Search

Supplementary figure legends for Supplementary Figures 1-3. In addition, supplementary Methods with detailed protocols and sequence information.

Glioma stem cells (GSC) and epithelial–mesenchymal transition (EMT) are strongly associated with therapy resistance and tumor recurrence, but the underlying mechanisms are incompletely understood. Here, we show that S100A4 is a novel biomarker of GSCs. S100A4+ cells in gliomas are enriched with cancer cells that have tumor-initiating and sphere-forming abilities, with the majority located in perivascular niches where GSCs are found. Selective ablation of S100A4-expressing cells was sufficient to block tumor growth in vitro and in vivo. We also identified S100A4 as a critical regulator of GSC self-renewal in mouse and patient-derived glioma tumorspheres. In contrast with previous reports of S100A4 as a reporter of EMT, we discovered that S100A4 is an upstream regulator of the master EMT regulators SNAIL2 and ZEB along with other mesenchymal transition regulators in glioblastoma. Overall, our results establish S100A4 as a central node in a molecular network that controls stemness and EMT in glioblastoma, suggesting S100A4 as a candidate therapeutic target. Cancer Res; 77(19); 5360–73. ©2017 AACR.

Supplementary Table 2 listing significantly (q,0.05 and FC>2) in S100A4-KD cells (common to both siRNA compared to scrambled control siRNA).

Supplementary data showing expression and lontg-term self-renewal of S100A4+/GFP+ cells in spontaneous and transplanted tumors, including reactivation of S100A4 in S100A4-KD tumors. IPA and GSEA analyses showing pathways enriched in S100A4 expressing cells.

Supplementary Figure Legends 1-7 from Stromal Transforming Growth Factor-β Signaling Mediates Prostatic Response to Androgen Ablation by Paracrine Wnt Activity

Supplementary Figure 6 from Stromal Transforming Growth Factor-β Signaling Mediates Prostatic Response to Androgen Ablation by Paracrine Wnt Activity

Supplementary Figure 1 from Stromal Transforming Growth Factor-β Signaling Mediates Prostatic Response to Androgen Ablation by Paracrine Wnt Activity

Mechanisms of androgen dependence of the prostate are critical to understanding prostate cancer progression to androgen independence associated with disease mortality. Transient elevation of transforming growth factor-β (TGF-β) occurs after androgen ablation. To determine the role of TGF-β on prostate response to androgen ablation, conditional TGF-β type II receptor knockout mouse models of the epithelia (Tgfbr2NKX3.1KO) and stromal fibroblasts (Tgfbr2fspKO) were used. After castration, the prostates of Tgfbr2NKX3.1KO mice had apoptosis levels similar to those expected for control Tgfbr2floxE2/floxE2 mice. Prostates of Tgfbr2fspKO mice, however, had reduced regression and high levels of proliferation associated with canonical Wnt activity throughout the glandular epithelia regardless of androgen status. In contrast, Tgfbr2floxE2/floxE2 prostates had epithelial canonical Wnt activity only in the surviving proximal ducts after castration. In vitro studies showed that androgen antagonist, bicalutamide, transiently elevated both Tgfbr2floxE2/floxE2 and Tgfbr2fspKO stromal expression of Wnt-2, Wnt-3a, and Wnt-5a. The neutralization of Wnt signaling by the expression of secreted frizzled related protein-2 (SFRP-2) resulted in decreased LNCaP prostate epithelial cell proliferation in stromal conditioned media transfer experiments. In vivo tissue recombination studies using Tgfbr2fspKO prostatic stromal cells in combination with wild-type or SV40 large T antigen expressing epithelia resulted in prostates that were refractile to androgen ablation. The expression of SFRP-2 restored the Tgfbr2fspKO-associated prostate responsiveness to androgen ablation. These studies reveal a novel TGF-β, androgen, and Wnt paracrine signaling axis that enables prostatic regression of the distal ducts after androgen ablation while supporting proximal duct survival. [Cancer Res 2008;68(12):4709–18]

Supplementary Figure 4 from Stromal Transforming Growth Factor-β Signaling Mediates Prostatic Response to Androgen Ablation by Paracrine Wnt Activity

Supplementary Figure 2 from Stromal Transforming Growth Factor-β Signaling Mediates Prostatic Response to Androgen Ablation by Paracrine Wnt Activity

Supplementary Figure 3 from Stromal Transforming Growth Factor-β Signaling Mediates Prostatic Response to Androgen Ablation by Paracrine Wnt Activity

