The core SWI/SNF catalytic subunit Brg1 regulates nephron progenitor cell proliferation and differentiation.

Chromatin-remodeling complexes play critical roles in establishing gene expression patterns in response to developmental signals. How these epigenetic regulators determine the fate of progenitor cells during development of specific organs is not well understood. We found that genetic deletion of Brg1 (Smarca4), the core enzymatic protein in SWI/SNF, in nephron progenitor cells leads to severe renal hypoplasia. Nephron progenitor cells were depleted in Six2-Cre, Brg1flx/flx mice due to reduced cell proliferation. This defect in self-renewal, together with impaired differentiation resulted in a profound nephron deficit in Brg1 mutant kidneys. Sall1, a transcription factor that is required for expansion and maintenance of nephron progenitors, associates with SWI/SNF. Brg1 and Sall1 bind promoters of many progenitor cell genes and regulate expression of key targets that promote their proliferation.

Truncated SALL1 Impedes Primary Cilia Function in Townes-Brocks Syndrome.

Townes-Brocks syndrome (TBS) is characterized by a spectrum of malformations in the digits, ears, and kidneys. These anomalies overlap those seen in a growing number of ciliopathies, which are genetic syndromes linked to defects in the formation or function of the primary cilia. TBS is caused by mutations in the gene encoding the transcriptional repressor SALL1 and is associated with the presence of a truncated protein that localizes to the cytoplasm. Here, we provide evidence that SALL1 mutations might cause TBS by means beyond its transcriptional capacity. By using proximity proteomics, we show that truncated SALL1 interacts with factors related to cilia function, including the negative regulators of ciliogenesis CCP110 and CEP97. This most likely contributes to more frequent cilia formation in TBS-derived fibroblasts, as well as in a CRISPR/Cas9-generated model cell line and in TBS-modeled mouse embryonic fibroblasts, than in wild-type controls. Furthermore, TBS-like cells show changes in cilia length and disassembly rates in combination with aberrant SHH signaling transduction. These findings support the hypothesis that aberrations in primary cilia and SHH signaling are contributing factors in TBS phenotypes, representing a paradigm shift in understanding TBS etiology. These results open possibilities for the treatment of TBS.

Analysis of FGF20-regulated genes in organ of Corti progenitors by translating ribosome affinity purification.

BACKGROUND: Understanding the mechanisms that regulate hair cell (HC) differentiation in the organ of Corti (OC) is essential to designing genetic therapies for hearing loss due to HC loss or damage. We have previously identified Fibroblast Growth Factor 20 (FGF20) as having a key role in HC and supporting cell differentiation in the mouse OC. To investigate the genetic landscape regulated by FGF20 signaling in OC progenitors, we employ Translating Ribosome Affinity Purification combined with Next Generation RNA Sequencing (TRAPseq) in the Fgf20 lineage. RESULTS: We show that TRAPseq targeting OC progenitors effectively enriched for RNA from this rare cell population. TRAPseq identified differentially expressed genes (DEGs) downstream of FGF20, including Etv4, Etv5, Etv1, Dusp6, Hey1, Hey2, Heyl, Tectb, Fat3, Cpxm2, Sall1, Sall3, and cell cycle regulators such as Cdc20. Analysis of Cdc20 conditional-null mice identified decreased cochlea length, while analysis of Sall1-null and Sall1-ΔZn2-10 mice, which harbor a mutation that causes Townes-Brocks syndrome, identified a decrease in outer hair cell number. CONCLUSIONS: We present two datasets: genes with enriched expression in OC progenitors, and DEGs downstream of FGF20 in the embryonic day 14.5 cochlea. We validate select DEGs via in situ hybridization and in vivo functional studies in mice.

A Sall1-NuRD interaction regulates multipotent nephron progenitors and is required for loop of Henle formation.

