Focal segmental glomerular sclerosis (FSGS) is an irreversible renal pathology characterized by podocyte detachment from the glomerular basement membrane, hyalinosis, and sclerosis. Clinically, it manifests with proteinuria and progressive loss of glomerular filtration. Primary idiopathic FSGS can occur in isolation and frequently progresses to end-stage renal disease, requiring dialysis or kidney transplantation. In 30-50% of these patients, proteinuria and FSGS recur in the renal allograft, suggesting the presence of a podocyte-toxic factor(s) in the recipient's serum. Currently, there is no reliable way to quantify the serum activity or predict the subset of FSGS patients at risk for recurrence after transplantation. We describe a novel in vitro method that measures the podocyte-toxic activity of sera from FSGS patients using cultured human podocytes; we compare this with the effect of compounds such as adriamycin. Using immunofluorescence microscopy followed by computerized image-processing analysis, we show that incubation of human podocytes with adriamycin leads to a dose-dependent disassembly of focal adhesion complexes (FACs). We then demonstrate that sera from patients with posttransplant recurrent or idiopathic FSGS cause a similar FAC disturbance. In contrast, sera from nonrecurrent FSGS patients do not affect FACs. In some FSGS patients, toxic effects of serum can be prevented by blockade of the tumor necrosis factor-α pathway. We propose that this method may be useful as a diagnostic tool to identify FSGS patients with serum podocyte-toxic activity that presumably places them at increased risk for recurrence in the renal allograft.

Focal segmental glomerulosclerosis (FSGS) is frequently found in biopsies of patients with steroid resistant nephrotic syndrome (SRNS). The pathogenesis of SRNS/FSGS is often unknown and the disease will recur in up to 50% of patients post-transplant, indicating the presence of circulating podocyte-toxic factor(s). Several studies have reported clinical improvement after anti-TNFα therapy. However, prediction of the clinical outcome in SRNS/FSGS is difficult, and novel predictive biomarkers are needed. An image-based assay, which measures disassembly of focal adhesion complexes in cultured podocytes, was used to ascertain the presence of podocyte toxic activity in SRNS/FSGS sera. Expression of TNFα pathway genes was analysed in the Nephroseq FSGS cohort and in cultured podocytes treated with SRNS/FSGS sera. Podocyte toxic activity was detected in 48/96 SRNS/FSGS patients. It did not correlate with serum TNFα levels, age, sex, ethnicity or glomerular filtration rate. In ~25% of the toxic samples, the toxicity was strongly inhibited by blockade of TNFα signaling. Transcriptional profiling of human FSGS biopsies and podocytes treated with FSGS sera revealed significant increases in expression of TNFα pathway genes. We identified patients with serum podocyte toxic activity who may be at risk for FSGS recurrence, and those patients in whom serum podocyte toxicity may be reversed by TNFα blockade. Activation of TNFα pathway genes occurs in podocytes of FSGS patients suggesting a causative effect of this pathway in response to circulating factor(s). In vitro analyses of patient sera may stratify patients according to prognostic outcomes and potential responses to specific clinical interventions.

Our approach allowed categorization of patients into three subgroups: (1) patients with monogenic SRNS; (2) patients with idiopathic SRNS and marked serum podocyte toxicity; and (3) patients without identifiable genetic cause nor evidence of serum podocyte toxicity. Post-transplant SRNS recurrence risk appears to be low in groups 1 and 3, but high in group 2.

Candida glabrata is the second most important human fungal pathogen. Despite its formal name, C. glabrata is in fact more closely related to the nonpathogenic budding yeast Saccharomyces cerevisiae. However, less is known about the biology of this pathogen. Zinc cluster proteins form a large family of transcriptional regulators involved in the regulation of numerous processes such as the control of the metabolism of sugars, amino acids, fatty acids, as well as drug resistance. The C. glabrata genome encodes 41 known or putative zinc cluster proteins, and the majority of them are uncharacterized. We have generated a panel of strains carrying individual deletions of zinc cluster genes. Using a novel approach relying on tetracycline for conditional expression in C. glabrata at the translational level, we show that only two zinc cluster genes are essential. We have performed phenotypic analysis of nonessential zinc cluster genes. Our results show that two deletion strains are thermosensitive whereas two strains are sensitive to caffeine, an inhibitor of the target of rapamycin pathway. Increased salt tolerance has been observed for eight deletion strains, whereas one strain showed reduced tolerance to salt. We have also identified a number of strains with increased susceptibility to the antifungal drugs fluconazole and ketoconazole. Interestingly, one deletion strain showed decreased susceptibility to the antifungal micafungin. In summary, we have assigned phenotypes to more than half of the zinc cluster genes in C. glabrata. Our study provides a resource that will be useful to better understand the biological role of these transcription factors.

ABSTRACTTheCandida albicans MYO5gene encodes myosin I, a protein required for the formation of germ tubes and true hyphae. Because the polarized growth of opaque-phase cells in response to pheromone results in mating projections that can resemble germ tubes, we examined the role of Myo5p in this process. We localized green fluorescent protein (GFP)-tagged Myo5p in opaque-phase cells ofC. albicansduring both bud and shmoo formation. In vegetatively growing opaque cells, Myo5p is found at sites of bud emergence and bud growth, while in pheromone-stimulated cells, Myo5p localizes at the growing tips of shmoos. Intriguingly, cells homozygous forMTLain which theMYO5gene was deleted failed to switch efficiently from the white phase to the opaque phase, although ectopic expression ofWOR1from theMET3promoter can convertmyo5mutants into mating-competent opaque cells. However, whenWOR1expression was shut off, themyo5-defective cells rapidly lost both their opaque phenotype and mating competence, suggesting that Myo5p is involved in the maintenance of the opaque state. WhenMYO5is expressed conditionally in opaque cells, the opaque phenotype, as well as the mating ability of the cells, becomes unstable under repressive conditions, and quantitative real-time PCR demonstrated that the shutoff ofMYO5expression correlates with a dramatic reduction inWOR1expression. It appears that while myosin I is not directly required for mating inC. albicans, it is involved inWOR1expression and the white-opaque transition and thus is indirectly implicated in mating.