PURPOSE Infusing continuity of care into medical student clerkships may accelerate professional development, preserve patient-centered attitudes, and improve primary care training. However, prospective, randomized studies of longitudinal curricula are lacking. METHOD All entering Northwestern University Feinberg School of Medicine students in 2015 and 2016 were randomized to the Education Centered Medical Home (ECMH), a 4-year, team-based primary care clerkship; or a mentored individual preceptorship (IP) for 2 years followed by a traditional 4-week primary care clerkship. Students were surveyed 4 times (baseline, M1, M2, and M3 year [through 2018]); surveys included the Maslach Burnout Inventory (MBI); the Communication, Curriculum, and Culture (C3) survey assessing the hidden curriculum; and the Attitudes Toward Health Care Teams (ATHCT) scale. The authors analyzed results using an intent-to-treat approach. RESULTS Three hundred twenty-nine students were randomized; 316 (96%) participated in surveys. Seventy percent of all respondents would recommend the ECMH to incoming first-year students. ECMH students reported a more positive learning environment (overall quality, 4.4 ECMH vs 4.0 IP, P < .001), greater team-centered attitudes (ATHCT scale, 3.2 vs 3.0, P = .007), less exposure to negative aspects of the hidden curriculum (C3 scale, 4.6 vs 4.3, P < .001), and comparable medical knowledge acquisition. ECMH students established more continuity relationships with patients (2.2 vs 0.3, P < .001) and reported significantly higher professional efficacy (MBI-PE, 4.1 vs 3.9, P = .02). CONCLUSIONS In this randomized medical education trial, the ECMH provided superior primary care training across multiple outcomes compared with a traditional clerkship-based model, including improved professional efficacy.

In current circumstances one rightfully wonders if persistence of SARS-Cov-2 will fundamentally alter the landscape of medical education and hospital training. Absent a vaccine, the prevalence of this virus adds to annual respiratory illnesses caused by seasonal influenza, respiratory syncytial virus, rhinoviruses, and other coronaviruses. Faced with a looming new-normal, many educators are ruminating on how best to ensure rigorous medical training that produces a steady stream of competent physicians. By way of background, the umbrella of medical education covers a highly structured curriculum in a variety of pre-clinical and clinical environments whose architecture and requirements are set by the Liaison Committee for Medical Education (LCME) ( 1 ) and the Accreditation Council for Graduate Medical Education (ACGME) ( 2 ). These requirements reflect established habits for producing quality outcomes. Capricious changes to these requirements can alter the carat of each uncut gem matriculating to medical school. And for this reason, students follow inviolate course work to their doctorate. Graduates can apply for state licensure to become physicians after receiving accredited training as interns and residents. Only later when seen serving a public good are physicians fully vested professionals. Such training can last 7-10 years. Modern training encompasses a well-thought-out system of educational milestones …

The inherent plasticity and resiliency of fibroblasts make this cell type a conventional tool for basic research. But where do they come from, are all fibroblasts the same, and how do they function in disease? The first fibroblast lineages in mammalian development emerge from the ooze of primary mesenchyme during gastrulation. They are cells that efficiently create and negotiate the extracellular matrix of the mesoderm in order to migrate and meet their developmental fate. Mature fibroblasts in epithelial tissues live in the interstitial spaces between basement membranes that spatially delimit complex organ structures. While the function of resident fibroblasts in healthy tissues is largely conjecture, the accumulation of fibroblasts in pathologic lesions offers insight into biologic mechanisms that control their function; fibroblasts are poised to coordinate fibrogenesis in tissue injury, neoplasia, and aging. Here, we examine the developmental origin and plasticity of fibroblasts, their molecular and functional definitions, the epigenetic control underlying their identity and activation, and the evolution of their immune regulatory functions. These topics are reviewed through the lens of fate mapping using genetically engineered mouse models and from the perspective of single-cell RNA sequencing. Recent observations suggest dynamic and heterogeneous functions for fibroblasts that underscore their complex molecular signatures and utility in injured tissues.

Glioma stem cells (GSC) and epithelial–mesenchymal transition (EMT) are strongly associated with therapy resistance and tumor recurrence, but the underlying mechanisms are incompletely understood. Here, we show that S100A4 is a novel biomarker of GSCs. S100A4+ cells in gliomas are enriched with cancer cells that have tumor-initiating and sphere-forming abilities, with the majority located in perivascular niches where GSCs are found. Selective ablation of S100A4-expressing cells was sufficient to block tumor growth in vitro and in vivo. We also identified S100A4 as a critical regulator of GSC self-renewal in mouse and patient-derived glioma tumorspheres. In contrast with previous reports of S100A4 as a reporter of EMT, we discovered that S100A4 is an upstream regulator of the master EMT regulators SNAIL2 and ZEB along with other mesenchymal transition regulators in glioblastoma. Overall, our results establish S100A4 as a central node in a molecular network that controls stemness and EMT in glioblastoma, suggesting S100A4 as a candidate therapeutic target. Cancer Res; 77(19); 5360–73. ©2017 AACR.

The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodelling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, HAND2, MEF2C and TBX5, can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodelling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules.

The mechanisms by which angiotensin II (Ang II) promotes renal fibrosis remain incompletely understood. Ang II both stimulates TGFβ signaling and activates the EGF receptor (EGFR), but the relative contribution of these pathways to renal fibrogenesis is unknown. Using a murine model with EGFR-deficient proximal tubules, we demonstrate that upstream activation of EGFR-dependent ERK signaling is critical for mediating sustained TGFβ expression in renal fibrosis. Persistent activation of the Ang II receptor stimulated ROS-dependent phosphorylation of Src, leading to sustained EGFR-dependent signaling for TGFβ expression. Either genetic or pharmacologic inhibition of EGFR significantly decreased TGFβ-mediated fibrogenesis. We conclude that TGFβ-mediated tissue fibrosis relies on a persistent feed-forward mechanism of EGFR/ERK activation through an unexpected signaling pathway, highlighting EGFR as a potential therapeutic target for modulating tissue fibrogenesis.

