Bone Marrow Mesenchymal Stem Cells Induce Metabolic Plasticity in Estrogen Receptor-Positive Breast Cancer.

Cancer cells reprogram energy metabolism through metabolic plasticity, adapting ATP-generating pathways in response to treatment or microenvironmental changes. Such adaptations enable cancer cells to resist standard therapy. We employed a coculture model of estrogen receptor-positive (ER+) breast cancer and mesenchymal stem cells (MSC) to model interactions of cancer cells with stromal microenvironments. Using single-cell endogenous and engineered biosensors for cellular metabolism, coculture with MSCs increased oxidative phosphorylation, intracellular ATP, and resistance of cancer cells to standard therapies. Cocultured cancer cells had increased MCT4, a lactate transporter, and were sensitive to the MCT1/4 inhibitor syrosingopine. Combining syrosingopine with fulvestrant, a selective estrogen receptor degrading drug, overcame resistance of ER+ breast cancer cells in coculture with MSCs. Treatment with antiestrogenic therapy increased metabolic plasticity and maintained intracellular ATP levels, while MCT1/4 inhibition successfully limited metabolic transitions and decreased ATP levels. Furthermore, MCT1/4 inhibition decreased heterogenous metabolic treatment responses versus antiestrogenic therapy. These data establish MSCs as a mediator of cancer cell metabolic plasticity and suggest metabolic interventions as a promising strategy to treat ER+ breast cancer and overcome resistance to standard clinical therapies. IMPLICATIONS: This study reveals how MSCs reprogram metabolism of ER+ breast cancer cells and point to MCT4 as potential therapeutic target to overcome resistance to antiestrogen drugs.

Estrogen receptor inhibition mediates radiosensitization of ER-positive breast cancer models.

Endocrine therapy (ET) is an effective first-line therapy for women with estrogen receptor-positive (ER + ) breast cancers. While both ionizing radiation (RT) and ET are used for the treatment of women with ER+ breast cancer, the most effective sequencing of therapy and the effect of ET on tumor radiosensitization remains unclear. Here we sought to understand the effects of inhibiting estrogen receptor (ER) signaling in combination with RT in multiple preclinical ER+ breast cancer models. Clonogenic survival assays were performed using variable pre- and post-treatment conditions to assess radiosensitization with estradiol, estrogen deprivation, tamoxifen, fulvestrant, or AZD9496 in ER+ breast cancer cell lines. Estrogen stimulation was radioprotective (radiation enhancement ratios [rER]: 0.51-0.82). Conversely, when given one hour prior to RT, ER inhibition or estrogen depletion radiosensitized ER+ MCF-7 and T47D cells (tamoxifen rER: 1.50-1.60, fulvestrant rER: 1.76-2.81, AZD9496 rER: 1.33-1.48, estrogen depletion rER: 1.47-1.51). Combination treatment resulted in an increase in double-strand DNA (dsDNA) breaks as a result of inhibition of non-homologous end joining-mediated dsDNA break repair with no effect on homologous recombination. Treatment with tamoxifen or fulvestrant in combination with RT also increased the number of senescent cells but did not affect apoptosis or cell cycle distribution. Using an MCF-7 xenograft model, concurrent treatment with tamoxifen and RT was synergistic and resulted in a significant decrease in tumor volume and a delay in time to tumor doubling without significant toxicity. These findings provide preclinical evidence that concurrent treatment with ET and RT may be an effective radiosensitization strategy.

CRYßB2 enhances tumorigenesis through upregulation of nucleolin in triple negative breast cancer.