The formation of the proper number of nephrons requires a tightly regulated balance between renal progenitor cell self-renewal and differentiation. The molecular pathways that regulate the transition from renal progenitor to renal vesicle are not well understood. Here, we show that Sall1interacts with the nucleosome remodeling and deacetylase complex (NuRD) to inhibit premature differentiation of nephron progenitor cells. Disruption of Sall1-NuRD in vivo in knock-in mice (ΔSRM) resulted in accelerated differentiation of nephron progenitors and bilateral renal hypoplasia. Transcriptional profiling of mutant kidneys revealed a striking pattern in which genes of the glomerular and proximal tubule lineages were either unchanged or upregulated, and those in the loop of Henle and distal tubule lineages were downregulated. These global changes in gene expression were accompanied by a significant decrease in THP-, NKCC2- and AQP1-positive loop of Henle nephron segments in mutant ΔSRM kidneys. These findings highlight an important function of Sall1-NuRD interaction in the regulation of Six2-positive multipotent renal progenitor cells and formation of the loop of Henle.

Disparate levels of beta-catenin activity determine nephron progenitor cell fate.

Formation of a functional kidney depends on the balance between renewal and differentiation of nephron progenitors. Failure to sustain this balance can lead to kidney failure or stem cell tumors. For nearly 60 years, we have known that signals from an epithelial structure known as the ureteric bud were essential for maintaining this balance. More recently it was discovered that one molecule, Wnt9b, was necessary for both renewal and differentiation of the nephron progenitor cells. How one ligand signaling through one transcription factor promoted two seemingly contradictory cellular processes was unclear. In this study, we show that Wnt9b/beta-catenin signaling alone is sufficient to promote both renewal and differentiation. Moreover, we show that discrete levels of beta-catenin can promote these two disparate fates, with low levels fostering progenitor renewal and high levels driving differentiation. These results provide insight into how Wnt9b regulates distinct target genes that balance nephron progenitor renewal and differentiation.

SALL1 functions as a tumor suppressor in breast cancer by regulating cancer cell senescence and metastasis through the NuRD complex.

BACKGROUND: SALL1 is a multi-zinc finger transcription factor that regulates organogenesis and stem cell development, but the role of SALL1 in tumor biology and tumorigenesis remains largely unknown. METHODS: We analyzed SALL1 expression levels in human and murine breast cancer cells as well as cancer tissues from different types of breast cancer patients. Using both in vitro co-culture system and in vivo breast tumor models, we investigated how SALL1 expression in breast cancer cells affects tumor cell growth and proliferation, metastasis, and cell fate. Using the gain-of function and loss-of-function strategies, we dissected the molecular mechanism responsible for SALL1 tumor suppressor functions. RESULTS: We demonstrated that SALL1 functions as a tumor suppressor in breast cancer, which is significantly down-regulated in the basal like breast cancer and in estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2) triple negative breast cancer patients. SALL1 expression in human and murine breast cancer cells inhibited cancer cell growth and proliferation, metastasis, and promoted cell cycle arrest. Knockdown of SALL1 in breast cancer cells promoted cancer cell growth, proliferation, and colony formation. Our studies revealed that tumor suppression was mediated by recruitment of the Nucleosome Remodeling and Deacetylase (NuRD) complex by SALL1, which promoted cancer cell senescence. We further demonstrated that the mechanism of inhibition of breast cancer cell growth and invasion by SALL1-NuRD depends on the p38 MAPK, ERK1/2, and mTOR signaling pathways. CONCLUSION: Our studies indicate that the developmental control gene SALL1 plays a critical role in tumor suppression by recruiting the NuRD complex and thereby inducing cell senescence in breast cancer cells.

Notch-Tnf signalling is required for development and homeostasis of arterial valves.

