Osteopontin deletion attenuates cyst growth but exacerbates fibrosis in mice with cystic kidney disease.

Osteopontin (OPN) is a multi-functional glycoprotein that coordinates the innate immune response, prevents nanocrystal formation in renal tubule fluid, and is a biomarker for kidney injury. OPN expression is markedly increased in cystic epithelial cells of polycystic kidney disease (PKD) kidneys; however, its role in PKD progression remains unclear. We investigated the in vitro effects of recombinant OPN on the proliferation of tubular epithelial cells from PKD and normal human kidneys and in vivo effects of OPN deletion on kidney cyst formation, fibrosis, and mineral metabolism in pcy/pcy mice, a non-orthologous model of autosomal-dominant PKD. In vitro studies revealed that OPN enhanced the proliferation of PKD cells but had no effect on normal kidney cells. Deletion of OPN in pcy/pcy mice significantly reduced kidney cyst burden; however, this was accompanied by increased fibrosis and no change in kidney function. The loss of OPN had no effect on kidney macrophage numbers, cyst epithelial cell proliferation, or apoptosis. Furthermore, there was no difference in kidney mineral deposition or mineral metabolism parameters between pcy/pcy mice with and without OPN expression. Global deletion of OPN reduced kidney cyst burden, while paradoxically exacerbating kidney fibrosis in mice with cystic kidney disease.

Key questions and gaps in understanding adipose tissue macrophages and early-life metabolic programming.

The global obesity epidemic, with its associated comorbidities and increased risk of early mortality, underscores the urgent need for enhancing our understanding of the origins of this complex disease. It is increasingly clear that metabolism is programmed early in life and that metabolic programming can have life-long health consequences. As a critical metabolic organ sensitive to early-life stimuli, proper development of adipose tissue (AT) is crucial for life-long energy homeostasis. Early-life nutrients, especially fatty acids (FAs), significantly influence the programming of AT and shape its function and metabolism. Of growing interest are the dynamic responses during pre- and postnatal development to proinflammatory omega-6 (n6) and anti-inflammatory omega-3 (n3) FA exposures in AT. In the US maternal diet, the ratio of "pro-inflammatory" n6- to "anti-inflammatory" n3-FAs has grown dramatically due to the greater prevalence of n6-FAs. Notably, AT macrophages (ATMs) form a significant population within adipose stromal cells, playing not only an instrumental role in AT formation and maintenance but also acting as key mediators of cell-to-cell lipid and cytokine signaling. Despite rapid advances in ATM and immunometabolism fields, research has focused on responses to obesogenic diets and during adulthood. Consequently, there is a significant gap in identifying the mechanisms contributing metabolic health, especially regarding lipid exposures during the establishment of ATM physiology. Our review highlights the current understanding of ATM diversity, their critical role in AT, their potential role in early-life metabolic programming, and the broader implications for metabolism and health.

Spatial Transcriptomics and Single-Nucleus Multi-omics Analysis Revealing the Impact of High Maternal Folic Acid Supplementation on Offspring Brain Development.

Folate, an essential vitamin B9, is crucial for diverse biological processes including neurogenesis. Folic acid (FA) supplementation during pregnancy is a standard practice for preventing neural tube defects (NTDs). However, concerns are growing over the potential risks of excessive maternal FA intake. Here, we employed mouse model and spatial transcriptomics and single-nucleus multi-omics approaches to investigate the impact of high maternal FA supplementation during the periconceptional period on offspring brain development. Maternal high FA supplementation affected gene pathways linked to neurogenesis and neuronal axon myelination across multiple brain regions, as well as gene expression alterations related to learning and memory in thalamic and ventricular regions. Single-nucleus multi-omics analysis revealed that maturing excitatory neurons in the dentate gyrus (DG) are particularly vulnerable to high maternal FA intake, leading to aberrant gene expressions and chromatin accessibility in pathways governing ribosomal biogenesis critical for synaptic formation. Our findings provide new insights into specific brain regions, cell types, gene expressions and pathways that can be affected by maternal high FA supplementation.

Protective role for kidney TREM2high macrophages in obesity- and diabetes-induced kidney injury.

Diabetic kidney disease (DKD), the most common cause of kidney failure, is a frequent complication of diabetes and obesity, and yet to date, treatments to halt its progression are lacking. We analyze kidney single-cell transcriptomic profiles from DKD patients and two DKD mouse models at multiple time points along disease progression-high-fat diet (HFD)-fed mice aged to 90-100 weeks and BTBR ob/ob mice (a genetic model)-and report an expanding population of macrophages with high expression of triggering receptor expressed on myeloid cells 2 (TREM2) in HFD-fed mice. TREM2high macrophages are enriched in obese and diabetic patients, in contrast to hypertensive patients or healthy controls in an independent validation cohort. Trem2 knockout mice on an HFD have worsening kidney filter damage and increased tubular epithelial cell injury, all signs of worsening DKD. Together, our studies suggest that strategies to enhance kidney TREM2high macrophages may provide therapeutic benefits for DKD.