Expression of ß-crystallin B2 (CRYßB2) is elevated in African American (AA) breast tumors. The underlying mechanisms of CRYßB2-induced malignancy and the association of CRYßB2 protein expression with survival have not yet been described. Here, we report that the expression of CRYßB2 in breast cancer cells increases stemness, growth, and metastasis. Transcriptomics data revealed that CRYßB2 upregulates genes that are functionally associated with unfolded protein response, oxidative phosphorylation, and DNA repair, while down-regulating genes related to apoptosis. CRYßB2 in tumors promotes de-differentiation, an increase in mesenchymal markers and cancer-associated fibroblasts, and enlargement of nucleoli. Proteome microarrays identified a direct interaction between CRYßB2 and the nucleolar protein, nucleolin. CRYßB2 induces nucleolin, leading to the activation of AKT and EGFR signaling. CRISPR studies revealed a dependency on nucleolin for the pro-tumorigenic effects of CRYßB2. Triple-negative breast cancer (TNBC) xenografts with upregulated CRYßB2 are distinctively sensitive to the nucleolin aptamer, AS-1411. Lastly, in AA patients, higher levels of nucleolar CRYßB2 in primary TNBC correlates with decreased survival. In summary, CRYßB2 is upregulated in breast tumors of AA patients and induces oncogenic alterations consistent with an aggressive cancer phenotype. CRYßB2 increases sensitivity to nucleolin inhibitors and may promote breast cancer disparity.

177Lu-labeled low-molecular-weight agents for PSMA-targeted radiopharmaceutical therapy.

PURPOSE: To develop a prostate-specific membrane antigen (PSMA)-targeted radiotherapeutic for metastatic castration-resistant prostate cancer (mCRPC) with optimized efficacy and minimized toxicity employing the ß-particle radiation of 177Lu. METHODS: We synthesized 14 new PSMA-targeted, 177Lu-labeled radioligands (177Lu-L1-177Lu-L14) using different chelating agents and linkers. We evaluated them in vitro using human prostate cancer PSMA(+) PC3 PIP and PSMA(-) PC3 flu cells and in corresponding flank tumor models. Efficacy and toxicity after 8 weeks were evaluated at a single administration of 111 MBq for 177Lu-L1, 177Lu-L3, 177Lu-L5 and 177Lu-PSMA-617. Efficacy of 177Lu-L1 was further investigated using different doses, and long-term toxicity was determined in healthy immunocompetent mice. RESULTS: Radioligands were produced in high radiochemical yield and purity. Cell uptake and internalization indicated specific uptake only in PSMA(+) PC3 cells. 177Lu-L1, 177Lu-L3 and 177Lu-L5 demonstrated comparable uptake to 177Lu-PSMA-617 and 177Lu-PSMA-I&T in PSMA-expressing tumors up to 72 h post-injection. 177Lu-L1, 177Lu-L3 and 177Lu-L5 also demonstrated efficient tumor regression at 8 weeks. 177Lu-L1 enabled the highest survival rate. Necropsy studies of the treated group at 8 weeks revealed subacute damage to lacrimal glands and testes. No radiation nephropathy was observed 1 year post-treatment in healthy mice receiving 111 MBq of 177Lu-L1, most likely related to the fast renal clearance of this agent. CONCLUSIONS: 177Lu-L1 is a viable clinical candidate for radionuclide therapy of PSMA-expressing malignancies because of its high tumor-targeting ability and low off-target radiotoxic effects.

Development of [18F]FPy-WL12 as a PD-L1 Specific PET Imaging Peptide.