AIMS: Congenital anomalies of arterial valves are common birth defects, leading to valvar stenosis. With no pharmaceutical treatment that can prevent the disease progression, prosthetic replacement is the only choice of treatment, incurring considerable morbidity and mortality. Animal models presenting localized anomalies and stenosis of congenital arterial valves similar to that of humans are critically needed research tools to uncover developmental molecular mechanisms underlying this devastating human condition. METHODS AND RESULTS: We generated and characterized mouse models with conditionally altered Notch signalling in endothelial or interstitial cells of developing valves. Mice with inactivation of Notch1 signalling in valvar endothelial cells (VEC) developed congenital anomalies of arterial valves including bicuspid aortic valves and valvar stenosis. Notch1 signalling in VEC was required for repressing proliferation and activating apoptosis of valvar interstitial cells (VIC) after endocardial-to-mesenchymal transformation (EMT). We showed that Notch signalling regulated Tnfα expression in vivo, and Tnf signalling was necessary for apoptosis of VIC and post-EMT development of arterial valves. Furthermore, activation or inhibition of Notch signalling in cultured pig aortic VEC-promoted or suppressed apoptosis of VIC, respectively. CONCLUSION: We have now met the need of critical animal models and shown that Notch-Tnf signalling balances proliferation and apoptosis for post-EMT development of arterial valves. Our results suggest that mutations in its components may lead to congenital anomaly of aortic valves and valvar stenosis in humans.

Sall1 balances self-renewal and differentiation of renal progenitor cells.

The formation of the proper number of functional nephrons requires a delicate balance between renal progenitor cell self-renewal and differentiation. The molecular factors that regulate the dramatic expansion of the progenitor cell pool and differentiation of these cells into nephron precursor structures (renal vesicles) are not well understood. Here we show that Sall1, a nuclear transcription factor, is required to maintain the stemness of nephron progenitor cells. Transcriptional profiling of Sall1 mutant cells revealed a striking pattern, marked by the reduction of progenitor genes and amplified expression of renal vesicle differentiation genes. These global changes in gene expression were accompanied by ectopic differentiation at E12.5 and depletion of Six2+Cited1+ cap mesenchyme progenitor cells. These findings highlight a novel role for Sall1 in maintaining the stemness of the progenitor cell pool by restraining their differentiation into renal vesicles.

Phenotypic and genotypic aspects of Townes-Brock syndrome: case report of patient in southern Brazil with a new SALL1 hotspot region nonsense mutation.

BACKGROUND: Townes-Brocks syndrome (TBS) is a rare autosomal dominant condition characterized by renal, anal, limb, and auditory abnormalities. TBS diagnosis can be challenging in settings where genetic analysis is not readily available. TBS traits overlap with those of Goldenhar and VACTERL syndromes. CASE PRESENTATION: Here, we present the case of a 5-year-old Brazilian boy born with an anorectal abnormality, limb and external ears malformations, genitourinary anomalies, and a congenital heart defect. Genetic analysis revealed a SALL1 nonsense mutation. The case is discussed in the context of the current literature. CONCLUSIONS: Because of the variability in TBS clinical presentation, genetic analysis is key to the differential diagnosis of TBS relative to phenotypically similar syndromes.

A mouse model of Townes-Brocks syndrome expressing a truncated mutant Sall1 protein is protected from acute kidney injury.

It has been postulated that developmental pathways are reutilized during repair and regeneration after injury, but functional analysis of many genes required for kidney formation has not been performed in the adult organ. Mutations in SALL1 cause Townes-Brocks syndrome (TBS) and nonsyndromic congenital anomalies of the kidney and urinary tract, both of which lead to childhood kidney failure. Sall1 is a transcriptional regulator that is expressed in renal progenitor cells and developing nephrons in the embryo. However, its role in the adult kidney has not been investigated. Using a mouse model of TBS (Sall1TBS), we investigated the role of Sall1 in response to acute kidney injury. Our studies revealed that Sall1 is expressed in terminally differentiated renal epithelia, including the S3 segment of the proximal tubule, in the mature kidney. Sall1TBS mice exhibited significant protection from ischemia-reperfusion injury and aristolochic acid-induced nephrotoxicity. This protection from acute injury is seen despite the presence of slowly progressive chronic kidney disease in Sall1TBS mice. Mice containing null alleles of Sall1 are not protected from acute kidney injury, indicating that expression of a truncated mutant protein from the Sall1TBS allele, while causative of congenital anomalies, protects the adult kidney from injury. Our studies further revealed that basal levels of the preconditioning factor heme oxygenase-1 are elevated in Sall1TBS kidneys, suggesting a mechanism for the relative resistance to injury in this model. Together, these studies establish a functional role for Sall1 in the response of the adult kidney to acute injury.