The Journal of the American Society of Nephrology (JASN) gives authors submitting original research the option of suggesting qualified reviewers or those they wish to exclude. This historical habit often leaves us wondering whether author preferences correlate with reviewer recommendations and whether differences related to reviewer selection affect decisions by editors. In a self-study presented here, we found that author-suggested reviewers, as a group, make more positive recommendations than editor-suggested reviewers (P = 0.01), although the difference disappears when recommendations are compared with those of editor-suggested reviewers of the same manuscript (P = 0.081). The distribution of recommendations by author-excluded reviewers, as a group, did not differ from those by editor-suggested reviewers; however, author-excluded reviewers impart significantly more negative recommendations than other reviewers of the same manuscript (P = 0.029). We further explored whether such differences result from individual reviewer tendencies to give generally more positive or more negative recommendations than editor-suggested reviewers and found no such tendency. Finally, editorial decisions on manuscripts reviewed by author-suggested or author-excluded reviewers do not differ from those decisions on manuscripts assigned but not reviewed by them. JASN's policy of editors making decisions independent from individual reviewer recommendations minimizes the effect of selection bias on publication decisions.

Although often used interchangeably, dehydration and volume depletion are not synonyms. Dehydration refers to loss of total-body water, producing hypertonicity, which now is the preferred term in lieu of dehydration, whereas volume depletion refers to a deficit in extracellular fluid volume. In particular, hypertonicity implies intracellular volume contraction, whereas volume depletion implies blood volume contraction. Using a case of hyperglycemic hypertonic nonketosis as an example, we examine the changing composition of body fluid spaces to explore the distinction between dehydration and hypertonicity from volume depletion.

The pathologic paradigm for renal progression is advancing tubulointerstitial fibrosis. Whereas mechanisms underlying fibrogenesis have grown in scope and understanding in recent decades, effective human treatment to directly halt or even reverse fibrosis remains elusive. Here, we examine key features mediating the molecular and cellular basis of tubulointerstitial fibrosis and highlight new insights that may lead to novel therapies. How to prevent chronic kidney disease from progressing to renal failure awaits even deeper biochemical understanding.

Background In Goodpasture's disease, circulating autoantibodies bind to the noncollagenous-1 (NC1) domain of type IV collagen in the glomerular basement membrane (GBM). The specificity and molecular architecture of epitopes of tissue-bound autoantibodies are unknown. Alport's post-transplantation nephritis, which is mediated by alloantibodies against the GBM, occurs after kidney transplantation in some patients with Alport's syndrome. We compared the conformations of the antibody epitopes in Goodpasture's disease and Alport's post-transplantation nephritis with the intention of finding clues to the pathogenesis of anti-GBM glomerulonephritis. Methods We used an enzyme-linked immunosorbent assay to determine the specificity of circulating autoantibodies and kidney-bound antibodies to NC1 domains. Circulating antibodies were analyzed in 57 patients with Goodpasture's disease, and kidney-bound antibodies were analyzed in 14 patients with Goodpasture's disease and 2 patients with Alport's post-transplantation nephritis. The molecular architecture of key epitope regions was deduced with the use of chimeric molecules and a three-dimensional model of the alpha345NC1 hexamer. Results In patients with Goodpasture's disease, both autoantibodies to the alpha3NC1 monomer and antibodies to the alpha5NC1 monomer (and fewer to the alpha4NC1 monomer) were bound in the kidneys and lungs, indicating roles for the alpha3NC1 and alpha5NC1 monomers as autoantigens. High antibody titers at diagnosis of anti-GBM disease were associated with ultimate loss of renal function. The antibodies bound to distinct epitopes encompassing region E(A) in the alpha5NC1 monomer and regions E(A) and E(B) in the alpha3NC1 monomer, but they did not bind to the native cross-linked alpha345NC1 hexamer. In contrast, in patients with Alport's post-transplantation nephritis, alloantibodies bound to the E(A) region of the alpha5NC1 subunit in the intact hexamer, and binding decreased on dissociation. Conclusions The development of Goodpasture's disease may be considered an autoimmune "conformeropathy" that involves perturbation of the quaternary structure of the alpha345NC1 hexamer, inducing a pathogenic conformational change in the alpha3NC1 and alpha5NC1 subunits, which in turn elicits an autoimmune response. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases.)

