Approach to weight management in patients with advanced chronic kidney disease in a real-life clinical setting.

Objective: Excess adiposity represents a risk factor for chronic kidney disease (CKD) and progression to end-stage kidney disease. Anti-Obesity Medications (AOMs) are vastly underutilized in patients with advanced CKD because of concerns related to safety and efficacy. This study was conducted to evaluate the real-world approach to weight management and the efficacy and safety of AOMs in people with advanced CKD. Methods: This is a retrospective analysis of individuals with Body Mass Index (BMI) ≥ 27 kg/m2 and eGFR ≤ 30 mL/min/1.73 m2 referred to an academic medical weight-management program between 01/2015 and 09/2022. Evaluation of weight-management approaches, body weight change, treatment-related side effects, and reasons for treatment discontinuation were reported. Results: Eighty-nine patients met inclusion criteria, 16 were treated with intensive lifestyle modifications (ILM) alone and 73 with AOMs (all treated with glucagon-like peptide-1 receptor agonist [GLP1-RA] +/- other AOMs) along with ILM. Patients treated with AOMs had a longer duration of on-treatment follow-up (median 924 days) compared to (93 days) the ILM group. Over 75% of patients treated with AOMs lost ≥5% body weight versus 25% of those treated with ILM. Only 15% of patients treated with AOMs discontinued therapy due to treatment-related side effects. Conclusion: In patients with obesity and advanced CKD, GLP-1RA-based anti-obesity treatment was well-tolerated, effective, and led to durable weight reduction.

NDUFS4 regulates cristae remodeling in diabetic kidney disease.

The mitochondrial electron transport chain (ETC) is a highly adaptive process to meet metabolic demands of the cell, and its dysregulation has been associated with diverse clinical pathologies. However, the role and nature of impaired ETC in kidney diseases remains poorly understood. Here, we generate diabetic mice with podocyte-specific overexpression of Ndufs4, an accessory subunit of mitochondrial complex I, as a model investigate the role of ETC integrity in diabetic kidney disease (DKD). We find that conditional male mice with genetic overexpression of Ndufs4 exhibit significant improvements in cristae morphology, mitochondrial dynamics, and albuminuria. By coupling proximity labeling with super-resolution imaging, we also identify the role of cristae shaping protein STOML2 in linking NDUFS4 with improved cristae morphology. Together, we provide the evidence on the central role of NDUFS4 as a regulator of cristae remodeling and mitochondrial function in kidney podocytes. We propose that targeting NDUFS4 represents a promising approach to slow the progression of DKD.

NDUFS4 Regulates Cristae Remodeling in Diabetic Kidney Disease.

The mitochondrial electron transport chain (ETC) is a highly adaptive process to meet metabolic demands of the cell, and its dysregulation has been associated with diverse clinical pathologies. However, the role and nature of impaired ETC in kidney diseases remains poorly understood. Here, we generated diabetic mice with podocyte-specific overexpression of Ndufs4, an accessory subunit of mitochondrial complex I, as a model to investigate the role of ETC integrity in diabetic kidney disease (DKD). We find that these conditional mice exhibit significant improvements in cristae morphology, mitochondrial dynamics, and albuminuria. By coupling proximity labeling with super-resolution imaging, we also identify the role of cristae shaping proteins in linking NDUFS4 with improved cristae morphology. Taken together, we discover the central role of NDUFS4 as a powerful regulator of cristae remodeling, respiratory supercomplexes assembly, and mitochondrial ultrastructure in vitro and in vivo. We propose that targeting NDUFS4 represents a promising approach to slow the progression of DKD.

Evaluating awareness of skin cancer in skin of color organ transplant recipients.

Organ transplant recipients are at high risk for skin cancer. Currently, more than half of the transplant waiting list is composed of skin of color patients. Skin cancer in skin of color is associated with higher morbidity and mortality and has a different clinical presentation and risk factors. Yet, skin cancer prevention resources and efforts are primarily focused on non-skin of color patients. A cross-sectional pilot survey was administered to assess and compare skin cancer attitudes, behaviors, and knowledge especially risk factors and features specific to skin of color between skin of color and non-skin of color organ transplant recipients. Patients from a patient list obtained from the University of Texas Southwestern Medical Center organ transplant center were randomized on Excel and contacted by phone with the choice to participate by phone or online. 219 of 403 patients completed the survey. Skin of color organ transplant recipients was significantly more likely to never practice recommended skin cancer preventative behaviors (p = 0.002, 0.006, 0.02), to hold a lower perceived self-risk (p = 0.02), to worry less about getting skin cancer (p = 0.003), and to have false perceptions about risk factors (p = 0.001, 0.005) in either univariable or multivariable analysis. However, they were more likely (38%, p = 0.02) to recognize human papillomavirus as a risk factor. The knowledge gaps identified can guide the development of skin cancer educational resources that are more comprehensive and relevant to skin of color recipients. This can lead to better outcomes and reduce racial health disparities.