Expression of programmed cell death ligand 1 (PD-L1) within tumors is an important biomarker for guiding immune checkpoint therapies; however, immunohistochemistry-based methods of detection fail to provide a comprehensive picture of PD-L1 levels in an entire patient. To facilitate quantification of PD-L1 in the whole body, we developed a peptide-based, high-affinity PD-L1 imaging agent labeled with [18F]fluoride for positron emission tomography (PET) imaging. The parent peptide, WL12, and the nonradioactive analog of the radiotracer, 19FPy-WL12, inhibit PD-1/PD-L1 interaction at low nanomolar concentrations (half maximal inhibitory concentration [IC50], 26-32 nM). The radiotracer, [18F]FPy-WL12, was prepared by conjugating 2,3,5,6-tetrafluorophenyl 6-[18F]fluoronicotinate ([18F]FPy-TFP) to WL12 and assessed for specificity in vitro in 6 cancer cell lines with varying PD-L1 expression. The uptake of the radiotracer reflected the PD-L1 expression assessed by flow cytometry. Next, we performed the in vivo evaluation of [18F]FPy-WL12 in mice bearing cancer xenografts by PET imaging, ex vivo biodistribution, and blocking studies. In vivo data demonstrated a PD-L1-specific uptake of [18F]FPy-WL12 in tumors that is reduced in mice receiving a blocking dose. The majority of [18F]FPy-WL12 radioactivity was localized in the tumors, liver, and kidneys indicating the need for optimization of the labeling strategy to improve the in vivo pharmacokinetics of the radiotracer.

Evaluation of PSMA-Targeted PAMAM Dendrimer Nanoparticles in a Murine Model of Prostate Cancer.

The prostate-specific membrane antigen (PSMA) is a validated target for detection and management of prostate cancer (PC). It has also been utilized for targeted drug delivery through antibody-drug conjugates and polymeric micelles. Polyamidoamine (PAMAM) dendrimers are emerging as a versatile platform in a number of biomedical applications due to their unique physicochemical properties, including small size, large number of reactive terminal groups, bulky interior void volume, and biocompatibility. Here, we report the synthesis of generation 4 PSMA-targeted PAMAM dendrimers [G4(MP-KEU)] and evaluation of their targeting properties in vitro and in vivo using an experimental model of PC. A facile, one-pot synthesis gave nearly neutral nanoparticles with a narrow size distribution of 5 nm in diameter and a molecular weight of 27.3 kDa. They exhibited in vitro target specificity with a dissociation constant ( Kd) of 0.32 ± 0.23 µm and preferential accumulation in PSMA+ PC3 PIP tumors versus isogenic PSMA- PC3 flu tumors. Positron emission tomography-computed tomography imaging and ex vivo biodistribution studies of dendrimers radiolabeled with 64Cu, [64Cu]G4(MP-KEU), demonstrated high accumulation in PSMA+ PC3 PIP tumors at 24 h post-injection (45.83 ± 20.09% injected dose per gram of tissue, %ID/g), demonstrating a PSMA+ PC3 PIP/PSMA- PC3 flu ratio of 7.65 ± 3.35. Specific accumulation of G4(MP-KEU) and [64Cu]G4(MP-KEU) in PSMA+ PC3 PIP tumors was inhibited by the known small-molecule PSMA inhibitor, ZJ-43. On the contrary, G4(Ctrl), control dendrimers without PSMA-targeting moieties, showed comparable low accumulation of ∼1%ID/g in tumors irrespective of PSMA expression, further confirming PSMA+ tumor-specific uptake of G4(MP-KEU). These results suggest that G4(MP-KEU) may represent a suitable scaffold by which to target PSMA-expressing tissues with imaging and therapeutic agents.

Evaluation of 111In-DOTA-5D3, a Surrogate SPECT Imaging Agent for Radioimmunotherapy of Prostate-Specific Membrane Antigen.