Not just for lymphoid cells: the role of the noncanonical NF-κB signaling pathway in early and late myelopoiesis with a focus on hypereosinophilic disorders.

The noncanonical NF-κB pathway is involved in lymphoid organ development, B-cell maturation, and cytokine production. However, new research has demonstrated that this pathway is also key for the orderly and sequential maturation of myeloid cells, including neutrophils and eosinophils. When this pathway is disrupted or constitutively activated, aberrations in hematopoietic stem and progenitor cell survival and proliferation, as well as subsequent granulopoiesis and eosinophilopoiesis, are affected. Disturbance of such a coordinated and delicate process can manifest in devastating clinical disease, including acute and chronic myeloid leukemias, preleukemic processes such as myelodysplastic syndrome, or hyperinflammatory conditions like hypereosinophilic syndrome. In this review, we discuss the molecular machinery within the noncanonical NF-κB pathway, crosstalk with the canonical NF-κB pathway, murine models of noncanonical signaling, and how aberrations in this pathway manifest in leukemic or hyperinflammatory disease with a focus on hypereosinophilic syndrome. Potential and promising drug therapies will also be discussed, emphasizing the noncanonical NF-κB pathway as a potential target for improved treatment for patients with leukemia or idiopathic hypereosinophilic syndrome. The hope is that review of such mechanisms and treatments may eventually result in findings that aid physicians in rapidly diagnosing and more accurately classifying patients with such complex and overlapping hematopoietic diseases.

Nephronophthisis-associated FBW7 mediates cyst-dependent decline of renal function in ADPKD.

Nephronophthisis (NPHP) and autosomal dominant Polycystic Kidney Disease (ADPKD) are two genetically distinct forms of Polycystic Kidney Disease (PKD), yet both diseases present with kidney cysts and a gradual decline in renal function. Prevailing dogma in PKD is that changes in kidney architecture account for the decline in kidney function, but the molecular/cellular basis of such coupling is unknown. To address this question, we induced a form of proteome reprogramming by deleting Fbxw7 encoding FBW7, the recognition receptor of the SCF FBW7 E3 ubiquitin ligase in different segments of the kidney tubular system. Deletion of Fbxw7 in the medulla led to a juvenile-adult NPHP-like phenotype, where the decline in renal function was due to SOX9-mediated interstitial fibrosis rather than cystogenesis. In contrast, the decline of renal function in ADPKD is coupled to cystic expansion via the abnormal accumulation of FBW7 in the proximal tubules and other cell types in the renal cortex. We propose that FBW7 functions at the apex of a protein network that determines renal function in ADPKD by sensing architectural changes induced by cystic expansion.

Macrophage Ontogeny, Phenotype, and Function in Ischemia Reperfusion-Induced Injury and Repair.

AKI is characterized by a sudden, and usually reversible, decline in kidney function. In mice, ischemia-reperfusion injury (IRI) is commonly used to model the pathophysiologic features of clinical AKI. Macrophages are a unifying feature of IRI as they regulate both the initial injury response as well as the long-term outcome following resolution of injury. Initially, macrophages in the kidney take on a proinflammatory phenotype characterized by the production of inflammatory cytokines, such as CCL2 (monocyte chemoattractant protein 1), IL-6, IL-1 ß , and TNF- α . Release of these proinflammatory cytokines leads to tissue damage. After resolution of the initial injury, macrophages take on a reparative role, aiding in tissue repair and restoration of kidney function. By contrast, failure to resolve the initial injury results in prolonged inflammatory macrophage accumulation and increased kidney damage, fibrosis, and the eventual development of CKD. Despite the extensive amount of literature that has ascribed these functions to M1/M2 macrophages, a recent paradigm shift in the macrophage field now defines macrophages on the basis of their ontological origin, namely monocyte-derived and tissue-resident macrophages. In this review, we focus on macrophage phenotype and function during IRI-induced injury, repair, and transition to CKD using both the classic (M1/M2) and novel (ontological origin) definition of kidney macrophages.

Isolation and Flow Cytometry Analysis of Macrophages from the Kidney.