Mitochondrial Regulation of Diabetic Kidney Disease.

The role and nature of mitochondrial dysfunction in diabetic kidney disease (DKD) has been extensively studied. Yet, the molecular drivers of mitochondrial remodeling in DKD are poorly understood. Diabetic kidney cells exhibit a cascade of mitochondrial dysfunction ranging from changes in mitochondrial morphology to significant alterations in mitochondrial biogenesis, biosynthetic, bioenergetics and production of reactive oxygen species (ROS). How these changes individually or in aggregate contribute to progression of DKD remain to be fully elucidated. Nevertheless, because of the remarkable progress in our basic understanding of the role of mitochondrial biology and its dysfunction in DKD, there is great excitement on future targeted therapies based on improving mitochondrial function in DKD. This review will highlight the latest advances in understanding the nature of mitochondria dysfunction and its role in progression of DKD, and the development of mitochondrial targets that could be potentially used to prevent its progression.

PGC1α is required for the renoprotective effect of lncRNA Tug1 in vivo and links Tug1 with urea cycle metabolites.

lncRNA taurine-upregulated gene 1 (Tug1) is a promising therapeutic target in the progression of diabetic nephropathy (DN), but the molecular basis of its protection remains poorly understood. Here, we generate a triple-mutant diabetic mouse model coupled with metabolomic profiling data to interrogate whether Tug1 interaction with peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) is required for mitochondrial remodeling and progression of DN in vivo. We find that, compared with diabetic conditional deletion of Pgc1α in podocytes alone (db/db; Pgc1αPod-f/f), diabetic Pgc1α knockout combined with podocyte-specific Tug1 overexpression (db/db; TugPodTg; Pgc1αPod-f/f) reverses the protective phenotype of Tug1 overexpression, suggesting that PGC1α is required for the renoprotective effect of Tug1. Using unbiased metabolomic profiling, we find that altered urea cycle metabolites and mitochondrial arginase 2 play an important role in Tug1/PGC1α-induced mitochondrial remodeling. Our work identifies a functional role of the Tug1/PGC1α axis on mitochondrial metabolic homeostasis and urea cycle metabolites in experimental models of diabetes.

Role for carbohydrate response element-binding protein (ChREBP) in high glucose-mediated repression of long noncoding RNA Tug1.

Long noncoding RNAs (lncRNAs) have been shown to play key roles in a variety of biological activities of the cell. However, less is known about how lncRNAs respond to environmental cues and what transcriptional mechanisms regulate their expression. Studies from our laboratory have shown that the lncRNA Tug1 (taurine upregulated gene 1) is crucial for the progression of diabetic kidney disease, a major microvascular complication of diabetes. Using a combination of proximity labeling with the engineered soybean ascorbate peroxidase (APEX2), ChIP-qPCR, biotin-labeled oligonucleotide pulldown, and classical promoter luciferase assays in kidney podocytes, we extend our initial observations in the current study and now provide a detailed analysis on a how high-glucose milieu downregulates Tug1 expression in podocytes. Our results revealed an essential role for the transcription factor carbohydrate response element binding protein (ChREBP) in controlling Tug1 transcription in the podocytes in response to increased glucose levels. Along with ChREBP, other coregulators, including MAX dimerization protein (MLX), MAX dimerization protein 1 (MXD1), and histone deacetylase 1 (HDAC1), were enriched at the Tug1 promoter under high-glucose conditions. These observations provide the first characterization of the mouse Tug1 promoter's response to the high-glucose milieu. Our findings illustrate a molecular mechanism by which ChREBP can coordinate glucose homeostasis with the expression of the lncRNA Tug1 and further our understanding of dynamic transcriptional regulation of lncRNAs in a disease state.

Well Plate Integrated Topography Gradient Screening Technology for Studying Cell-Surface Topography Interactions.