5D3 is a new high-affinity murine monoclonal antibody specific for prostate-specific membrane antigen (PSMA). PSMA is a target for the imaging and therapy of prostate cancer. 111In-labeled antibodies have been used as surrogates for 177Lu/90Y-labeled therapeutics. We characterized 111In-DOTA-5D3 by SPECT/CT imaging, tissue biodistribution studies, and dosimetry. Methods: Radiolabeling, stability, cell uptake, and internalization of 111In-DOTA-5D3 were performed by established techniques. Biodistribution and SPECT imaging were done on male nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice bearing human PSMA(+) PC3 PIP and PSMA(-) PC3 flu prostate cancer xenografts on the upper right and left flanks, respectively, at 2, 24, 48, 72, and 192 h after injection. Biodistribution was also evaluated in tumor-free, healthy male CD-1 mice. Blocking studies were performed by coinjection of a 10-fold and 50-fold excess of 5D3 followed by biodistribution at 24 h to determine PSMA binding specificity. The absorbed radiation doses were calculated on the basis of murine biodistribution data, which were translated to a human adult man using organ weights as implemented in OLINDA/EXM. Results:111In-DOTA-5D3 was synthesized with specific activity of approximately 2.24 ± 0.74 MBq/µg (60.54 ± 20 µCi/µg). Distribution of 111In-DOTA-5D3 in PSMA(+) PC3 PIP tumor peaked at 24 h after injection and remained high until 72 h. Uptake in normal tissues, including the blood, spleen, liver, heart, and lungs, was highest at 2 h after injection. Coinjection of 111In-DOTA-5D3 with a 10- and 50-fold excess of nonradiolabeled antibody significantly reduced PSMA(+) PC3 PIP tumor and salivary gland uptake at 24 h but did not reduce uptake in kidneys and lacrimal glands. Significant clearance of 111In-DOTA-5D3 from all organs occurred at 192 h. The highest radiation dose was received by the liver (0.5 mGy/MBq), followed by the spleen and kidneys. Absorbed radiation doses to the salivary and lacrimal glands and bone marrow were low. Conclusion:111In-DOTA-5D3 is a new radiolabeled antibody for imaging and a surrogate for therapy of malignant tissues expressing PSMA.

Peptide-based PET quantifies target engagement of PD-L1 therapeutics.

Immune checkpoint therapies have shown tremendous promise in cancer therapy. However, tools to assess their target engagement, and hence the ability to predict their efficacy, have been lacking. Here, we show that target engagement and tumor-residence kinetics of antibody therapeutics targeting programmed death ligand-1 (PD-L1) can be quantified noninvasively. In computational docking studies, we observed that PD-L1-targeted monoclonal antibodies (atezolizumab, avelumab, and durvalumab) and a high-affinity PD-L1-binding peptide, WL12, have common interaction sites on PD-L1. Using the peptide radiotracer [64Cu]WL12 in vivo, we employed positron emission tomography (PET) imaging and biodistribution studies in multiple xenograft models and demonstrated that variable PD-L1 expression and its saturation by atezolizumab, avelumab, and durvalumab can be quantified independently of biophysical properties and pharmacokinetics of antibodies. Next, we used [64Cu]WL12 to evaluate the impact of time and dose on the unoccupied fraction of tumor PD-L1 during treatment. These quantitative measures enabled, by mathematical modeling, prediction of antibody doses needed to achieve therapeutically effective occupancy (defined as >90%). Thus, we show that peptide-based PET is a promising tool for optimizing dose and therapeutic regimens employing PD-L1 checkpoint antibodies, and can be used for improving therapeutic efficacy.

Peptide-Based 68Ga-PET Radiotracer for Imaging PD-L1 Expression in Cancer.

Tumors create and maintain an immunosuppressive microenvironment that promotes cancer cell escape from immune surveillance. The immune checkpoint protein programmed death-ligand 1 (PD-L1) is expressed in many cancers and is an important contributor to the maintenance of the immunosuppressive tumor microenvironment. PD-L1 is a prominent target for cancer immunotherapy. Guidance of anti-PD-L1 therapy is currently effected through measurement of PD-L1 through biopsy and immunohistochemistry. Here, we report a peptide-based imaging agent, [68Ga]WL12, to detect PD-L1 expression in tumors noninvasively by positron emission tomography (PET). WL12, a cyclic peptide comprising 14 amino acids, binds to PD-L1 with high affinity (IC50≈ 23 nM). Synthesis of [68Ga]WL12 provided radiochemical purity >99% after purification. Biodistribution in immunocompetent mice demonstrated 11.56 ± 3.18, 4.97 ± 0.8, 1.9 ± 0.1, and 1.33 ± 0.21 percentage of injected dose per gram (%ID/g) in hPD-L1, MDAMB231, SUM149, and CHO tumors, respectively, at 1 h postinjection, with high binding specificity noted with coinjection of excess, nonradiolabeled WL12. PET imaging demonstrated high tissue contrast in all tumor models tested.