Background and objectives: α- and β-dystroglycan (DG), which link the actin cytoskeleton of the podocyte to the glomerular basement membrane, are maintained in FSGS but decreased in minimal change disease (MCD). Fibrosis has been linked to increased fibroblast-specific protein-1 (FSP1) and epithelial–mesenchymal transition. We studied DG, FSP1, and podocyte differentiation in FSGS variants and cases of suspected FSGS. Design, setting, participants, & measurements: We studied renal biopsies with FSGS, not otherwise specified (NOS), tip lesion, or collapsing variants (COLL), versus secondary FSGS or cases without segmental sclerotic lesions where a diagnosis of MCD versus FSGS could not be established (undefined [UNDEF]) and compared the expression of DG, FSP1, and podocyte Wilms9 tumor antigen (WT1). Results: WT1 is markedly decreased in NOS versus normal and correlates with the extent of sclerosis. α- and β-DG are maintained in most primary and secondary FSGS cases. In contrast, α-DG is significantly decreased in UNDEF, supporting a diagnosis of MCD. Furthermore, follow-up shows remission or decreased proteinuria in four of six of these UNDEF cases in response to therapy. Interstitial FSP1 is numerically highest in COLL but is only rarely found in tubules or podocytes in any other forms of FSGS. Conclusions: We conclude that increased FSP1 may be a marker of the aggressive course of collapsing FSGS. Furthermore, DG staining is a useful adjunct to assist in distinction of FSGS versus MCD in biopsies without defining lesions.

Lung fibroblasts are key mediators of fibrosis resulting in accumulation of excessive interstitial collagen and extracellular matrix, but their origins are not well defined.We aimed to elucidate the contribution of lung epithelium-derived fibroblasts via epithelial-mesenchymal transition (EMT) in the intratracheal bleomycin model.Primary type II alveolar epithelial cells were cultured from Immortomice and exposed to transforming growth factor-beta(1) and epidermal growth factor. Cell fate reporter mice that permanently mark cells of lung epithelial lineage with beta-galactosidase were developed to study EMT, and bone marrow chimeras expressing green fluorescent protein under the control of the fibroblast-associated S100A4 promoter were generated to examine bone marrow-derived fibroblasts. Mice were given intratracheal bleomycin (0.08 unit). Immunostaining was performed for S100A4, beta-galactosidase, green fluorescent protein, and alpha-smooth muscle actin.In vitro, primary type II alveolar epithelial cells undergo phenotypic changes of EMT when exposed to transforming growth factor-beta(1) and epidermal growth factor with loss of prosurfactant protein C and E-cadherin and gain of S100A4 and type I procollagen. In vivo, using cell fate reporter mice, approximately one-third of S100A4-positive fibroblasts were derived from lung epithelium 2 weeks after bleomycin administration. From bone marrow chimera studies, one-fifth of S100A4-positive fibroblasts were derived from bone marrow at this same time point. Myofibroblasts rarely derived from EMT or bone marrow progenitors.Both EMT and bone marrow progenitors contribute to S100A4-positive fibroblasts in bleomycin-induced lung fibrosis. However, neither origin is a principal contributor to lung myofibroblasts.

Background Depletion of glomerular podocytes is an important feature of progressive diabetic nephropathy. Although the most plausible explanation for this podocyte depletion is detachment from the glomerular basement membrane after cellular apoptosis, the mechanism is unclear. Fibroblast-specific protein 1 (FSP1; encoded by the S100A4 gene) is a member of the S100 family of calcium-binding proteins and is constitutively expressed in the cytoplasm of tissue fibroblasts or epithelial cells converted into fibroblasts by means of epithelial-mesenchymal transition. Study Design Retrospective cross-sectional analysis. Settings & Participants 109 patients with type 2 diabetes mellitus, of whom 43 (39%) underwent kidney biopsy. Predictor Clinical stage (4 categories) and histological grade (5 categories) of diabetic nephropathy. Outcome FSP1 expression in podocytes in urine and glomeruli in kidney biopsy specimens. Measurements Immunohistochemistry, real-time polymerase chain reaction, and in situ hybridization. Results 38 of 109 patients (35%) were normoalbuminuric, 16 (15%) had microalbuminuria, 8 (7%) had macroalbuminuria, and 47 (43%) had decreased kidney function. Approximately 95% of podocytes in urine sediment were not apoptotic, and 86% expressed FSP1. The number of FSP1-positive podocytes in urine sediment was significantly larger in patients with macroalbuminuria than in those with normoalbuminuria ( P = 0.03). Intraglomerular expression of FSP1 occurred almost exclusively in podocytes from patients with diabetes, and the number of FSP1-positive podocytes was larger in glomeruli showing diffuse mesangiopathy than in those showing focal mesangiopathy ( P = 0.01). The number also was larger in glomeruli with nodular lesions than in those without nodular lesions ( P Limitations Nonrepresentative study population. Conclusions These results suggest that the appearance of FSP1 in podocytes of patients with diabetes is associated with more severe clinical and pathological findings of diabetic nephropathy, perhaps because of induction of podocyte detachment through epithelial-mesenchymal transition–like phenomena.

A 61-year-old woman was admitted to this hospital because of a 2-month history of abdominal pain and weight loss and an elevated serum creatinine level. On admission, physical examination was normal except for mild abdominal tenderness. Abdominal imaging revealed no specific abnormalities. Laboratory testing revealed anemia. The creatinine level initially fell after the administration of intravenous fluids but then rose and remained elevated. On the 8th day, erythema and pain developed in the left eye. A diagnostic procedure was performed.