Renal macrophages help maintain homeostasis, participate in tissue injury and repair, and play a vital role in immune surveillance [1-3]. Kidney macrophages can be broken down into two subsets, infiltrating macrophages, which can be further broken down into Ly6Chi and Ly6Clo cells, and kidney resident macrophages. While recent studies have shed light on the differing origins and niches of these cells, a more thorough understanding of kidney macrophage populations and how they may respond to various conditions is needed. This protocol describes how to efficiently isolate murine kidney macrophage populations for flow cytometry analysis.

Sex-Linked Growth Disorder and Aberrant Pituitary Gene Expression in Nestin-Cre-Mediated Egr1 Conditional Knockout Mice.

Genes that regulate hormone release are essential for maintaining metabolism and energy balance. Egr1 encodes a transcription factor that regulates hormone production and release, and a decreased in growth hormones has been reported in Egr1 knockout mice. A reduction in growth hormones has also been observed in Nestin-Cre mice, a model frequently used to study the nervous system. Currently, it is unknown how Egr1 loss or the Nestin-Cre driver disrupt pituitary gene expression. Here, we compared the growth curves and pituitary gene expression profiles of Nestin-Cre-mediated Egr1 conditional knockout (Egr1cKO) mice with those of their controls. Reduced body weight was observed in both the Nestin-Cre and Egr1cKO mice, and the loss of Egr1 had a slightly more severe impact on female mice than on male mice. RNA-seq data analyses revealed that the sex-related differences were amplified in the Nestin-Cre-mediated Egr1 conditional knockout mice. Additionally, in the male mice, the influence of Egr1cKO on pituitary gene expression may be overridden by the Nestin-Cre driver. Differentially expressed genes associated with the Nestin-Cre driver were significantly enriched for genes related to growth factor activity and binding. Altogether, our results demonstrate that Nestin-Cre and the loss of Egr1 in the neuronal cell lineage have distinct impacts on pituitary gene expression in a sex-specific manner.

Immune checkpoint activity regulates polycystic kidney disease progression.

Innate and adaptive immune cells modulate the severity of autosomal dominant polycystic kidney disease (ADPKD), a common kidney disease with inadequate treatment options. ADPKD has parallels with cancer, in which immune checkpoint inhibitors have been shown to reactivate CD8+ T cells and slow tumor growth. We have previously shown that in PKD, CD8+ T cell loss worsens disease. This study used orthologous early-onset and adult-onset ADPKD models (Pkd1 p.R3277C) to evaluate the role of immune checkpoints in PKD. Flow cytometry of kidney cells showed increased levels of programmed cell death protein 1 (PD-1)/cytotoxic T lymphocyte associated protein 4 (CTLA-4) on T cells and programmed cell death ligand 1 (PD-L1)/CD80 on macrophages and epithelial cells in Pkd1RC/RC mice versus WT, paralleling disease severity. PD-L1/CD80 was also upregulated in ADPKD human cells and patient kidney tissue versus controls. Genetic PD-L1 loss or treatment with an anti-PD-1 antibody did not impact PKD severity in early-onset or adult-onset ADPKD models. However, treatment with anti-PD-1 plus anti-CTLA-4, blocking 2 immune checkpoints, improved PKD outcomes in adult-onset ADPKD mice; neither monotherapy altered PKD severity. Combination therapy resulted in increased kidney CD8+ T cell numbers/activation and decreased kidney regulatory T cell numbers correlative with PKD severity. Together, our data suggest that immune checkpoint activation is an important feature of and potential novel therapeutic target in ADPKD.

Cx3cr1 controls kidney resident macrophage heterogeneity.

Kidney macrophages are comprised of both monocyte-derived and tissue resident populations; however, the heterogeneity of kidney macrophages and factors that regulate their heterogeneity are poorly understood. Herein, we performed single cell RNA sequencing (scRNAseq), fate mapping, and parabiosis to define the cellular heterogeneity of kidney macrophages in healthy mice. Our data indicate that healthy mouse kidneys contain four major subsets of monocytes and two major subsets of kidney resident macrophages (KRM) including a population with enriched Ccr2 expression, suggesting monocyte origin. Surprisingly, fate mapping data using the newly developed Ms4a3Cre Rosa Stopf/f TdT model indicate that less than 50% of Ccr2+ KRM are derived from Ly6chi monocytes. Instead, we find that Ccr2 expression in KRM reflects their spatial distribution as this cell population is almost exclusively found in the kidney cortex. We also identified Cx3cr1 as a gene that governs cortex specific accumulation of Ccr2+ KRM and show that loss of Ccr2+ KRM reduces the severity of cystic kidney disease in a mouse model where cysts are mainly localized to the kidney cortex. Collectively, our data indicate that Cx3cr1 regulates KRM heterogeneity and niche-specific disease progression.