New high-throughput technologies for cell-material interaction studies provide researchers with powerful tools to speed up research in the field of biomaterial-cell interactions. However, sharing technologies is often difficult due to the necessity of specific knowledge and experiences. Engineered surfaces can elucidate effects of surface topography on cell behavior, which is of critical value for gaining control over cellular processes. Here, the translation of a gradient-based high-throughput cell screening approach for aligned nano/micro topographies interacting with cells is presented. An aligned topography 96-well plate is created by upscaling of highly specific gradient technology. The resulting cell culture dishes are compatible with general laboratory and imaging equipment, and the platform allows for studying cell behavior with regard to adhesion and alignment. The challenge lies in increasing the dimensions of the previous 1 × 1 cm gradient topography substrate, to be able to cover the span of a 96-well plate and translate it into a standardized cell-screening tool. Adhesion experiments of human bone marrow derived mesenchymal stem cells confirm the standardization, compatibility, and usability of the technology. In the process of using multi-system imaging and analysis, it becomes apparent that future challenges need to include universally applied data analysis approaches.

Shaping Up Mitochondria in Diabetic Nephropathy.

Mitochondrial medicine has experienced significant progress in recent years and is expected to grow significantly in the near future, yielding many opportunities to translate novel bench discoveries into clinical medicine. Multiple lines of evidence have linked mitochondrial dysfunction to a variety of metabolic diseases, including diabetic nephropathy (DN). Mitochondrial dysfunction presumably precedes the emergence of key histologic and biochemical features of DN, which provides the rationale to explore mitochondrial fitness as a novel therapeutic target in patients with DN. Ultimately, the success of mitochondrial medicine is dependent on a better understanding of the underlying biology of mitochondrial fitness and function. To this end, recent advances in mitochondrial biology have led to new understandings of the potential effect of mitochondrial dysfunction in a myriad of human pathologies. We have proposed that molecular mechanisms that modulate mitochondrial dynamics contribute to the alterations of mitochondrial fitness and progression of DN. In this comprehensive review, we highlight the possible effects of mitochondrial dysfunction in DN, with the hope that targeting specific mitochondrial signaling pathways may lead to the development of new drugs that mitigate DN progression. We will outline potential tools to improve mitochondrial fitness in DN as a novel therapeutic strategy. These emerging views suggest that the modulation of mitochondrial fitness could serve as a key target in ameliorating progression of kidney disease in patients with diabetes.

MTHFD2 links RNA methylation to metabolic reprogramming in renal cell carcinoma.

One-carbon metabolism plays a central role in a broad array of metabolic processes required for the survival and growth of tumor cells. However, the molecular basis of how one-carbon metabolism may influence RNA methylation and tumorigenesis remains largely unknown. Here we show MTHFD2, a mitochondrial enzyme involved in one-carbon metabolism, contributes to the progression of renal cell carcinoma (RCC) via a novel epitranscriptomic mechanism that involves HIF-2α. We found that expression of MTHFD2 was significantly elevated in human RCC tissues, and MTHFD2 knockdown strongly reduced xenograft tumor growth. Mechanistically, using an unbiased methylated RNA immunoprecipitation sequencing (meRIP-Seq) approach, we found that MTHFD2 plays a critical role in controlling global N6-methyladenosine (m6A) methylation levels, including the m6A methylation of HIF-2α mRNA, which results in enhanced translation of HIF-2α. Enhanced HIF-2α translation, in turn, promotes the aerobic glycolysis, linking one-carbon metabolism to HIF-2α-dependent metabolic reprogramming through RNA methylation. Our findings also suggest that MTHFD2 and HIF-2α form a positive feedforward loop in RCC, promoting metabolic reprograming and tumor growth. Taken together, our results suggest that MTHFD2 links RNA methylation status to the metabolic state of tumor cells in RCC.

Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice.

Phosphorylation of Dynamin-related protein1 (Drp1) represents an important regulatory mechanism for mitochondrial fission. Here we established the role of Drp1 Serine 600 (S600) phosphorylation on mitochondrial fission in vivo, and assessed the functional consequences of targeted elimination of the Drp1S600 phosphorylation site in progression of diabetic nephropathy (DN). We generated a knockin mouse in which S600 was mutated to alanine (Drp1S600A). We found that diabetic Drp1S600A mice exhibited improved biochemical and histological features of DN along with reduced mitochondrial fission and diminished mitochondrial ROS in vivo. Importantly, we observed that the effect of Drp1S600 phosphorylation on mitochondrial fission in the diabetic milieu was stimulus- but not cell type-dependent. Mechanistically, we showed that mitochondrial fission in high glucose conditions occurs through concomitant binding of phospho-Drp1S600 with mitochondrial fission factor (Mff) and actin-related protein 3 (Arp3), ultimately leading to accumulation of F-actin and Drp1 on the mitochondria. Taken together, these findings establish that a single phosphorylation site in Drp1 can regulate mitochondrial fission and progression of DN in vivo, and highlight the stimulus-specific consequences of Drp1S600 phosphorylation on mitochondrial dynamics.

