Multiomics Analyses Identify AKR1A1 as a Biomarker for Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease. Because many genes associate with DKD, multiomics approaches were used to narrow the list of functional genes, gene products, and related pathways providing insights into the pathophysiological mechanisms of DKD. The Kidney Precision Medicine Project human kidney single-cell RNA-sequencing (scRNA-seq) data set and Mendeley Data on human kidney cortex biopsy proteomics were used. The R package Seurat was used to analyze scRNA-seq data and data from a subset of proximal tubule cells. PathfindR was applied for pathway analysis in cell type-specific differentially expressed genes and the R limma package was used to analyze differential protein expression in kidney cortex. A total of 790 differentially expressed genes were identified in proximal tubule cells, including 530 upregulated and 260 downregulated transcripts. Compared with differentially expressed proteins, 24 genes or proteins were in common. An integrated analysis combining protein quantitative trait loci, genome-wide association study hits (namely, estimated glomerular filtration rate), and a plasma metabolomics analysis was performed using baseline metabolites predictive of DKD progression in our longitudinal Diabetes Heart Study samples. The aldo-keto reductase family 1 member A1 gene (AKR1A1) was revealed as a potential molecular hub for DKD cellular dysfunction in several cross-linked pathways featured by deficiency of this enzyme.

ß2-adrenoreceptor Signaling Increases Therapy Resistance in Prostate Cancer by Upregulating MCL1.

There is accumulating evidence that continuous activation of the sympathetic nervous system due to psychosocial stress increases resistance to therapy and accelerates tumor growth via ß2-adrenoreceptor signaling (ADRB2). However, the effector mechanisms appear to be specific to tumor type. Here we show that activation of ADRB2 by epinephrine, increased in response to immobilization stress, delays the loss of MCL1 apoptosis regulator (MCL1) protein expression induced by cytotoxic drugs in prostate cancer cells; and thus, increases resistance of prostate cancer xenografts to cytotoxic therapies. The effect of epinephrine on MCL1 protein depended on protein kinase A (PKA) activity, but was independent from androgen receptor expression. Furthermore, elevated blood epinephrine levels correlated positively with an increased MCL1 protein expression in human prostate biopsies. In summary, we demonstrate that stress triggers an androgen-independent antiapoptotic signaling via the ADRB2/PKA/MCL1 pathway in prostate cancer cells. IMPLICATIONS: Presented results justify clinical studies of ADRB2 blockers as therapeutics and of MCL1 protein expression as potential biomarker predicting efficacy of apoptosis-targeting drugs in prostate cancer.

Ursodeoxycholate Restores Biliary Excretion of Methotrexate in Rats with Ethinyl Estradiol Induced-Cholestasis by Restoring Canalicular Mrp2 Expression.

The in vivo relevance of ursodeoxycholate (UDCA) treatment (100 mg/kg/day, per oral tid for 5 days before cholestasis induction followed by the same dosing for 5 days) on hepatic function was investigated in rats with 17α-ethinylestradiol (EE, 10 mg/kg, subcutaneous for 5 days)-induced experimental cholestasis. The bile flow rate and the expression level of hepatic multidrug resistance-associated protein 2 (Mrp 2) that were decreased in cholestasis were restored after UDCA treatment. Consistent with this, the biliary excretion clearance (CLexc,bile) of a representative Mrp2 substrate-methotrexate (MTX)-was decreased in cholestatic rats but was restored after UDCA treatment. Consequently, the plasma concentrations of MTX, which were increased by cholestasis, were decreased to control levels by UDCA treatment. Thus, the restoration of CLexc,bile appears to be associated with the increase in Mrp2 expression on the canalicular membrane by UDCA treatment followed by Mrp2-mediated biliary excretion of MTX. On the other hand, the hepatic uptake clearance (CLup,liver) of MTX was unchanged by cholestasis or UDCA treatment, suggestive of the absence of any association between the uptake process and the overall biliary excretion of MTX. Since UDCA has been known to induce the expression of canalicular MRP2 in humans, UDCA treatment might be effective in humans to maintain or accelerate the hepatobiliary elimination of xenobiotics or metabolic conjugates that are MRP2 substrates.

Novel Molecular Multilevel Targeted Antitumor Agents.