Probable paraneoplastic leukocytosis in a dog with a gastrointestinal stromal tumor.

A 9-year-old female spayed Boston Terrier presented for diagnostic investigation of lethargy, poor appetite, weight loss, and a marked leukocytosis. Significant muscle wasting and a palpable abdominal mass were present on physical examination. Abdominal imaging revealed the mass to be of small intestinal origin; consequently, an intestinal resection and anastomosis were performed without complication. The histopathologic diagnosis was a gastrointestinal stromal tumor, verified by immunohistochemical positivity to CD117 (KIT). Two weeks after discharge, the leukocytosis had resolved. Though the exact molecular mediator of the severe leukocytosis was undetermined, resolution following tumor removal suggests a paraneoplastic cause. To the authors' knowledge, this is the first reported case of probable paraneoplastic leukocytosis secondary to a gastrointestinal stromal tumor in the dog. Gastrointestinal tract imaging should be performed when this uncommon hematologic abnormality is present.

A kidney resident macrophage subset is a candidate biomarker for renal cystic disease in preclinical models.

Although renal macrophages have been shown to contribute to cyst development in polycystic kidney disease (PKD) animal models, it remains unclear whether there is a specific macrophage subpopulation involved. Here, we analyzed changes in macrophage populations during renal maturation in association with cystogenesis rates in conditional Pkd2 mutant mice. We observed that CD206+ resident macrophages were minimal in a normal adult kidney but accumulated in cystic areas in adult-induced Pkd2 mutants. Using Cx3cr1 null mice, we reduced macrophage number, including CD206+ macrophages, and showed that this significantly reduced cyst severity in adult-induced Pkd2 mutant kidneys. We also found that the number of CD206+ resident macrophage-like cells increased in kidneys and in the urine from autosomal-dominant PKD (ADPKD) patients relative to the rate of renal functional decline. These data indicate a direct correlation between CD206+ resident macrophages and cyst formation, and reveal that the CD206+ resident macrophages in urine could serve as a biomarker for renal cystic disease activity in preclinical models and ADPKD patients. This article has an associated First Person interview with the first author of the paper.

Cholesterol Granuloma of the Cranial Mediastinum in a Dog.

A 10 yr old female spayed Pomeranian presented with a history of dyspnea and coughing and was diagnosed with a cranial mediastinal mass presumed to be a thymoma. Surgical removal was elected and occurred without intraoperative complications. Histopathology revealed the lesion to be a cholesterol granuloma. The patient developed a brief period of increased respiratory difficulty 3 days postoperatively. Thoracic radiographs showed mild pleural effusion and the patient improved with supportive care. Five months postoperatively, repeat thoracic radiographs revealed no evidence of recurrence or respiratory pathology. This case report describes a cholesterol granuloma in a unique location and reviews the pathogenesis/pathophysiology of this type of mass.

A Comprehensive Immune Cell Atlas of Cystic Kidney Disease Reveals the Involvement of Adaptive Immune Cells in Injury-Mediated Cyst Progression in Mice.

BACKGROUND: Inducible disruption of cilia-related genes in adult mice results in slowly progressive cystic disease, which can be greatly accelerated by renal injury. METHODS: To identify in an unbiased manner modifier cells that may be influencing the differential rate of cyst growth in injured versus non-injured cilia mutant kidneys at a time of similar cyst severity, we generated a single-cell atlas of cystic kidney disease. We conducted RNA-seq on 79,355 cells from control mice and adult-induced conditional Ift88 mice (hereafter referred to as cilia mutant mice) that were harvested approximately 7 months post-induction or 8 weeks post 30-minute unilateral ischemia reperfusion injury. RESULTS: Analyses of single-cell RNA-seq data of CD45+ immune cells revealed that adaptive immune cells differed more in cluster composition, cell proportion, and gene expression than cells of myeloid origin when comparing cystic models with one another and with non-cystic controls. Surprisingly, genetic deletion of adaptive immune cells significantly reduced injury-accelerated cystic disease but had no effect on cyst growth in non-injured cilia mutant mice, independent of the rate of cyst growth or underlying genetic mutation. Using NicheNet, we identified a list of candidate cell types and ligands that were enriched in injured cilia mutant mice compared with aged cilia mutant mice and non-cystic controls that may be responsible for the observed dependence on adaptive immune cells during injury-accelerated cystic disease. CONCLUSIONS: Collectively, these data highlight the diversity of immune cell involvement in cystic kidney disease.