The chromatin landscape of healthy and injured cell types in the human kidney.

There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. Comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measure dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We establish a spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we note distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3, KLF6, and KLF10 regulates the transition between health and injury, while in thick ascending limb cells this transition is regulated by NR2F1. Further, combined perturbation of ELF3, KLF6, and KLF10 distinguishes two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.

Sall1-dependent signals affect Wnt signaling and ureter tip fate to initiate kidney development.

Development of the metanephric kidney depends on precise control of branching of the ureteric bud. Branching events represent terminal bifurcations that are thought to depend on unique patterns of gene expression in the tip compared with the stalk and are influenced by mesenchymal signals. The metanephric mesenchyme-derived signals that control gene expression at the ureteric bud tip are not well understood. In mouse Sall1 mutants, the ureteric bud grows out and invades the metanephric mesenchyme, but it fails to initiate branching despite tip-specific expression of Ret and Wnt11. The stalk-specific marker Wnt9b and the beta-catenin downstream target Axin2 are ectopically expressed in the mutant ureteric bud tips, suggesting that upregulated canonical Wnt signaling disrupts ureter branching in this mutant. In support of this hypothesis, ureter arrest is rescued by lowering beta-catenin levels in the Sall1 mutant and is phenocopied by ectopic expression of a stabilized beta-catenin in the ureteric bud. Furthermore, transgenic overexpression of Wnt9b in the ureteric bud causes reduced branching in multiple founder lines. These studies indicate that Sall1-dependent signals from the metanephric mesenchyme are required to modulate ureteric bud tip Wnt patterning in order to initiate branching.

Progressive renal injury from transgenic expression of human carbonic anhydrase IV folding mutants is enhanced by deficiency of p58IPK.

Mutations in the human carbonic anhydrase IV (hCAIV) have been associated with retinal degeneration in an autosomal-dominant form of retinitis pigmentosa (RP17). Prior in vitro cell culture studies confirmed that all of the RP17-associated hCAIV mutations cause protein misfolding, leading to endoplasmic reticulum (ER) stress-induced apoptosis in cells expressing the mutant proteins. To evaluate the physiological impacts of these folding mutants in other carbonic anhydrase IV-producing tissues, we generated two transgenic mouse lines expressing R219S or R14W hCAIV under control of the endogenous hCAIV promoter. Expression of either of these mutant proteins in kidneys caused progressive renal injury in male transgenic mice as evidenced by an age-dependent increase in the tubule cell apoptosis starting at approximately 20 weeks of age and vacuolization throughout the renal cortex in older mice. Up-regulation of the ER chaperone, BiP, was observed in the cells of the renal cortex of the male transgenic mice, suggesting ER stress as a causal factor for the renal injury. The renal injury inflicted by expression of the folding mutants was markedly enhanced by haploinsufficiency of the ER cochaperone p58(IPK). The transgenic mice expressing the hCAIV folding mutants on a p58(IPK) heterozygous background showed extensive renal tubular apoptosis by approximately 10 weeks of age in both male and female mice. These data indicate that expression of the RP17-associated folding mutants of hCAIV can adversely affect tissues beyond the retina and their in vivo proteotoxicity is sensitive to modulation of the protein folding environment of the ER.

Deletion of NuRD component Mta2 in nephron progenitor cells causes developmentally programmed FSGS.