Chronic hypoxia accelerates renal fibrosis. The chief mediator of the hypoxic response is hypoxia-inducible factor 1 (HIF-1) and its oxygen-sensitive component HIF-1α. HIF-1 regulates a wide variety of genes, some of which are closely associated with tissue fibrosis. To determine the specific role of HIF-1 in renal fibrosis, we generated a knockout mouse in which tubular epithelial expression of von Hippel-Lindau tumor suppressor (VHL), which acts as a ubiquitin ligase to promote proteolysis of HIF-1α, was targeted. We investigated the effect of VHL deletion (i.e., stable expression of HIF-1α) histologically and used the anti-HIF-1α agent [3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole] (YC-1) to test whether inhibition of HIF-1α could represent a novel approach to treating renal fibrosis. The area of renal fibrosis was significantly increased in a 5/6 renal ablation model of VHL−/−mice and in all VHL−/−mice at least 60 wk of age. Injection of YC-1 inhibited the progression of renal fibrosis in unilateral ureteral obstruction model mice. In conclusion, HIF-1α appears to be a critical contributor to the progression of renal fibrosis and could be a useful target for its treatment.

Mechanisms of androgen dependence of the prostate are critical to understanding prostate cancer progression to androgen independence associated with disease mortality. Transient elevation of transforming growth factor-β (TGF-β) occurs after androgen ablation. To determine the role of TGF-β on prostate response to androgen ablation, conditional TGF-β type II receptor knockout mouse models of the epithelia (Tgfbr2NKX3.1KO) and stromal fibroblasts (Tgfbr2fspKO) were used. After castration, the prostates of Tgfbr2NKX3.1KO mice had apoptosis levels similar to those expected for control Tgfbr2floxE2/floxE2 mice. Prostates of Tgfbr2fspKO mice, however, had reduced regression and high levels of proliferation associated with canonical Wnt activity throughout the glandular epithelia regardless of androgen status. In contrast, Tgfbr2floxE2/floxE2 prostates had epithelial canonical Wnt activity only in the surviving proximal ducts after castration. In vitro studies showed that androgen antagonist, bicalutamide, transiently elevated both Tgfbr2floxE2/floxE2 and Tgfbr2fspKO stromal expression of Wnt-2, Wnt-3a, and Wnt-5a. The neutralization of Wnt signaling by the expression of secreted frizzled related protein-2 (SFRP-2) resulted in decreased LNCaP prostate epithelial cell proliferation in stromal conditioned media transfer experiments. In vivo tissue recombination studies using Tgfbr2fspKO prostatic stromal cells in combination with wild-type or SV40 large T antigen expressing epithelia resulted in prostates that were refractile to androgen ablation. The expression of SFRP-2 restored the Tgfbr2fspKO-associated prostate responsiveness to androgen ablation. These studies reveal a novel TGF-β, androgen, and Wnt paracrine signaling axis that enables prostatic regression of the distal ducts after androgen ablation while supporting proximal duct survival. [Cancer Res 2008;68(12):4709–18]

Hippus is a prominent, repetitive oscillation of the pupils. Although regarded by some as a normal variant of pupillary unrest, the clinical importance of hippus has not been investigated systematically in hospitalized patients.We conducted a retrospective cohort study of 117 hospitalized patients demonstrating hippus. To mitigate observer bias, 486 control patients were selected using 2 adjacent admissions by the same attending physician before and after each index case. The primary outcomes were mortality during the admission and within 30 days of discharge.Patients with bedside hippus were more likely to die within 30 days of observation (P.00005). Independent risk factors for death by 30 days were altered mental status (odds ratio [OR] 4.11; 95% confidence interval [CI], 2.05-8.25, P.001), hippus (OR 2.99; 95% CI, 1.46-6.11, P = .003), cirrhosis (P = .029), and renal disease (P = .054); angiotensin-system inhibitors were protective (P = .012). Patients with hippus were more likely to have altered mental status (OR 11.23; 95% CI, 6.27-20.09, P.001), a history of trauma (OR 3.76; 95% CI, 1.65-8.59, P = .002), cirrhosis (P = .038), renal disease (P = .051), and a history of using iron supplements (P = .016).The recognition of hippus in hospitalized patients is a clinically important predictor of early mortality.

Once in a while, we should compulse over well-established paradigms, and I have been thinking about the terminality of differentiated epithelial cells. There are many believers in terminal differentiation, but if the concept encourages a sense of immutability, then does it cripple new ideas

Anniversaries are times to look back at the path and provenance of what was achieved and to preview what may lie ahead. As JASN celebrates its 25th anniversary of continuous publication this month, its Editors-in-Chief reflect on these issues and quite warmly thank all of JASN ’s authors, readers

Providing a research environment that entwines new medical knowledge with education, training, and clinical care is one of the most important things a medical school can do to improve human health. With new regulations governing compliance with effort reporting on grants, expenditure timing, conflicts of interest, model systems, and clinical trials, research administration has become increasingly more costly, requires specialized knowledge, and can no longer languish without expectations of high-level performance. While our school of medicine is built around a responsibility-centered budget with the university, several years ago we migrated individual units in the school from responsibility-centered budgeting to one where appropriations are made from the dean's office in service of larger academic goals. Many of our units were realigned under new management structures using pods or groupings for research administration to both maximize cost-effectiveness and take advantage of cross-trained administrative talent. We also adopted new management metrics based on transparent data. The most promising approach to growing and managing the research enterprise is wisely investing resources to meet the long-range financial plan of the school and configure that plan to support the goals of the university. In our experience, these objectives are best met through a targeted appropriations model that feeds the intellectual curiosity of faculty in service of our trainees and patients.