Plasmonic Gold Nanohole Array for Surface-Enhanced Raman Scattering Detection of DNA Methylation.

Surface-enhanced Raman spectroscopy (SERS), which utilizes nanogaps between noble-metal nanostructures as hot spots to yield ultrasensitive SERS signals, is an outstanding label-free and straightforward tool for DNA methylation analysis. Herein, a plasmonic gold nanohole array (PGNA) with well-controlled hot spots and an open surface was designed as a SERS substrate for DNA methylation detection. A finite-difference time-domain (FDTD) simulation was first employed to investigate the electric field distributions of the PGNA as a function of the geometric parameters. The plasmonic response was tuned to 785 cm-1 to match the ring breathing vibrational band of cytosine, the intensity change of which was revealed to be a marker of DNA methylation. Then, guided by the FDTD simulation results, the PGNA was fabricated via the electron beam lithography (EBL) technique. The fabricated PGNA had an open and easily accessible surface topology, a SERS enhancement factor of ∼106, and a relative standard deviation (RSD) of 7.1% for 500 repetitions over an area of 20 × 20 µm2 using 1 µM Rhodamine 6G as the Raman reporter. The fabricated PGNA was further used as a platform for determining DNA methylation. The proposed method exhibited a sensitivity for detecting 1% of methylation changes. Moreover, insight into the dynamic information on methylation events was obtained by combining principal component analysis (PCA) with 2D correlation spectroscopy analysis. Finally, clear discrimination of the different methylation sites, such as 5-methylcytosine and N6-methyladenine, was demonstrated.

Sensitive Bacterial Detection via Dielectrophoretic-Enhanced Mass Transport Using Surface-Plasmon-Resonance Biosensors.

The performance of surface plasmon resonance (SPR)-based bacterial biosensors is often compromised as a result of diffusion-limited mass transport of bacteria to the sensing surface. In this work, dually functional interdigitated electrodes (IDEs) were developed to sustain SPR and increase bacterial mass transport through external application of dielectrophoresis (DEP). IDEs were defined into 50 nm Au films with fixed electrode gaps ( EG = 5 µm) and varied electrode widths ( EW = 10, 20, and 100 µm),  referred to as interdigitated SPR (iSPR) chips. The iSPR chips with EW = 100 µm effectively supported SPR, with comparable sensitivity to those of conventional SPR chips. The surfaces of iSPR chips ( EW = 100 µm) were modified with mannose to target the FimH adhesin of Escherichia coli and increase cellular adhesion. An LOD of ∼3.0 × 102 CFU/mL E. coli was achieved on mannosylated iSPR chips under positive-DEP conditions, which is about a 5 order of magnitude improvement compared with those of mannosylated conventional SPR chips without DEP. Furthermore, secondary antibody amplification enabled selective enhancement of dilute (103 CFU/mL) E. coli suspensions, whereas no amplification was observed for concentrated (108 CFU/mL) nontarget ( Staphylococcus epidermidis) bacterial suspensions. The results presented here indicate the great potential of the incorporation of DEP into SPR biosensors for rapid, sensitive, and specific detection of bacteria with broad applications in areas of biomedical diagnostics, environmental monitoring, food safety, and homeland security.

Surface-Enhanced Raman Scattering for Rapid Detection and Characterization of Antibiotic-Resistant Bacteria.

As the prevalence of antibiotic-resistant bacteria continues to rise, biosensing technologies are needed to enable rapid diagnosis of bacterial infections. Furthermore, understanding the unique biochemistry of resistance mechanisms can facilitate the development of next generation therapeutics. Surface-enhanced Raman scattering (SERS) offers a potential solution to real-time diagnostic technologies, as well as a route to fundamental, mechanistic studies. In the current review, SERS-based approaches to the detection and characterization of antibiotic-resistant bacteria are covered. The commonly used nanomaterials (nanoparticles and nanostructured surfaces) and surface modifications (antibodies, aptamers, reporters, etc.) for SERS bacterial detection and differentiation are discussed first, and followed by a review of SERS-based detection of antibiotic-resistant bacteria from environmental/food processing and clinical sources. Antibiotic susceptibility testing and minimum inhibitory concentration testing with SERS are then summarized. Finally, recent developments of SERS-based chemical imaging/mapping of bacteria are reviewed.

T Cell-Activating Mesenchymal Stem Cells as a Biotherapeutic for HCC.