Low nephron endowment at birth is a risk factor for chronic kidney disease. The prevalence of this condition is increasing due to higher survival rates of preterm infants and children with multi- organ birth defect syndromes that affect the kidney and urinary tract. We created a mouse model of congenital low nephron number due to deletion of Mta2 in nephron progenitor cells. Mta2 is a core component of the Nucleosome Remodeling and Deacetylase (NuRD) chromatin remodeling complex. These mice developed albuminuria at 4 weeks of age followed by focal segmental glomerulosclerosis (FSGS) at 8 weeks, with progressive kidney injury and fibrosis. Our studies reveal that altered mitochondrial metabolism in the post-natal period leads to accumulation of neutral lipids in glomeruli at 4 weeks of age followed by reduced mitochondrial oxygen consumption. We found that NuRD cooperated with Zbtb7a/7b to regulate a large number of metabolic genes required for fatty acid oxidation and oxidative phosphorylation. Analysis of human kidney tissue also supported a role for reduced mitochondrial lipid metabolism and ZBTB7A/7B in FSGS and CKD. We propose that an inability to meet the physiological and metabolic demands of post-natal somatic growth of the kidney promotes the transition to CKD in the setting of glomerular hypertrophy due to low nephron endowment.

The chromatin landscape of healthy and injured cell types in the human kidney.

There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. However, comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measured dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We established a comprehensive and spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we noted distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3 , KLF6 , and KLF10 regulated the transition between health and injury, while in thick ascending limb cells this transition was regulated by NR2F1 . Further, combined perturbation of ELF3 , KLF6 , and KLF10 distinguished two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.

Tamm-Horsfall protein-deficient thick ascending limbs promote injury to neighboring S3 segments in an MIP-2-dependent mechanism.

Tamm-Horsfall protein (THP) is a glycoprotein expressed exclusively in thick ascending limbs (TAL) of the kidney. We recently described a novel protective role of THP against acute kidney injury (AKI) via downregulation of inflammation in the outer medulla. Our current study investigates the mechanistic relationships among the status of THP, inflammation, and tubular injury. Using an ischemia-reperfusion model in wild-type and THP-/- mice, we demonstrate that it is the S3 proximal segments but not the THP-deficient TAL that are the main targets of tubular injury during AKI. The injured S3 segments that are surrounded by neutrophils in THP-/- mice have marked overexpression of neutrophil chemoattractant MIP-2 compared with wild-type counterparts. Neutralizing macrophage inflammatory protein-2 (MIP-2) antibody rescues S3 segments from injury, decreases neutrophil infiltration, and improves kidney function in THP-/- mice. Furthermore, using immunofluorescence volumetric imaging of wild-type mouse kidneys, we show that ischemia alters the intracellular translocation of THP in the TAL cells by partially shifting it from its default apical surface domain to the basolateral domain, the latter being contiguous to the basolateral surface of S3 segments. Concomitant with this is the upregulation, in the basolateral surface of S3 segments, of the scavenger receptor SRB-1, a putative receptor for THP. We conclude that TAL affects the susceptibility of S3 segments to injury at least in part by regulating MIP-2 expression in a THP-dependent manner. Our findings raise the interesting possibility of a direct role of basolaterally released THP on regulating inflammation in S3 segments.

Early nephrologist involvement in hospital-acquired acute kidney injury: a pilot study.