A PSMA-targeted theranostic agent for photodynamic therapy.

Prostate-specific membrane antigen (PSMA) is over-expressed in the epithelium of prostate cancer and in the neovasculature of many non-prostate solid tumors. PSMA has been increasingly used as a target for cancer imaging and therapy. Here we describe a low-molecular-weight theranostic photosensitizer, YC-9, for PSMA-targeted optical imaging and photodynamic therapy (PDT). YC-9 was synthesized by conjugating IRDye700DX N-hydroxysuccinimide (NHS) ester with a PSMA targeting Lys-Glu urea through a lysine-suberate linker in suitable yield. Optical imaging in vivo demonstrated PSMA-specific tumor uptake of YC-9 with rapid clearance from non-target tissues. PSMA-specific cell kill was demonstrated with YC-9in vitro through PDT in PSMA+ PC3-PIP and PSMA- PC3-flu cells. In vivo PDT in mice bearing PSMA+ PC3-PIP tumors at 4h post-injection of YC-9 (A total of four PDT sessions were performed, 48h apart) resulted in significant tumor growth delay, while tumors in control groups continued to grow. PDT with YC-9 significantly increased the median survival of the PSMA+ PC3-PIP tumor mice (56.5days) compared to control groups [23.5-30.0days, including untreated, light alone, YC-9 alone (without light) and non-targeted IRDye700DX PDT treatment groups], without noticeable toxicity at the doses used. This study proves in principle that YC-9 is a promising therapeutic agent for targeted PDT of PSMA-expressing tissues, such as prostate tumors, and may also be useful against non-prostate tumors by virtue of neovascular PSMA expression.

Rapid PD-L1 detection in tumors with PET using a highly specific peptide.

Molecular imaging can report on the status of the tumor immune microenvironment and guide immunotherapeutic strategies to enhance the efficacy of immune modulation therapies. Imaging agents that can rapidly report on targets of immunomodulatory therapies are few. The programmed death ligand 1 (PD-L1) is an immune checkpoint protein over-expressed in several cancers and contributes to tumor immune suppression. Tumor PD-L1 expression is indicative of tumor response to PD-1 and PD-L1 targeted therapies. Herein, we report a highly specific peptide-based positron emission tomography (PET) imaging agent for PD-L1. We assessed the binding modes of the peptide WL12 to PD-L1 by docking studies, developed a copper-64 labeled WL12 ([64Cu]WL12), and performed its evaluation in vitro, and in vivo by PET imaging, biodistribution and blocking studies. Our results show that [64Cu]WL12 can be used to detect tumor PD-L1 expression specifically and soon after injection of the radiotracer, to fit within the standard clinical workflow of imaging within 60 min of administration.

PD-L1 Detection in Tumors Using [(64)Cu]Atezolizumab with PET.

The programmed death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) pair is a major immune checkpoint pathway exploited by cancer cells to develop and maintain immune tolerance. With recent approvals of anti-PD-1 and anti-PD-L1 therapeutic antibodies, there is an urgent need for noninvasive detection methods to quantify dynamic PD-L1 expression in tumors and to evaluate the tumor response to immune modulation therapies. To address this need, we assessed [(64)Cu]atezolizumab for the detection of PD-L1 expression in tumors. Atezolizumab (MPDL3208A) is a humanized, human and mouse cross-reactive, therapeutic PD-L1 antibody that is being investigated in several cancers. Atezolizumab was conjugated with DOTAGA and radiolabeled with copper-64. The resulting [(64)Cu]atezolizumab was assessed for in vitro and in vivo specificity in multiple cell lines and tumors of variable PD-L1 expression. We performed PET-CT imaging, biodistribution, and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-hPD-L1) and in controls (CHO). Specificity of [(64)Cu]atezolizumab was further confirmed in orthotopic tumor models of human breast cancer (MDAMB231 and SUM149) and in a syngeneic mouse mammary carcinoma model (4T1). We observed specific binding of [(64)Cu]atezolizumab to tumor cells in vitro, correlating with PD-L1 expression levels. Specific accumulation of [(64)Cu]atezolizumab was also observed in tumors with high PD-L1 expression (CHO-hPD-L1 and MDAMB231) compared to tumors with low PD-L1 expression (CHO, SUM149). Collectively, these studies demonstrate the feasibility of using [(64)Cu]atezolizumab for the detection of PD-L1 expression in different tumor types.