A multifunctional fusion protein, IL-13.E13K-D2-NLS, effectively recognizes glioblastoma (GBM) cells and delivers its portion to the cell nucleus. IL-13.E13K-D2-NLS is composed of a cancer cell targeting ligand (IL-13.E13K), specialized cytosol translocation bacterial toxin domain 2 of Pseudomonas exotoxin A (D2) and SV40 T antigen nuclear localization signal (NLS). We have now tested whether we can produce proteins that would serve as a delivery vehicle to lysosomes and mitochondria as well. Moreover, we examined whether IL-13.E13K-D2-NLS can deliver anti-cancer drugs like doxorubicin to their nuclear site of action in cancer cells. We have thus constructed two novel proteins: IL-13.E13K-D2-LLS which incorporates lysosomal localization signal (LLS) of a human lysosomal associated membrane protein (LAMP-1) for targeting to lysosomes and IL-13-D2-KK2, which incorporates a pro-apoptotic peptide (KLAKLAK)2 (KK2) exerting its action in mitochondria. Furthermore, we have produced IL-13.E13K-D2-NLS and IL-13.E13K-D2-LLS versions containing a cysteine for site-specific conjugation with a modified doxorubicin, WP936. We found that single-chain recombinant proteins IL-13.E13K-D2-LLS and IL-13-D2-KK2 are internalized and localized mostly to the lysosomal and mitochondrial compartments, respectively, without major trafficking to cells' nuclei. We also determined that IL-13.E13K-D2-NLS-cys[WP936], IL-13.E13K-D2-LAMP-cys[WP936] and IL-13-D2-KK2 were cytotoxic to GBM cells overexpressing IL-13RA2, while much less cytotoxic to GBM cell lines expressing low levels of the receptor. IL-13.E13K-D2-NLS-cys[WP936] was the most potent of the tested anti-tumor agents including free WP936. We believe that our receptor-directed intracellular organelle-targeted proteins can be employed for numerous specific and safer treatment applications when drugs have specific intracellular sites of their action.

Combination of the PI3K inhibitor ZSTK474 with a PSMA-targeted immunotoxin accelerates apoptosis and regression of prostate cancer.

The phosphoinositide 3-kinase (PI3K) pathway is activated in most advanced prostate cancers, yet so far treatments with PI3K inhibitors have been at best tumorostatic in preclinical cancer models and do not show significant antitumor efficacy in clinical trials. Results from tissue culture experiments in prostate cancer cells suggest that PI3K inhibitors should be combined with other cytotoxic agents; however, the general toxicity of such combinations prevents translating these experimental data into preclinical and clinical models. We investigated the emerging concept of tumor-targeted synthetic lethality in prostate cancer cells by using the pan-PI3K inhibitor ZSTK474 and the immunotoxin J591PE, a protein chimera between the single-chain variable fragment of the monoclonal antibody J591 against the prostate-specific membrane antigen (PSMA) and the truncated form of the Pseudomonas aeruginosa exotoxin A (PE38QQR). The combination of ZSTK474 and J591PE increased apoptosis within 6 hours and cell death (monitored at 24-48 hours) in the PSMA-expressing cells LNCaP, C4-2, and C4-2Luc but not in control cells that do not express PSMA (PC3 and BT549 cells). Mechanistic analysis suggested that induction of apoptosis requires Bcl-2-associated death promoter (BAD) dephosphorylation and decreased expression of myeloid leukemia cell differentiation protein 1 (MCL-1). A single injection of ZSTK474 and J591PE into engrafted prostate cancer C4-2Luc cells led to consistent and stable reduction of luminescence within 6 days. These results suggest that the combination of a PI3K inhibitor and a PSMA-targeted protein synthesis inhibitor toxin represents a promising novel strategy for advanced prostate cancer therapy that should be further investigated.

Genomic distribution of three repetitive DNAs in cultivated hexaploid Diospyros spp. (D. kaki and D. virginiana) and their wild relatives.

To understand the genomic organization of Diospyros species with different ploidy levels, we cloned three different repetitive DNAs and compared their genomic distributions in ten Diospyros species, including hexaploid D. kaki and D. virginiana. Genomic Southern hybridization demonstrated that the EcoRV-repetitive DNA was present in tandem in the genomes of D. glandulosa (2n=2x=30), D. oleifera (2n=2x=30), D. lotus (2n=2x=30), D. virginiana (2n=6x=90) and D. kaki (2n=6x=90). All of these species except D. virginiana also contained the HincII-repetitive DNA in tandem. Fluorescent in situ hybridization showed that the EcoRV- and HincII-repetitive DNAs were predominantly located at the proximal or centromeric regions of chromosomes. The DraI-repetitive sequence cloned from D. ehretioides (2n=2x=30) was not found in the other Diospyros species tested. This suggests that D. ehretioides has a genomic organization different from that of the other Diospyros species. Speciation of hexaploid Diospyros species is also discussed with respect to the genomic distribution of the three repetitive DNAs cloned.