For determination of the molecular mechanisms underlying the induction of epithelial cell hypertrophy by angiotensin II (Ang II), a well-characterized porcine renal proximal tubular cell line LLCPKcl4, which does not express endogenous Ang II receptor subtypes, was transfected with cDNA encoding Ang II subtype 1 receptor (AT1R/Cl4). Ang II transactivated the EGF receptor (EGFR) in these AT1R/Cl4 cells, which was blocked by the selective AT1R antagonist losartan but not by the selective AT2R antagonist PD123319. Ang II did not transactivate EGFR in empty vector-transfected LLCPKcl4 cells (Vector/Cl4). Ang II elicited release of soluble heparin-binding EGF-like growth factor (HB-EGF) from AT1R/Cl4 cells, and Ang II-induced EGFR activation was prevented by pretreatment with the specific HB-EGF inhibitor CRM197 or the metalloproteinase inhibitors batimastat or phenanthroline, none of which had any effect on EGFR activation by exogenously administered EGF. Ang II stimulated protein synthesis and cell hypertrophy in AT1R/Cl4 cells without increasing cell number, and signaling studies revealed that Ang II stimulated phosphorylation of the 40S ribosomal protein S6 and the eukaryotic translation initiation factor 4E-binding protein 1, the two downstream target proteins of the mammalian target of rapamycin, which is a central regulator of protein synthesis and cell size. Ang II-induced mammalian target of rapamycin activation, [3H]leucine incorporation, and cellular hypertrophy were inhibited by pretreatment with either batimastat or CRM197 or by pretreatment with rapamycin or the EGFR tyrosine kinase inhibitor AG1478. Ang II also stimulated Smad 2/3 phosphorylation, which was blocked by a selective TGF-beta receptor I kinase inhibitor but not by CRM197. With blockade of TGF-beta receptor, Ang II-mediated hypertrophy was converted into cell proliferation, which was blocked by CRM197. In summary, this is the first demonstration that HB-EGF shedding-dependent EGFR transactivation, along with activation of TGF-beta signaling pathways, mediates Ang II-induced renal tubular epithelial cell hypertrophy.

Fibroblasts are one of the most important and episodically active cell types in the kidney. Under normal conditions, these cells provide a delicate collagenous matrix that partitions the interstitial spaces between nephrons, blood vessels and the renal capsule. Fibroblasts also remodel the interstitium as kidneys grow with age. This episodic activity of various fibroblast populations has a biological basis. Most fibroblasts are created locally through a process called epithelial-mesenchymal transition (EMT) and, once formed, they can proliferate in response to local mitogens. EMT is driven by an alteration in the balance of local cytokine concentrations that reverses the differentiation of selected epithelia along tubular nephrons. During persistent injury and inflammation, fibroblasts further increase their numbers and secrete excess interstitial collagens, and EMT is particularly aggressive in this setting. The mechanisms by which fibroblasts simultaneously destroy normal interstitial architecture and disable epithelial nephrons are more comprehensible today. Recent therapeutic clues for attenuating fibroblast formation during renal fibrogenesis also suggest an advantage in shifting local cytokine balance to favor mesenchymal-epithelial transition. This review examines these issues and identifies new targets for the treatment of one of the most difficult problems facing clinical nephrology.

Various disciplines within nephrology investigate the mechanisms by which kidneys fail. Progress in the areas of glomerular hemodynamics, proteinuria, tubular biology, interstitial nephritis, fibroblast formation, and fibrosis have added kernels of information that together support a unified theory of renal progression. Prevention of progression to end-stage disease has largely focused on control of systemic and glomerular hypertension. Current success in delaying a decline in glomerular filtration rate underlines the promise of a more comprehensive approach. New knowledge about the cell biology of progression also suggests that other adjunctive therapies may be possible. We describe the progress and highlight those spheres where new-targeted interventions may arise.

Rapidly progressive glomerulonephritis in Goodpasture disease is mediated by autoantibodies binding to the non-collagenous NC1 domain of α3(IV) collagen in the glomerular basement membrane. Goodpasture epitopes in the native autoantigen are cryptic (sequestered) within the NC1 hexamers of the α3α4α5(IV) collagen network. The biochemical mechanism for crypticity and exposure for autoantibody binding is not known. We now report that crypticity is a feature of the quaternary structure of two distinct subsets of α3α4α5(IV) NC1 hexamers: autoantibody-reactive M-hexamers containing only monomer subunits and autoantibody-impenetrable D-hexamers composed of both dimer and monomer subunits. Goodpasture antibodies only breach the quaternary structure of M-hexamers, unmasking the cryptic epitopes, whereas D-hexamers are resistant to autoantibodies under native conditions. The epitopes of D-hexamers are structurally sequestered by dimer reinforcement of the quaternary complex, which represents a new molecular solution for conferring immunologic privilege to a potential autoantigen. Dissociation of non-reinforced M-α3α4α5(IV) hexamers by Goodpasture antibodies is a novel mechanism whereby pathogenic autoantibodies gain access to cryptic B cell epitopes. These findings provide fundamental new insights into immune privilege and the molecular mechanisms underlying the pathogenesis of human autoimmune Goodpasture disease.