[(18)F]Fluoroethyl Triazole Substituted PSMA Inhibitor Exhibiting Rapid Normal Organ Clearance.

Prostate-specific membrane antigen (PSMA) is overexpressed in the epithelium of prostate cancer and nonprostate solid tumor neovasculature. PSMA is increasingly utilized as a target for cancer imaging and therapy. Here, we report the synthesis and in vivo biodistribution of a low-molecular-weight PSMA-based imaging agent, 2-[3-(1-carboxy-5-{3-[1-(2-[(18)F]fluoroethyl)-1H-1,2,3-triazol-yl]propanamido}pentyl)ureido]pentanedioic acid ([(18)F]YC-88), containing an [(18)F]fluoroethyl triazole moiety. [(18)F]YC-88 was synthesized from 2-[(18)F]fluoroethyl azide and the corresponding alkyne precursor in two steps using either a one- or two-pot procedure. Biodistribution and positron emission tomography (PET) imaging were performed in immunocompromised mice using isogenic PSMA(+) PC3 PIP and PSMA(-) PC3 flu xenografts. YC-88 exhibited high affinity for PSMA as evidenced by a Ki value of 12.9 nM. The non-decay corrected radiochemical yields of [(18)F]YC-88 averaged 14 ± 1% (n = 5). Specific radioactivities ranged from 320 to 2,460 Ci/mmol (12-91 GBq/µmol) with an average of 940 Ci/mmol (35 GBq/µmol, n = 5). In an immunocompromised mouse model, [(18)F]YC-88 clearly delineated PSMA(+) PC3 PIP prostate tumor xenografts on imaging with PET. At 1 h postinjection, 47.58 ± 5.19% injected dose per gram of tissue (% ID/g) was evident within the PSMA(+) PC3 PIP tumor, with a ratio of 170:1 of uptake within PSMA(+) PC3 PIP to PSMA(-) PC3 flu tumor placed in the opposite flank. The tumor-to-kidney ratio at 2 h postinjection was 4:1. At or after 30 min postinjection, minimal nontarget tissue uptake of [(18)F]YC-88 was observed. Compared to [(18)F]DCFPyL, which is currently in clinical trials, the uptake of [(18)F]YC-88 within the kidney, liver, and spleen was significantly lower at all time-points studied. At 30 min and 1 h postinjection, salivary gland uptake of [(18)F]YC-88 was significantly less than that of [(18)F]DCFPyL. [(18)F]YC-88 is a new PSMA-targeted PET agent synthesized utilizing click chemistry that demonstrates high PSMA(+) tumor uptake in a xenograft model. Because of its low uptake in the kidney, rapid clearance from nontarget organs, and relatively simple one-pot, two-step radiosynthesis, [(18)F]YC-88 is a viable new PET radiotracer for imaging PSMA-expressing lesions.

A humanized antibody for imaging immune checkpoint ligand PD-L1 expression in tumors.

