Chronic kidney disease (CKD) affects 8 to 16% people worldwide, with an increasing incidence and prevalence of end-stage kidney disease (ESKD). The effective management of CKD is confounded by the inability to identify patients at high risk of progression while in early stages of CKD. To address this challenge, a renal biopsy transcriptome-driven approach was applied to develop noninvasive prognostic biomarkers for CKD progression. Expression of intrarenal transcripts was correlated with the baseline estimated glomerular filtration rate (eGFR) in 261 patients. Proteins encoded by eGFR-associated transcripts were tested in urine for association with renal tissue injury and baseline eGFR. The ability to predict CKD progression, defined as the composite of ESKD or 40% reduction of baseline eGFR, was then determined in three independent CKD cohorts. A panel of intrarenal transcripts, including epidermal growth factor (EGF), a tubule-specific protein critical for cell differentiation and regeneration, predicted eGFR. The amount of EGF protein in urine (uEGF) showed significant correlation (P < 0.001) with intrarenal EGF mRNA, interstitial fibrosis/tubular atrophy, eGFR, and rate of eGFR loss. Prediction of the composite renal end point by age, gender, eGFR, and albuminuria was significantly (P < 0.001) improved by addition of uEGF, with an increase of the C-statistic from 0.75 to 0.87. Outcome predictions were replicated in two independent CKD cohorts. Our approach identified uEGF as an independent risk predictor of CKD progression. Addition of uEGF to standard clinical parameters improved the prediction of disease events in diverse CKD populations with a wide spectrum of causes and stages.

Exposure of human tumor cell lines to moderate doses of anticancer agents induces terminal proliferation arrest accompanied by morphologic and enzymatic changes that resemble senescence of normal cells. We have investigated the role of p53 and p21 waf1/cip1 in the induction of this response in drug-treated tumor cells. Doxorubicin treatment induced the senescence-like phenotype (SLP) and its associated terminal growth arrest in wild-type HCT116 colon carcinoma cells; this response was strongly decreased but not abolished in HCT116 lines with homozygous knockout of p53 or p21. Transduction of HT1080 ®brosarcoma cells with a genetic inhibitor of p53 also decreased the induction of SLP and increased drug-induced mitotic cell death. To determine if drugstimulated p21 expression was responsible for senescence-like growth arrest, we have expressed dierent levels of p21 from an inducible promoter. While highlevel overexpression of p21 was sucient to induce SLP in HT1080 cells, the levels of p21 expressed in doxorubicin-treated cells could account for only a fraction of doxorubicin-induced SLP. Our results indicate that p53 and p21 act as positive regulators of senescence-like terminal proliferation arrest, but their function is neither sucient nor absolutely required for this treatment response in tumor cells.

The MDR1 P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is a transmembrane ATPase efflux pump for various lipophilic compounds, including many anti-cancer drugs. mAb UIC2, reactive with the extracellular moiety of Pgp, inhibits Pgp-mediated efflux. UIC2 reactivity with Pgp was increased by the addition of several Pgp-transported compounds or ATP-depleting agents, and by mutational inactivation of both nucleotide-binding domains (NBDs) of Pgp. UIC2 binding to Pgp mutated in both NBDs was unaffected in the presence of Pgp transport substrates or in ATP-depleted cells, whereas the reactivities of the wild-type Pgp and Pgps mutated in a single NBD were increased by these treatments to the level of the double mutant. These results indicate the existence of different Pgp conformations associated with different stages of transport-associated ATP hydrolysis and suggest trapping in a transient conformation as a mechanism for antibody-mediated inhibition of Pgp.P-glycoprotein (Pgp), the product of the human MDR1 gene, acts as a broad specificity plasma membrane efflux pump for many hydrophobic compounds (1, 2) and recently was shown to function as a short chain lipid translocase (3). Pgp is a member of a superfamily of ATP-binding cassette (ABC) transporters, characterized by the presence of conserved ABC domains containing consensus nucleotide-binding domain (NBD) sequence motifs (4). ABC transporters of a subgroup that includes the MDR1 Pgp, a closely related MDR2 gene product that acts as a phospholipid translocase (5, 6), the yeast STE6 protein that transports the a pheromone (7), and the cystic fibrosis transmembrane conductance regulator (8), are characterized by a common architecture. These proteins are composed of two halves separated by a ''linker'' region; each half comprises a hydrophobic region with six predicted membrane-spanning segments and the ABC domain.Expression of the MDR1 Pgp in tumor cells is associated with a clinically important phenotype of crossresistance to many structurally diverse anti-cancer drugs, which are pumped out by Pgp. Pgp was shown to bind its transport substrates (9), an event that most probably occurs in the lipid bilayer of the plasma membrane (10), and to hydrolyze ATP (11). The ATPase activity of Pgp is strongly stimulated by the addition of Pgp transport substrates (12). The stoichiometry, temporal sequence, and structural transitions linking the binding and transport of a Pgp substrate with the binding and hydrolysis of ATP are as yet unknown.We previously have developed a mouse mAb UIC2, specific for the extracellular moiety of the human MDR1 Pgp (13). In contrast to several other mAbs that react with Pgp on the surface of intact cells, the addition of UIC2 to tissue culture media decreases the activity of Pgp toward all the tested Pgp transport substrates (13-16). The conformational epitope that is recognized by UIC2 is distinct from the epitopes of the other mAbs, because only UIC2 fails to react with a mutant Pgp that carries a d...

Objectives Major histocompatibility complex (MHC) class II-mediated priming of T and B lymphocytes is a central element of autoimmunity in systemic lupus erythematosus (SLE) and lupus nephritis. The cysteine protease cathepsin S degrades the invariant peptide chain during MHC II assembly with antigenic peptide in antigen-presenting cells; therefore, we hypothesised that cathepsin S inhibition would be therapeutic in SLE. Methods We developed a highly specific small molecule, orally available, cathepsin S antagonist, RO5461111, with suitable pharmacodynamic and pharmacokinetic properties that efficiently suppressed antigen-specific T cell and B cell priming in vitro and in vivo.Results When given to MRL-Fas(lpr) mice with SLE and lupus nephritis, RO5461111 significantly reduced the activation of spleen dendritic cells and the subsequent expansion and activation of CD4 T cells and CD4/CD8 double-negative T cells. Cathepsin S inhibition impaired the spatial organisation of germinal centres, suppressed follicular B cell maturation to plasma cells and Ig class switch. This reversed hypergammaglobulinemia and significantly suppressed the plasma levels of numerous IgG (but not IgM) autoantibodies below baseline, including anti-dsDNA. This effect was associated with less glomerular IgG deposits, which protected kidneys from lupus nephritis. Conclusions Together, cathepsin S promotes SLE by driving MHC class II-mediated T and B cell priming, germinal centre formation and B cell maturation towards plasma cells. These afferent immune pathways can be specifically reversed with the cathepsin S antagonist RO5461111, which prevents lupus nephritis progression even when given after disease onset. This novel therapeutic strategy could correct a common pathomechanism of SLE and other immune complexrelated autoimmune diseases.