Antibodies against macrophages that overlap in specificity with fibroblasts. Background Fibroblasts can be misidentified as macrophages because both cell types share antigens that are associated with popular antibodies targeting the monocyte/macrophage lineage. With the recent description of fibroblast-specific protein 1 (FSP1), we revisited the specificity of antibodies directed against macrophages to determine systematically which antibodies best distinguish both cell types in fibrotic tissues. Methods Tissue fibrosis was produced in mice carrying the GFP transgene encoding green fluorescent protein under the control of the FSP1 promoter. Single cell suspensions from these marked tissues were submitted for flow cytometry using antibodies against Mac-1, Mac-2, Mac-3, F4/80, CD68, major histocompatibility complex (MHC) class II, and CD45, and cDNA amplification of mRNA encoding the above target antigens was performed using specific primer sets in sorted pools of cells. Fibrotic tissues were also stained by immunohistochemistry with the same antibodies and examined under confocal microscopy. Results Comparison overlap between FSP1 + fibroblasts with each of the macrophage markers demonstrated that all antimacrophage antibodies (Mac-1, Mac-2, Mac-3, CD68, MHC class II, and CD45) except one (F4/80) recognize both cell types. Conclusion Antibodies directed against F4/80 clearly distinguish macrophages from FSP1 + fibroblasts in fibrotic tissues and is the preferred antibody in mice.

Loss of functioning nephrons stimulates the growth of residual kidney tissue to augment work capacity and maintain normal renal function. This growth largely occurs by hypertrophy rather than from hyperplasia of the remaining nephrons. The signaling mechanisms that increase RNA and protein synthesis during compensatory renal hypertrophy are unknown. This study found that the remaining kidney hypertrophied 42% by 16 d after unilateral nephrectomy (UNX) in DBA/2 mice. Immunoblotting analysis revealed increased phosphorylation of the 40S ribosomal protein S6 (rpS6) and the eukaryotic translation initiation factor (eIF) 4E-binding protein 1 (4E-BP1), the two downstream effectors of the mammalian target of rapamycin (mTOR). The highly specific mTOR inhibitor rapamycin blocked UNX-increased phosphorylation of both rpS6 and 4E-BP1. UNX increased the content of not only 40S and 60S ribosomal subunits but also 80S monosomes and polysomes in the remaining kidney. Administration of rapamycin decreased UNX-induced polysome formation and shifted the polysome profile in the direction of monosomes and ribosomal subunits. Pretreatment of the mice with rapamycin inhibited UNX-induced hypertrophy. These studies demonstrate that activation of the mTOR signaling pathway in the remaining kidney after UNX plays an essential role in modulating RNA and protein synthesis during development of compensatory renal hypertrophy.

Stromal fibroblasts regulate epithelial cell behavior through direct and indirect cell-cell interactions. To clarify the role of TGF-beta signaling in stromal fibroblasts during mammary development and tumorigenesis, we conditionally knocked out the TGF-beta type II receptor gene in mouse mammary fibroblasts (Tgfbr2(fspKO)). Tgfbr2(fspKO) mice exhibit defective mammary ductal development, characterized in part by increased ductal epithelial cell turnover associated with an increase in stromal fibroblast abundance. Tgfbr2(fspKO) mammary fibroblasts transplanted with mammary carcinoma cells promote growth and invasion, which is associated with increased activating phosphorylation of the receptors: erbB1, erbB2, RON, and c-Met. Furthermore, the increased receptor phosphorylation correlates with increased secretion of the cognate ligands by Tgfbr2(fspKO) fibroblasts. Treatment of tumor cells with fibroblast-conditioned medium leads to increased tumor cell proliferation and motility, which are blocked by addition of pharmacologic inhibitors of TGF-alpha signaling or neutralizing antibodies to macrophage-stimulating protein (MSP), HGF, or c-Met. These studies characterize a significant role for stromal TGF-beta signaling in mammary tissue homeostasis and mammary tumor progression via regulation of TGF-alpha, MSP, and HGF signaling pathways.

It is widely accepted that the development of carcinoma--the most common form of human cancer--is due to the accumulation of somatic mutations in epithelial cells. The behaviour of carcinomas is also influenced by the tumour microenvironment, which includes extracellular matrix, blood vasculature, inflammatory cells and fibroblasts. Recent studies reveal that fibroblasts have a more profound influence on the development and progression of carcinomas than was previously appreciated. These new findings have important therapeutic implications.

