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.

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.

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.

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.

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.

Amelioration of diet-induced steatohepatitis in mice following combined therapy with ASO-Fsp27 and fenofibrate.

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease. NAFLD progresses from benign steatosis to steatohepatitis (NASH) to cirrhosis and is linked to hepatocellular carcinoma. No targeted treatment is currently approved for NAFLD/NASH. We previously showed that fat-specific protein 27 (FSP27), a lipid droplet-associated protein that controls triglyceride turnover in the hepatocyte, is required for fasting- and diet-induced triglyceride accumulation in the liver. However, silencing Fsp27 with antisense oligonucleotides (ASOs) did not improve hepatosteatosis in genetic nor nutritional mouse models of obesity. Herein, we tested the therapeutic potential of ASO-Fsp27 when used in combination with the PPARα agonist fenofibrate. C57BL/6 mice were fed a high-trans-fat, high-cholesterol, high-fructose diet for eight weeks to establish NASH, then kept on diet for six additional weeks while dosed with ASOs and fenofibrate, alone or in combination. Data show that ASO-Fsp27 and fenofibrate synergize to promote resistance to diet-induced obesity and hypertriglyceridemia and to reverse hepatic steatosis, inflammation, oxidative stress, and fibrosis. This multifactorial improvement of liver disease noted when combining both drugs suggests that a course of treatment that includes both reduced FSP27 activity and activation of PPARα could provide therapeutic benefit to patients with NAFLD/NASH.

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.

Therapeutic silencing of fat-specific protein 27 improves glycemic control in mouse models of obesity and insulin resistance.

Obesity is a component of the metabolic syndrome, mechanistically linked to diabetes, fatty liver disease, and cardiovascular disease. Proteins that regulate the metabolic fate of intracellular lipid droplets are potential therapeutic candidates to treat obesity and its related consequences. CIDEC (cell death-inducing DFFA-like effector C), also known in mice as Fsp27 (fat-specific protein 27), is a lipid droplet-associated protein that prevents lipid mobilization and promotes intracellular lipid storage. The consequences of complete loss of FSP27 on hepatic metabolism and on insulin resistance are controversial, as both healthy and deleterious lipodystrophic phenotypes have been reported in Fsp27-/- mice. To test whether therapeutic silencing of Fsp27 might be useful to improve obesity, fatty liver, and glycemic control, we used antisense oligonucleotides (ASOs) in both nutritional (high-fat diet) and genetic (leptin-deficient ob/ob) mouse models of obesity, hyperglycemia, and hepatosteatosis. We show that partial silencing Fsp27 in either model results in the robust decrease in visceral fat, improved insulin sensitivity and whole-body glycemic control, and tissue-specific changes in transcripts controlling lipid oxidation and synthesis. These data suggest that partial reduction of FSP27 activity (e.g., using ASOs) might be exploited therapeutically in insulin-resistant obese or overweight patients.

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 nucleosome remodeling and deacetylase complex in development and disease.

The nucleosome remodeling and deacetylase (NuRD) complex is one of the major chromatin remodeling complexes found in cells. It plays an important role in regulating gene transcription, genome integrity, and cell cycle progression. Through its impact on these basic cellular processes, increasing evidence indicates that alterations in the activity of this macromolecular complex can lead to developmental defects, oncogenesis, and accelerated aging. Recent genetic and biochemical studies have elucidated the mechanisms of NuRD action in modifying the chromatin landscape. These advances have the potential to lead to new therapeutic approaches to birth defects and cancer.

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.