B cell-derived chronic lymphocytic leukemia (B-CLL) represents a common malignancy whose cell derivation and pathogenesis are unknown. Recent studies have shown that >50% of CLLs display hypermutated ...immunoglobulin variable region (IgV) sequences and a more favorable prognosis, suggesting that they may represent a distinct subset of CLLs which have transited through germinal centers (GCs), the physiologic site of IgV hypermutation. To further investigate the phenotype of CLLs, their cellular derivation and their relationship to normal B cells, we have analyzed their gene expression profiles using oligonucleotide-based DNA chip microarrays representative of approximately 12,000 genes. The results show that CLLs display a common and characteristic gene expression profile that is largely independent of their IgV genotype. Nevertheless, a restricted number of genes (<30) have been identified whose differential expression can distinguish IgV mutated versus unmutated cases and identify them in independent panels of cases. Comparison of CLL profiles with those of purified normal B cell subpopulations indicates that the common CLL profile is more related to memory B cells than to those derived from naive B cells, CD5(+) B cells, and GC centroblasts and centrocytes. Finally, this analysis has identified a subset of genes specifically expressed by CLL cells of potential pathogenetic and clinical relevance.
Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina, and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes ...defined them as "ciliopathies.” However, disease mechanisms remain poorly understood. Here, we identify by whole-exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164, and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. Our findings link degenerative diseases of the kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.
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► Mutations of ZNF423 or CEP164 are causes of retinal-renal ciliopathies ► The gene products colocalize with TIP60 at both centrosomes and nuclear foci ► Knockdown of ZNF423 or CEP164 impairs DNA damage response signaling ► Knockdown of cep164 in zebrafish causes a ciliopathy phenotype and dysregulated DDR
Whole-exome resequencing of individuals with ciliopathies reveals mutations in genes that are involved in DNA damage response signaling, providing new insight into the pathogenic mechanisms behind this class of degenerative disorders.
While significant advances have been made in understanding renal pathophysiology, less is known about the role of glycosphingolipid (GSL) metabolism in driving organ dysfunction. Here, we used a ...small molecule inhibitor of glucosylceramide synthase to modulate GSL levels in three mouse models of distinct renal pathologies: Alport syndrome (Col4a3 KO), polycystic kidney disease (Nek8jck), and steroid-resistant nephrotic syndrome (Nphs2 cKO). At the tissue level, we identified a core immune-enriched transcriptional signature that was shared across models and enriched in human polycystic kidney disease. Single nuclei analysis identified robust transcriptional changes across multiple kidney cell types, including epithelial and immune lineages. To further explore the role of GSL modulation in macrophage biology, we performed in vitro studies with homeostatic and inflammatory bone marrow-derived macrophages. Cumulatively, this study provides a comprehensive overview of renal dysfunction and the effect of GSL modulation on kidney-derived cells in the setting of renal dysfunction.This study identifies shared immune-related gene signatures across kidney cell types in three distinct mouse models of renal disease and examines the role of glycosphingolipid metabolism in renal dysfunction.
Follicular lymphomas (FLs) are neoplastic counterparts of normal germinal center (GC) B cells. FLs are characterized by t(14;18) with deregulation of the Bcl-2 (BCL2) gene. The presence of t(14;18) ...and overexpression of Bcl-2 is necessary, but not sufficient, to cause this disease. An array containing 588 complementary DNAs (cDNAs) was used to compare the gene expression between GC B cells and FL cells. To specifically monitor genes expressed in normal GC B and FL cells and not the entire tissue compartment, normal and malignant B cells were purified from tissues. Using the array, 37 genes were up-regulated and 28 were down-regulated in FL cells as compared to normal GC B cells. The expression level of each differentially expressed gene was verified by quantitative polymerase chain reaction. Following these studies 24 genes were up-regulated and 8 genes down-regulated with a P value less than .1. Included among the genes that were up-regulated in FLs were cell cycle regulator proteins CDK10, p120, p21CIP1, and p16INK4A; transcription factors/regulators Pax-5 and Id-2, which are involved in normal B-cell development; and genes involved in cell-cell interactions, tumor necrosis factor, interleukin-2Rγ (IL-2Rγ), and IL-4Rα. Among the genes that were down-regulated in FLs wereMRP8 and MRP14, which are involved in adhesion. Interestingly, several of these genes are localized within chromosomal regions already described to be altered in FLs. These findings provide a basis for future studies into the pathogenesis and pathophysiology of FL and may lead to the identification of potential therapeutic targets as well as antigens for immunotherapeutic strategies.