BACKGROUND: The optimal timing of nephrology consultation in patients with hospital-acquired acute kidney injury (AKI) is unknown. STUDY DESIGN: Prospective controlled nonrandomized intervention study. SETTING & PARTICIPANTS: We screened daily serum creatinine (SCr) levels of 4,296 patients admitted to the St. Louis Veterans Affairs Medical Center between September and November 2008 (control group) and January to May 2009 (intervention group). 354 patients (8.2%) met the definition of in-hospital AKI (SCr level increase of 0.3 mg/dL over 48 hours); 176 of whom met all inclusion criteria; 85 and 91 patients were enrolled in the control (standard care) and intervention groups, respectively. INTERVENTION: Early renal service involvement (EARLI), defined as a 1-time nephrology consultation within 18 hours of the onset of AKI. OUTCOME: Primary outcome defined as 2.5-fold increase in SCr level from admission. MEASUREMENT: Daily SCr until discharge. RESULTS: The 2 groups had similar characteristics at baseline and at the time of AKI. The intervention was completed at a mean of 13.1 ± 0.8 hours from the onset of AKI. Nephrology recommendations in the EARLI group included specific diagnostic, therapeutic, and preventative components. The primary outcome occurred in 12.9% of patients in the control group compared with 3.3% of patients in the EARLI group (P = 0.02). Patients in the EARLI group had a lower peak SCr level of 1.8 ± 0.1 versus 2.1 ± 0.2 mg/dL in controls (P = 0.01). LIMITATIONS: Single-center nonrandomized study of mostly US male veterans. CONCLUSIONS: Early nephrologist involvement in patients with AKI may reduce the risk of a further decrease in kidney function. A larger randomized trial is needed to confirm the findings.

Rate of kidney function decline associates with mortality.

The effect of rate of decline of kidney function on risk for death is not well understood. Using the Department of Veterans Affairs national databases, we retrospectively studied a cohort of 4171 patients who had rheumatoid arthritis and early stage 3 chronic kidney disease (CKD; estimated GFR 45 to 60 ml/min) and followed them longitudinally to characterize predictors of disease progression and the effect of rate of kidney function decline on mortality. After a median of 2.6 years, 1604 (38%) maintained stable kidney function; 426 (10%), 1147 (28%), and 994 (24%) experienced mild, moderate, and severe progression of CKD, respectively (defined as estimated GFR decline of 0 to 1, 1 to 4, and >4 ml/min per yr). Peripheral artery disease predicted moderate progression of CKD progression. Black race, hypertension, diabetes, cardiovascular disease, and peripheral artery disease predicted severe progression of CKD. After a median of 5.7 years, patients with severe progression had a significantly increased risk for mortality (hazard ratio 1.54; 95% confidence interval 1.30 to 1.82) compared with those with mild progression; patients with moderate progression exhibited a similar trend (hazard ratio 1.10; 95% confidence interval 0.98 to 1.30). Our results demonstrate an independent and graded association between the rate of kidney function decline and mortality. Incorporating the rate of decline into the definition of CKD may transform a static definition into a dynamic one that more accurately describes the potential consequences of the disease for an individual.

Pharmacologic inhibition of RGD-binding integrins ameliorates fibrosis and improves function following kidney injury.

Fibrosis is a final common pathway for many causes of progressive chronic kidney disease (CKD). Arginine-glycine-aspartic acid (RGD)-binding integrins are important mediators of the pro-fibrotic response by activating latent TGF-ß at sites of injury and by providing myofibroblasts information about the composition and stiffness of the extracellular matrix. Therefore, blockade of RGD-binding integrins may have therapeutic potential for CKD. To test this idea, we used small-molecule peptidomimetics that potently inhibit a subset of RGD-binding integrins in a murine model of kidney fibrosis. Acute kidney injury leading to fibrosis was induced by administration of aristolochic acid. Continuous subcutaneous administration of CWHM-12, an RGD integrin antagonist, for 28 days improved kidney function as measured by serum creatinine. CWHM-12 significantly reduced Collagen 1 (Col1a1) mRNA expression and scar collagen deposition in the kidney. Protein and gene expression markers of activated myofibroblasts, a major source of extracellular matrix deposition in kidney fibrosis, were diminished by treatment. RNA sequencing revealed that inhibition of RGD integrins influenced multiple pathways that determine the outcome of the response to injury and of repair processes. A second RGD integrin antagonist, CWHM-680, administered once daily by oral gavage was also effective in ameliorating fibrosis. We conclude that targeting RGD integrins with such small-molecule antagonists is a promising therapeutic approach in fibrotic kidney disease.