Patient-Specific Pharmacokinetics and Dasatinib Nephrotoxicity.

BACKGROUND: Dasatinib has been associated with nephrotoxicity. We sought to examine the incidence of proteinuria on dasatinib and determine potential risk factors that may increase dasatinib-associated glomerular injury. METHODS: We examined glomerular injury through urine albumin-creatinine ratio (UACR) in 82 patients with chronic myelogenous leukemia who were on tyrosine-kinase inhibitor therapy for at least 90 days. t tests were used to compare mean differences in UACR, while regression analysis was used to assess the effects of drug parameters on proteinuria development while on dasatinib. We assayed plasma dasatinib pharmacokinetics using tandem mass spectroscopy and further described a case study of a patient who experienced nephrotic-range proteinuria while on dasatinib. RESULTS: Participants treated with dasatinib ( n =32) had significantly higher UACR levels (median 28.0 mg/g; interquartile range, 11.5-119.5) than participants treated with other tyrosine-kinase inhibitors ( n =50; median 15.0 mg/g; interquartile range, 8.0-35.0; P < 0.001). In total, 10% of dasatinib users exhibited severely increased albuminuria (UACR >300 mg/g) versus zero in other tyrosine-kinase inhibitors. Average steady-state concentrations of dasatinib were positively correlated with UACR ( ρ =0.54, P = 0.03) and duration of treatment ( P = 0.003). There were no associations with elevated BP or other confounding factors. In the case study, kidney biopsy revealed global glomerular damage with diffuse foot process effacement that recovered on termination of dasatinib treatment. CONCLUSIONS: Exposure to dasatinib was associated with a significant chance of developing proteinuria compared with other similar tyrosine-kinase inhibitors. Dasatinib plasma concentration significantly correlated with higher risk of developing proteinuria while receiving dasatinib. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/CJASN/2023_09_08_CJN0000000000000219.mp3.

Dasatinib nephrotoxicity correlates with patient-specific pharmacokinetics.

Introduction: Dasatinib has been associated with nephrotoxicity. We sought to examine the incidence of proteinuria on dasatinib and determine potential risk factors that may increase dasatinib-associated glomerular injury. Methods: We examine glomerular injury via urine albumin-to-creatinine ratio (UACR) in 101 chronic myelogenous leukemia patients who were on tyrosine-kinase inhibitor (TKI) therapy for at least 90 days. We assay plasma dasatinib pharmacokinetics using tandem mass spectroscopy, and further describe a case study of a patient who experienced nephrotic-range proteinuria while on dasatinib. Results: Patients treated with dasatinib (n= 32) had significantly higher UACR levels (median 28.0 mg/g, IQR 11.5 - 119.5) than patients treated with other TKIs (n=50; median 15.0 mg/g, IQR 8.0 - 35.0; p < 0.001). In total, 10% of dasatinib users exhibited severely increased albuminuria (UACR > 300 mg/g) versus zero in other TKIs. Average steady state concentrations of dasatinib were positively correlated with UACR (ρ = 0.54, p = 0.03) as well as duration of treatment ( p =0.003). There were no associations with elevated blood pressure or other confounding factors. In the case study, kidney biopsy revealed global glomerular damage with diffuse foot process effacement that recovered upon termination of dasatinib treatment. Conclusions: Exposure to dasatinib is associated a significant chance of developing proteinuria compared to other similar TKIs. Dasatinib plasma concentration significantly correlates with increased risk of developing proteinuria while receiving dasatinib. Screening for renal dysfunction and proteinuria is strongly advised for all dasatinib patients.

Nicotine, smoking, podocytes, and diabetic nephropathy.

Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease. Besides glycemic and blood pressure control, environmental factors such as cigarette smoking (CS) adversely affect the progression of DN. The effects of CS on DN progression have been attributed to combustion-generated molecules without consideration to the role of nicotine (NIC), responsible for the addictive properties of both CS and electronic cigarettes (ECs). Podocytes are essential to preserve the structure and function of the glomerular filtration barrier, and strong evidence indicates that early podocyte loss promotes DN progression. We performed experiments in human podocytes and in a mouse model of diabetes that develops nephropathy resembling human DN. We determined that NIC binding to podocytes in concentrations achieved with CS and ECs activated NADPH oxidase, which sets in motion a dysfunctional molecular network integrated by cyclooxygenase 2, known to induce podocyte injury; downregulation of AMP-activated protein kinase, important for maintaining cellular energy stores and antioxidation; and upregulation of CD36, which increased lipid uptake and promoted apoptosis. In diabetic mice, NIC increased proteinuria, a recognized marker of chronic kidney disease progression, accompanied by reduced glomerular podocyte synaptopodin, a crucial stabilizer of the podocyte cytoskeleton, and increased fibronectin expression. This novel study critically implicates NIC itself as a contributor to DN progression in CS and EC users.NEW & NOTEWORTHY In this study, we demonstrate that nicotine increases the production of reactive oxygen species, increases cyclooxygenase-2 expression, and upregulates Cd36 while inducing downregulation of AMP-activated protein kinase. In vivo nicotine increases proteinuria and fibronectin expression in diabetic mice. This study demonstrates that effects of nicotine on podocytes are responsible, at least in part, for the deleterious effects of smoking in the progression of chronic kidney disease, including diabetic nephropathy.

LIM-Nebulette Reinforces Podocyte Structural Integrity by Linking Actin and Vimentin Filaments.

BACKGROUND: Maintenance of the intricate interdigitating morphology of podocytes is crucial for glomerular filtration. One of the key aspects of specialized podocyte morphology is the segregation and organization of distinct cytoskeletal filaments into different subcellular components, for which the exact mechanisms remain poorly understood. METHODS: Cells from rats, mice, and humans were used to describe the cytoskeletal configuration underlying podocyte structure. Screening the time-dependent proteomic changes in the rat puromycin aminonucleoside-induced nephropathy model correlated the actin-binding protein LIM-nebulette strongly with glomerular function. Single-cell RNA sequencing and immunogold labeling were used to determine Nebl expression specificity in podocytes. Automated high-content imaging, super-resolution microscopy, atomic force microscopy (AFM), live-cell imaging of calcium, and measurement of motility and adhesion dynamics characterized the physiologic role of LIM-nebulette in podocytes. RESULTS: Nebl knockout mice have increased susceptibility to adriamycin-induced nephropathy and display morphologic, cytoskeletal, and focal adhesion abnormalities with altered calcium dynamics, motility, and Rho GTPase activity. LIM-nebulette expression is decreased in diabetic nephropathy and FSGS patients at both the transcript and protein level. In mice, rats, and humans, LIM-nebulette expression is localized to primary, secondary, and tertiary processes of podocytes, where it colocalizes with focal adhesions as well as with vimentin fibers. LIM-nebulette shRNA knockdown in immortalized human podocytes leads to dysregulation of vimentin filament organization and reduced cellular elasticity as measured by AFM indentation. CONCLUSIONS: LIM-nebulette is a multifunctional cytoskeletal protein that is critical in the maintenance of podocyte structural integrity through active reorganization of focal adhesions, the actin cytoskeleton, and intermediate filaments.

Application of Structure-based Methods to Analyze Resistance Mutations for Chemically Diverse Non-Nucleoside Reverse Transcriptase Inhibitors.