A significant population of patients with chronic kidney disease (CKD) develops cardiac hypertrophy, which can lead to heart failure and sudden cardiac death. Soluble klotho (sKL), the shed ...ectodomain of the transmembrane protein klotho, protects the heart against hypertrophic growth. We have shown that sKL protects the heart by regulating the formation and function of lipid rafts by targeting the sialic acid moiety of gangliosides, GM1/GM3. Reduction in circulating sKL contributes to an increased risk of cardiac hypertrophy in mice. sKL replacement therapy has been considered but its use is limited by the inability to mass produce the protein. Therefore, alternative methods to protect the heart are proposed. Glucosylation of ceramide catalyzed by glucosylceramide synthase is the entry step for the formation of gangliosides. Here we show that oral administration of a glucosylceramide synthase inhibitor (GCSi) reduces plasma and heart tissue glycosphingolipids, including gangliosides. Administration of GCSi is protective in two mouse models of cardiac stress-induction, one with isoproterenol overstimulation and the other with 5/6 nephrectomy-induced CKD. Treatment with GCSi does not alter the severity of renal dysfunction and hypertension in CKD. These results provide proof of principle for targeting glucosylceramide synthase to decrease gangliosides as a treatment for cardiac hypertrophy. They also support the hypothesis that sKL protects the heart by targeting gangliosides.
Polycystic kidney disease (PKD) represents a family of genetic disorders characterized by renal cystic growth and progression to kidney failure. No treatment is currently available for people with ...PKD, although possible therapeutic interventions are emerging. Despite genetic and clinical heterogeneity, PKDs have in common defects of cystic epithelia, including increased proliferation, apoptosis and activation of growth regulatory pathways. Sphingolipids and glycosphingolipids are emerging as major regulators of these cellular processes. We sought to evaluate the therapeutic potential for glycosphingolipid modulation as a new approach to treat PKD. Here we demonstrate that kidney glucosylceramide (GlcCer) and ganglioside GM3 levels are higher in human and mouse PKD tissue as compared to normal tissue, regardless of the causative mutation. Blockade of GlcCer accumulation with the GlcCer synthase inhibitor Genz-123346 effectively inhibits cystogenesis in mouse models orthologous to human autosomal dominant PKD (Pkd1 conditional knockout mice) and nephronophthisis (jck and pcy mice). Molecular analysis in vitro and in vivo indicates that Genz-123346 acts through inhibition of the two key pathways dysregulated in PKD: Akt protein kinase-mammalian target of rapamycin signaling and cell cycle machinery. Taken together, our data suggest that inhibition of GlcCer synthesis represents a new and effective treatment option for PKD.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Polycystic kidney diseases (PKDs) are genetic diseases characterized by renal cyst formation with increased cell proliferation, apoptosis, and transition to a secretory phenotype at the expense of ...terminal differentiation. Despite recent progress in understanding PKD pathogenesis and the emergence of potential therapies, the key molecular mechanisms promoting cystogenesis are not well understood. Here, we demonstrate that mechanisms including endoplasmic reticulum stress, oxidative damage, and compromised mitochondrial function all contribute to nephronophthisis-associated PKD. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is emerging as a critical mediator of these cellular processes. Therefore, we reasoned that pharmacological targeting of CaMKII may translate into effective inhibition of PKD in jck mice. Our data demonstrate that CaMKII is activated within cystic kidney epithelia in jck mice. Blockade of CaMKII with a selective inhibitor results in effective inhibition of PKD in jck mice. Mechanistic experiments in vitro and in vivo demonstrated that CaMKII inhibition relieves endoplasmic reticulum stress and oxidative damage and improves mitochondrial integrity and membrane potential. Taken together, our data support CaMKII inhibition as a new and effective therapeutic avenue for the treatment of cystic diseases.