Antibodies targeting the PD-1/PD-L1 immune checkpoint lead to tumor regression and improved survival in several cancers. PD-L1 expression in tumors may be predictive of response to checkpoint blockade therapy. Because tissue samples might not always be available to guide therapy, we developed and evaluated a humanized antibody for non-invasive imaging of PD-L1 expression in tumors. Radiolabeled [111In]PD-L1-mAb and near-infrared dye conjugated NIR-PD-L1-mAb imaging agents were developed using the mouse and human cross-reactive PD-L1 antibody MPDL3280A. We tested specificity of [111In]PD-L1-mAb and NIR-PD-L1-mAb in cell lines and in tumors with varying levels of PD-L1 expression. We performed SPECT/CT imaging, biodistribution and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-PDL1) and in controls (CHO). Results were confirmed in triple negative breast cancer (TNBC) (MDAMB231 and SUM149) and non-small cell lung cancer (NSCLC) (H2444 and H1155) xenografts with varying levels of PD-L1 expression. There was specific binding of [111In]PD-L1-mAb and NIR-PD-L1-mAb to tumor cells in vitro, correlating with PD-L1 expression levels. In mice bearing subcutaneous and orthotopic tumors, there was specific and persistent high accumulation of signal intensity in PD-L1 positive tumors (CHO-PDL1, MDAMB231, H2444) but not in controls. These results demonstrate that [111In]PD-L1-mAb and NIR-PD-L1-mAb can detect graded levels of PD-L1 expression in human tumor xenografts in vivo. As a humanized antibody, these findings suggest clinical translation of radiolabeled versions of MPDL3280A for imaging. Specificity of NIR-PD-L1-mAb indicates the potential for optical imaging of PD-L1 expression in tumors in relevant pre-clinical as well as clinical settings.

NFkappaB promotes inflammation, coagulation, and fibrosis in the aging glomerulus.

The peak prevalence of ESRD from glomerulosclerosis occurs at 70 to 79 years. To understand why old glomeruli are prone to failure, we analyzed the Fischer 344 rat model of aging under ad libitum-fed (rapid aging) and calorie-restricted (slowed aging) conditions. All glomerular cells contained genes whose expression changed "linearly" during adult life from 2 to 24 months: mesangial cells (e.g., MMP9), endothelial cells (e.g., ICAM and VCAM), parietal epithelial cells (e.g., ceruloplasmin), and podocytes (e.g., nephrin and prepronociceptin). Patterns of aging glomerular gene expression closely resembled atherosclerosis, including activation of endothelial cells, epithelial cells, and macrophages, as well as proinflammatory pathways related to cell adhesion, chemotaxis, blood coagulation, oxidoreductases, matrix metalloproteinases, and TGF-beta activation. We used a nonbiased data-mining approach to identify NFkappaB as the likely transcriptional regulator of these events. We confirmed NFkappaB activation by two independent methods: translocation of NFkappaB p50 to glomerular nuclei and ChIP assays demonstrating NFkappaB p50 binding to the kappaB motif of target genes in old versus young glomeruli. These data suggest that old glomeruli exhibit NFkappaB-associated up-regulation of a proinflammatory, procoagulable, and profibrotic phenotype compared with young glomeruli; these distinctions could explain their enhanced susceptibility to failure. Furthermore, these results provide a potential mechanistic explanation for the close relationship between ESRD and atherosclerotic organ failure as two parallel arms of age-associated NFkappaB-driven processes.

Antibodies to protein tyrosine phosphatase receptor type O (PTPro) increase glomerular albumin permeability (P(alb)).