BACKGROUND: Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are used in combination with antiretroviral therapy to suppress viral loads in HIV patients. The chemical design of NNRTIs has changed in recent years in response to resistance-associated mutations (RAMs) and resistance. NNRTIs are chemically diverse compounds that bind an allosteric site of HIV RT. Resistance- associated mutations (RAMs) identified in HIV patients are associated with NNRTI resistance. RAMs confer amino acid changes that alter both structural and physiochemical properties of the allosteric site. Ultimately, these changes reduce NNRTI affinity. Previously, we used a combination of computational and experimental methods to analyze and validate RAMs for 3 diarylpyrimidine (DAPY) NNRTIs. OBJECTIVE: The objective of this study is to apply these methods to other chemically diverse, non- DAPY NNRTIs. MATERIALS AND METHODS: We selected MIV-150 (experimental microbicide) and doravirine for this study. A computational and molecular modeling strategy was used to evaluate the effects of RAMs. Calculated changes in drug affinity and stability (ΔS + ΔA) were used to determine overall resistance levels: susceptible, low, intermediate, and high. The ΔS + ΔA values for K101P suggest that this mutation confers intermediate/high-level resistance to MIV-150, but remains susceptible to doravirine. Based on the determined resistance levels, we analyzed the models and used Molecular Dynamics (MD) to compare the interactions of MIV-150/doravirine with RT wild-type (WT) and RT (K101P). From MD, we found that key interactions were lost with RT (K101P), but were retained with doravirine. To experimentally validate our findings, we conducted a fluorescence-based reverse transcription assay for MIV-150 with RT (WT) and RT (K101P). IC50 values determined in assays showed a 101-fold change in potency for MIV-150, but essentially no change for doravirine. RESULTS: Our computational and experimental results are also consistent with antiviral data reported in the literature. CONCLUSION: We believe that this approach is effective for analyzing mutations to determine resistance profiles for chemically diverse NNRTIs in development.

Arctigenin attenuates diabetic kidney disease through the activation of PP2A in podocytes.

Arctigenin (ATG) is a major component of Fructus Arctii, a traditional herbal remedy that reduced proteinuria in diabetic patients. However, whether ATG specifically provides renoprotection in DKD is not known. Here we report that ATG administration is sufficient to attenuate proteinuria and podocyte injury in mouse models of diabetes. Transcriptomic analysis of diabetic mouse glomeruli showed that cell adhesion and inflammation are two key pathways affected by ATG treatment, and mass spectrometry analysis identified protein phosphatase 2 A (PP2A) as one of the top ATG-interacting proteins in renal cells. Enhanced PP2A activity by ATG reduces p65 NF-κB-mediated inflammatory response and high glucose-induced migration in cultured podocytes via interaction with Drebrin-1. Importantly, podocyte-specific Pp2a deletion in mice exacerbates DKD injury and abrogates the ATG-mediated renoprotection. Collectively, our results demonstrate a renoprotective mechanism of ATG via PP2A activation and establish PP2A as a potential target for DKD progression.

Structure-based methods to predict mutational resistance to diarylpyrimidine non-nucleoside reverse transcriptase inhibitors.

Resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) is a leading cause of HIV treatment failure. Often included in antiviral therapy, NNRTIs are chemically diverse compounds that bind an allosteric pocket of enzyme target reverse transcriptase (RT). Several new NNRTIs incorporate flexibility in order to compensate for lost interactions with amino acid conferring mutations in RT. Unfortunately, even successful inhibitors such as diarylpyrimidine (DAPY) inhibitor rilpivirine are affected by mutations in RT that confer resistance. In order to aid drug design efforts, it would be efficient and cost effective to pre-evaluate NNRTI compounds in development using a structure-based computational approach. As proof of concept, we applied a residue scan and molecular dynamics strategy using RT crystal structures to predict mutations that confer resistance to DAPYs rilpivirine, etravirine, and investigational microbicide dapivirine. Our predictive values, changes in affinity and stability, are correlative with fold-resistance data for several RT mutants. Consistent with previous studies, mutation K101P is predicted to confer high-level resistance to DAPYs. These findings were further validated using structural analysis, molecular dynamics, and an enzymatic reverse transcription assay. Our results confirm that changes in affinity and stability for mutant complexes are predictive parameters of resistance as validated by experimental and clinical data. In future work, we believe that this computational approach may be useful to predict resistance mutations for inhibitors in development.