Early infiltrating macrophage subtype correlates with late-stage phenotypic outcome in a mouse model of hepatorenal fibrocystic disease.

Hepatorenal fibrocystic disease (HRFCD) is a genetically inherited disorder related to primary cilia dysfunction in which patients display varying levels of fibrosis, bile duct expansion, and inflammation. In mouse models of HRFCD, the phenotype is greatly impacted by the genetic background in which the mutation is placed. Macrophages are a common factor associated with progression of HRFCD and are also strongly influenced by the genetic background. These data led us to hypothesize that macrophage subtypes that change in relation to the genetic background are responsible for the variable phenotypic outcomes in HRFCD. To test this hypothesis, we utilized a mouse model of HRFCD (Ift88Orpk mice) on the C57BL/6 and BALB/c inbred backgrounds that have well-documented differences in macrophage subtypes. Our analyses of infiltrating macrophage subtypes confirm that genetic strain influences the subtype of infiltrating macrophage present during normal postnatal liver development and in Ift88Orpk livers (Ly6clo in C57BL/6 vs Ly6chi in BALB/c). Each infiltrating macrophage subtype was similarly associated with a unique phenotypic outcome as analysis of liver tissue shows that C57BL/6 Ift88Orpk mice have increased bile duct expansion, but reduced levels of fibrosis compared to BALB/c Ift88Orpk livers. RNA sequencing data suggest that the ability to infiltrate macrophage subtypes to influence the phenotypic outcome may be due to unique ligand-receptor signaling between infiltrating macrophages and cilia dysfunctional biliary epithelium. To evaluate whether specific macrophage subtypes cause the observed phenotypic divergence, we analyzed the liver phenotype in BALB/c Ift88Orpk mice on a CCR2-/- background. Unexpectedly, the loss of Ly6chi macrophages, which were strongly enriched in BALB/c Ift88Orpk mice, did not significantly alter liver fibrosis. These data indicate that macrophage subtypes may correlate with HRFCD phenotypic outcome, but do not directly cause the pathology.

Kidney resident macrophages in the rat have minimal turnover and replacement by blood monocytes.

Kidney resident macrophages (KRMs) are involved in maintaining renal homeostasis and in controlling the pathological outcome of acute kidney injury and cystic kidney disease in mice. In adult mice, KRMs maintain their population through self-renewal with little or no input from the peripheral blood. Despite recent data suggesting that a transcriptionally similar population of KRM-like cells is present across species, the idea that they are self-renewing and minimally dependent on peripheral blood input in other species has yet to be proven due to the lack of an appropriate model and cross-species expression markers. In this study, we used our recently identified cross-species KRM cell surface markers and parabiosis surgery in inbred Lewis rats to determine if rat KRMs are maintained independent of peripheral blood input, similar to their mouse counterparts. Flow cytometry analysis indicated that parabiosis surgery in the rat results in the establishment of chimerism of T/B cells, neutrophils, and monocyte-derived infiltrating macrophages in the blood, spleen, and kidney 3 wk after parabiosis surgery. Analysis of KRMs using the cell surface markers CD81 and C1q indicated that these cells have minimal chimerism and, therefore, receive little input from the peripheral blood. These data indicate that KRM properties are conserved in at least two different species.NEW & NOTEWORTHY In this report, we performed parabiosis surgery on inbred Lewis rats and showed that rat kidney resident macrophages (KRMs), identified using our novel cross-species markers, are minimally dependent on peripheral blood input. Thus, for the first time, to our knowledge, we confirm that a hallmark of mouse KRMs is also present in KRMs isolated from another species.

Mammary carcinoma arising in an adenoma in a ewe.

A large, firm, multi-cystic mammary gland mass grew slowly over 4 y in a 12-y-old, female Finn-Shetland cross sheep. A diagnosis of epithelial malignancy was suspected following fine-needle aspiration cytology at 30 mo after initial observation. The sheep was euthanized when the flock was downsized 18 mo later. A field postmortem examination revealed a large mammary mass, but an absence of metastases to internal organs. Imprint cytology of the mammary tissue supported a benign proliferative process. Histologically, mammary tissue was obliterated by cystic, tubular, and papillary adenomatous arrangements of mammary epithelium, with an anaplastic component, consistent with mammary carcinoma arising in an adenoma. IHC showed strong nuclear positivity to the antibody against progesterone receptor and minimal positivity to the antibody against estrogen receptor alpha expression. Intrinsic subtyping for basal or luminal epithelial origin was attempted through adaptation of companion animal IHC classification panels; high- and low-molecular-weight cytokeratins (CK5, CK8, CK18) failed to stain, but p63 expression for basal epithelium was positive.