The outcome for advanced stage hepatocellular carcinoma (HCC) remains poor, highlighting the need for novel therapies. Genetically modified mesenchymal stem cells (MSCs) are actively being explored as cancer therapeutics due to their inherent ability to migrate to tumor sites. We reasoned that MSCs can be genetically modified to redirect T cells to Glypican-3 (GPC3)+ HCC, and genetically modified these with viral vectors encoding a GPC3/CD3 bispecific T cell engager (GPC3-ENG), a bispecifc T cell engager specific for an irrelevant antigen (EGFRvIII), and/or costimulatory molecules (CD80 and 41BBL). Coculture of GPC3+ cells, GPC3-ENG MSCs, and T cells resulted in T cell activation, as judged by interferon γ (IFNγ) production and killing of tumor cells by T cells. Modification of GPC3-ENG MSCs with CD80 and 41BBL was required for antigen-dependent interleukin-2 (IL-2) production by T cells and resulted in faster tumor cell killing by redirected T cells. In vivo, GPC3-ENG MSCs ± costimulatory molecules had antitumor activity in the HUH7 HCC xenograft model, resulting in a survival advantage. In conclusion, MSCs genetically modified to express GPC3-ENG ± costimulatory molecules redirect T cells to GPC3+ tumor cells and have potent antitumor activity. Thus, further preclinical exploration of our modified approach to GPC3-targeted immunotherapy for HCC is warranted.

The hallmarks of mitochondrial dysfunction in chronic kidney disease.

Recent advances have led to a greater appreciation of how mitochondrial dysfunction contributes to diverse acute and chronic pathologies. Indeed, mitochondria have received increasing attention as a therapeutic target in a variety of diseases because they serve as key regulatory hubs uniquely situated at crossroads between multiple cellular processes. This review provides an overview of the role of mitochondrial dysfunction in chronic kidney disease, with special emphasis on its role in the development of diabetic nephropathy. We examine the current understanding of the molecular mechanisms that cause mitochondrial dysfunction in the kidney and describe the impact of mitochondrial damage on kidney function. The new concept that mitochondrial shape and structure are closely linked with its function in the kidneys is discussed. Furthermore, the mechanisms that translate cellular cues and demands into mitochondrial remodeling and cellular damage, including the role of microRNAs and long noncoding RNAs, are examined with the final goal of identifying mitochondrial targets to improve treatment of patients with chronic kidney diseases.

Long-range surface plasmon resonance and surface-enhanced Raman scattering on X-shaped gold plasmonic nanohole arrays.

A multilayered architecture including a thin Au film supporting an X-shaped nanohole array and a thick continuous Au film separated by a Cytop dielectric layer is reported in this work. Long-range surface plasmon resonance (LR-SPR) was generated at the top Au/water interface, which also resulted in a long-range surface-enhanced Raman scattering (LR-SERS) effect. LR-SPR originates from the coupling of surface plasmons (SPs) propagating along the opposite sides of the thin Au film embedded in a symmetric refractive index environment with Cytop (n = 1.34) and water (n = 1.33). The finite-difference time-domain (FDTD) simulation method was used to investigate the optimal dimensions of the substrate by studying the reflectance spectra and electric field profiles. The calculated optimal structure was then fabricated via electron beam lithography, and its LR-SERS performance was demonstrated by detecting rhodamine 6G and 4-mercaptobenzoic acid in the refractive index-matched environment. We believe that this structure as a LR-SPR or LR-SERS substrate can have broad applications in biosensing.

Real-time in vivo mitochondrial redox assessment confirms enhanced mitochondrial reactive oxygen species in diabetic nephropathy.

While increased mitochondrial reactive oxygen species have been commonly implicated in a variety of disease states, their in vivo role in the pathogenesis of diabetic nephropathy remains controversial. Using a two-photon imaging approach with a genetically encoded redox biosensor, we monitored mitochondrial redox state in the kidneys of experimental models of diabetes in real-time in vivo. Diabetic (db/db) mice that express a redox-sensitive Green Fluorescent Protein biosensor (roGFP) specifically in the mitochondrial matrix (db/dbmt-roGFP) were generated, allowing dynamic monitoring of redox changes in the kidneys. These db/dbmt-roGFP mice exhibited a marked increase in mitochondrial reactive oxygen species in the kidneys. Yeast NADH-dehydrogenase, a mammalian Complex I homolog, was ectopically expressed in cultured podocytes, and this forced expression in roGFP-expressing podocytes prevented high glucose-induced increases in mitochondrial reactive oxygen species. Thus, in vivo monitoring of mitochondrial roGFP in diabetic mice confirms increased production of mitochondrial reactive oxygen species in the kidneys.

Comparative analysis of chimeric ZFP-, TALE- and Cas9-piggyBac transposases for integration into a single locus in human cells.

Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells.