus in the kidney balance the processes of water homeostasis, solute recovery, blood filtration, and metabolite excretion. When segment function is disrupted, a range of pathological features are presented. Little is known about nephron patterning during embryogenesis. In this study, we demonstrate that the early nephron is patterned by a gradient in ß-catenin activity along the axis of the nephron tubule. By modifying ß-catenin activity, we force cells within nephrons to differentiate according to the imposed ß-catenin activity level, thereby causing spatial shifts in nephron segments. The ß-catenin signalling gradient interacts with the BMP pathway which, through PTEN/PI3K/AKT signalling, antagonises ß-catenin activity and promotes segment identities associated with low ß-catenin activity. ß-catenin activity and PI3K signalling also integrate with Notch signalling to control segmentation: modulating ß-catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning.

Distinct bone morphogenetic proteins activate indistinguishable transcriptional responses in nephron epithelia including Notch target genes.

Endogenous Bone Morphogenetic Protein (BMP) signaling plays a significant role in the kidney's recovery from acute injury and exogenous administration of BMP7 has therapeutic potential in numerous rodent models of renal injury and disease. However, in the healthy kidney endogenous BMP7 ligand is vigorously counteracted by extracellular antagonists such as USAG1 and CHRDL1. Little is known about the degree of BMP signaling and the ligands driving it in the healthy adult kidney. In this study we characterize basal BMP signaling in the healthy tubular nephron, and show that BMP2 is expressed in proximal nephron epithelial cells. Comparative gene profiling of proximal tubule cell responses to BMP2 and BMP7 does not reveal any qualitative difference, suggesting that identical BMP gene targets may be activated in healthy and injured organs. Interestingly, our gene profiling analysis shows that BMP signaling activates a number of Notch regulated transcription factors, including HEY1. As in other biological systems, HEY1 functions as a negative feedback regulator of BMP2 expression in the proximal tubule. In summary, this work reveals endogenous BMP signaling patterns in the healthy human and mouse kidneys, and identifies novel gene targets, some of which are involved in the complex regulation of BMP signaling in the adult kidney.

Transgenic expression of nonclassically secreted FGF suppresses kidney repair.

FGF1 is a signal peptide-less nonclassically released growth factor that is involved in angiogenesis, tissue repair, inflammation, and carcinogenesis. The effects of nonclassical FGF export in vivo are not sufficiently studied. We produced transgenic mice expressing FGF1 in endothelial cells (EC), which allowed the detection of FGF1 export to the vasculature, and studied the efficiency of postischemic kidney repair in these animals. Although FGF1 transgenic mice had a normal phenotype with unperturbed kidney structure, they showed a severely inhibited kidney repair after unilateral ischemia/reperfusion. This was manifested by a strong decrease of postischemic kidney size and weight, whereas the undamaged contralateral kidney exhibited an enhanced compensatory size increase. In addition, the postischemic kidneys of transgenic mice were characterized by hyperplasia of interstitial cells, paucity of epithelial tubular structures, increase of the areas occupied by connective tissue, and neutrophil and macrophage infiltration. The continuous treatment of transgenic mice with the cell membrane stabilizer, taurine, inhibited nonclassical FGF1 export and significantly rescued postischemic kidney repair. It was also found that similar to EC, the transgenic expression of FGF1 in monocytes and macrophages suppresses kidney repair. We suggest that nonclassical export may be used as a target for the treatment of pathologies involving signal peptide-less FGFs.

Isolation and culture of cells from the nephrogenic zone of the embryonic mouse kidney.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering naïve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated http://www.gudmap.org/, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.