Purpose of review Tubulointerstitial fibrosis is the final common pathway to end-stage renal disease. Understanding the mechanisms of tubulointerstitial fibrosis is essential in establishing novel therapeutic strategies for the prevention or arrest of progressive kidney diseases. The present review focuses on a newly proposed mechanism of tubulointerstitial fibrosis, one that emphasizes the roles of epithelial-mesenchymal transition and cellular activation. Recent findings Among the cells that accumulate in the renal interstitium, fibroblasts are the principal effectors mediating tubulointerstitial fibrosis. By contrast, the phagocytosis of extracellular matrix and apoptotic cells by macrophages may actually exert a beneficial effect. Interstitial fibroblasts are more heterogeneous than expected, and during renal fibrosis new fibroblasts are derived mainly through epithelial-mesenchymal transition. The intracellular signaling pathways leading to initiation of epithelial-mesenchymal transition remain largely unknown, though recent studies have identified beta-catenin and Smad3 activation of lymphoid enhancer factor, integrin-linked kinase, and small GTPases and mitogen-activated protein kinases as key components. Transforming growth factor-beta is believed to be a critical fibrogenic factor, but recent studies have also focused on transforming growth factor-beta independent pathways as mechanisms of tubulointerstitial fibrosis. As the mechanisms underlying tubulointerstitial fibrosis leading to epithelial-mesenchymal transition have been identified, so have cytokines that efficiently antagonize renal fibrosis, particularly bone morphogenic protein-7 and hepatocyte growth factor. Summary In combination with traditional angiotensin converting enzyme inhibitors, newly identified cytokines may eventually form the basis for new therapeutic strategies aimed at inhibiting the progression of renal disease.

Stromal cells can have a significant impact on the carcinogenic process in adjacent epithelia. The role of transforming growth factor–β (TGF-β) signaling in such epithelial-mesenchymal interactions was determined by conditional inactivation of the TGF-β type II receptor gene in mouse fibroblasts ( Tgfbr2 fspKO ). The loss of TGF-β responsiveness in fibroblasts resulted in intraepithelial neoplasia in prostate and invasive squamous cell carcinoma of the forestomach, both associated with an increased abundance of stromal cells. Activation of paracrine hepatocyte growth factor (HGF) signaling was identified as one possible mechanism for stimulation of epithelial proliferation. Thus, TGF-β signaling in fibroblasts modulates the growth and oncogenic potential of adjacent epithelia in selected tissues.

Epithelial to mesenchymal transition (EMT) is a central mechanism for diversifying the cells found in complex tissues. This dynamic process helps organize the formation of the body plan, and while EMT is well studied in the context of embryonic development, it also plays a role in the genesis of fibroblasts during organ fibrosis in adult tissues. Emerging evidence from studies of renal fibrosis suggests that more than a third of all disease-related fibroblasts originate from tubular epithelia at the site of injury. This review highlights recent advances in the process of EMT signaling in health and disease and how it may be attenuated or reversed by selective cytokines and growth factors.

Selective depletion of fibroblasts preserves morphology and the functional integrity of peritoneum in transgenic mice with peritoneal fibrosing syndrome. Background A peritoneal fibrosing syndrome (PFS) can progressively reduce peritoneal ultrafiltration during chronic peritoneal dialysis in patients with renal failure. The pathogenesis of PFS is unclear and the role of peritoneal fibroblasts has not been evaluated experimentally. Methods We followed the fate of fibroblasts producing PFS in a mouse model using fibroblast-specific protein 1 (FSP1) as a marker. PFS was induced by daily peritoneal infusions of chlorhexidine gluconate (CHG) saline into transgenic mice expressing the thymidine kinase (∆tk) gene under the control of the FSP1 promoter (FSP1.∆tk mice). To demonstrate the role of fibroblasts in PFS, we treated these FSP1.∆tk mice with a nucleoside analogue to induce DNA chain termination and fibroblast death. Results Mice receiving peritoneal infusions of CHG saline every other day for 2 weeks developed increasing numbers of FSP1 + fibroblasts in the subserosal layers of the visceral peritoneum. Mac-3 + monocytes (macrophages) subsequently accumulated over the next 2 weeks in association with increased deposition of type I collagen and increased endothelial vascularity (CD31 + ) in these subserosal tissues. Since these peritoneal fibroblasts expressed monocyte chemoattractant protein-1 (MCP-1), heat shock protein 47 (HSP47), and vascular endothelial growth factor (VEGF), we suspect they were partially responsible for macrophage recruitment, matrix production, and the neoangiogenesis in the subserosal tissue. Treatment of PFS in FSP1.∆tk transgenic mice with a nucleoside analogue selectively reduced the numbers of peritoneal fibroblasts and attenuated the attendant changes in peritoneal histology. Rescuing the peritoneal membrane from chronic thickening and neoangiogenesis by reducing the number of fibroblasts also preserved ultrafiltration. Conclusion Peritoneal fibroblasts play a pivotal role in PFS, and their deletion using a fibroblasts-specific transgene was effective in preventing peritoneal fibrogenesis.

Goodpasture (GP) syndrome is a form of anti-glomerular basement membrane (GBM) disease, in which autoantibodies bind to alpha3(IV) collagen in GBM causing rapidly progressive glomerulonephritis and pulmonary hemorrhage. The conformational GP epitopes have been mapped to 2 regions within the noncollagenous (NC1) domain of the alpha3(IV) chain. Recently, we described the molecular organization of the autoantigen in the native alpha3alpha4alpha5(IV) collagen network of the GBM. The crystal structure of the NC1 domain has revealed how the GP epitopes are sequestered in the native GBM. Further insight into the pathogenesis of disease has been obtained from better animal models. These advances provide a foundation for the development of new specific therapies.