Alport mice serve as an animal model for renal fibrosis. MicroRNA-21 (miR-21) expression has been shown to be increased in the kidneys of Alport syndrome patients. Here, we investigated the ...nephroprotective effects of Lademirsen anti-miR-21 therapy. We used a fast-progressing
mouse model with a 129/SvJ background and an intermediate-progressing F1 hybrid mouse model with a mixed genetic background, with angiotensin-converting enzyme inhibitor (ACEi) monotherapy in combination with anti-miR-21 therapy. In the fast-progressing model, the anti miR-21 and ACEi therapies showed an additive effect in the reduction in fibrosis, the decline of proteinuria, the preservation of kidney function and increased survival. In the intermediate-progressing F1 model, the anti-miR-21 and ACEi therapies individually improved kidney pathology. Both also improved kidney function and survival; however, the combination showed a significant additive effect, particularly for survival. RNA sequencing (RNA-seq) gene expression profiling revealed that the anti-miR-21 and ACEi therapies modulate several common pathways. However, anti-miR-21 was particularly effective at normalizing the expression profiles of the genes involved in renal tubulointerstitial injury pathways. In conclusion, significant additive effects were detected for the combination of anti-miR-21 and ACEi therapies on kidney function, pathology and survival in Alport mouse models, as well as a strong differential effect of anti-miR-21 on the renal expression of fibrotic factors. These results support the addition of anti-miR-21 to the current standard of care (ACEi) in ongoing clinical trials in patients with Alport syndrome.
Polycystic kidney diseases (PKDs) comprise a subgroup of ciliopathies characterized by the formation of fluid-filled kidney cysts and progression to end-stage renal disease. A mechanistic ...understanding of cystogenesis is crucial for the development of viable therapeutic options. Here, we identify CDK5, a kinase active in post mitotic cells, as a new and important mediator of PKD progression. We show that long-lasting attenuation of PKD in the juvenile cystic kidneys (jck) mouse model of nephronophthisis by pharmacological inhibition of CDK5 using either R-roscovitine or S-CR8 is accompanied by sustained shortening of cilia and a more normal epithelial phenotype, suggesting this treatment results in a reprogramming of cellular differentiation. Also, a knock down of Cdk5 in jck cells using small interfering RNA results in significant shortening of ciliary length, similar to what we observed with R-roscovitine. Finally, conditional inactivation of Cdk5 in the jck mice significantly attenuates cystic disease progression and is associated with shortening of ciliary length as well as restoration of cellular differentiation. Our results suggest that CDK5 may regulate ciliary length by affecting tubulin dynamics via its substrate collapsin response mediator protein 2. Taken together, our data support therapeutic approaches aimed at restoration of ciliogenesis and cellular differentiation as a promising strategy for the treatment of renal cystic diseases.
Abstract
Bardet–Biedl syndrome (BBS) is a pleiotropic autosomal recessive ciliopathy affecting multiple organs. The development of potential disease-modifying therapy for BBS will require concurrent ...targeting of multi-systemic manifestations. Here, we show for the first time that monosialodihexosylganglioside accumulates in Bbs2−/− cilia, indicating impairment of glycosphingolipid (GSL) metabolism in BBS. Consequently, we tested whether BBS pathology in Bbs2−/− mice can be reversed by targeting the underlying ciliary defect via reduction of GSL metabolism. Inhibition of GSL synthesis with the glucosylceramide synthase inhibitor Genz-667161 decreases the obesity, liver disease, retinal degeneration and olfaction defect in Bbs2−/− mice. These effects are secondary to preservation of ciliary structure and signaling, and stimulation of cellular differentiation. In conclusion, reduction of GSL metabolism resolves the multi-organ pathology of Bbs2−/− mice by directly preserving ciliary structure and function towards a normal phenotype. Since this approach does not rely on the correction of the underlying genetic mutation, it might translate successfully as a treatment for other ciliopathies.
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