Glomerular capillary filtration barrier characteristics are determined in part by the slit-pore junctions of glomerular podocytes. Protein tyrosine phosphatase receptor-O (PTPro) is a transmembrane protein expressed on the apical surface of podocyte foot processes. Tyrosine phosphorylation of podocyte proteins including nephrin may control the filtration barrier. To determine whether PTPro activity is required to maintain glomerular macromolecular permeability, albumin permeability (P(alb)) was studied after incubation of glomeruli from normal animals with a series of monoclonal (mAb) and polyclonal antibodies. Reagents included mAbs to rabbit and rat PTPro and polyclonal rabbit immune IgG to rat PTPro. mAb 4C3, specific to the amino acid core of PTPro, decreased its phosphatase activity and increased P(alb) of rabbit glomeruli in a time- and concentration-dependent manner. In contrast, mAb P8E7 did not diminish phosphatase activity and did not alter P(alb). Preincubation of 4C3 with PTPro extracellular domain fusion protein blocked glomerular binding and abolished permeability activity. In parallel experiments, P(alb) of rat glomeruli was increased by two mAbs (1B4 and 1D1) or by polyclonal anti-rat PTPro. We conclude that PTPro interaction with specific antibodies acutely increases P(alb). The identity of the normal ligand for PTPro and of its substrate, as well as the mechanism by which phosphatase activity of this receptor affects the filtration barrier, remain to be determined.

Urine podocyte mRNAs mark progression of renal disease.

Because loss of podocytes associates with glomerulosclerosis, monitoring podocyte loss by measuring podocyte products in urine may be clinically useful. To determine whether a single episode of podocyte injury would cause persistent podocyte loss, we induced limited podocyte depletion using a diphtheria toxin receptor (hDTR) transgenic rat. We monitored podocyte loss by detecting nephrin and podocin mRNA in urine particulates with quantitative reverse transcriptase-PCR. Aquaporin 2 mRNA served as a kidney reference gene to account for variable kidney contribution to RNA amount and quality. We found that a single injection of diphtheria toxin resulted in an initial peak of proteinuria and podocyte mRNAs (podocin and nephrin) followed 8 d later by a second peak of proteinuria and podocyte mRNAs that were podocin positive but nephrin negative. Proteinuria that persisted for months correlated with podocin-positive, nephrin-negative mRNAs in urine. Animals with persistent podocyte mRNA in urine progressed to ESRD with global podocyte depletion and interstitial scarring. Podocytes in ectatic tubules expressed podocalyxin and podocin proteins but not nephrin, compatible with detached podocytes' having an altered phenotype. Parallel human studies showed that biopsy-proven glomerular injury associated with increased urinary podocin:aquaporin 2 and nephrin:aquaporin 2 molar ratios. We conclude that a single episode of podocyte injury can trigger glomerular destabilization, resulting in persistent podocyte loss and an altered phenotype of podocytes recovered from urine. Podocyte mRNAs in urine may be a useful clinical tool for the diagnosis and monitoring of glomerular diseases.

Nephron injury induced by diagnostic ultrasound imaging at high mechanical index with gas body contrast agent.

The right kidney of anesthetized rats was imaged with intermittent diagnostic ultrasound (1.5 MHz; 1-s trigger interval) under exposure conditions simulating those encountered in human perfusion imaging. The rats were infused intravenously with 10 microL/kg/min Definity (Bristol-Myers Squibb Medical Imaging, Inc., N. Billerica, MA, USA) while being exposed to mechanical index (MI) values of up to 1.5 for 1 min. Suprathreshold MI values ruptured glomerular capillaries, resulting in blood filling Bowman's space and proximal convoluted tubules of many nephrons. The re-establishment of a pressure gradient after hemostasis caused the uninjured portions of the glomerular capillaries to resume the production of urinary filtrate, which washed some or all of the erythrocytes out of Bowman's space and cleared blood cells from some nephrons into urine within six hours. However, many of the injured nephrons remained plugged with tightly packed red cell casts 24 h after imaging and also showed degeneration of tubular epithelium, indicative of acute tubular necrosis. The additional damage caused by the extravasated blood amplified that caused by the original cavitating gas body. Human nephrons are virtually identical to those of the rat and so it is probable that similar glomerular capillary rupture followed by transient blockage and/or epithelial degeneration will occur after clinical exposures using similar high MI intermittent imaging with gas body contrast agents. The detection of blood in postimaging urine samples using standard hematuria tests would confirm whether or not clinical protocols need to be developed to avoid this potential for iatrogenic injury.