Waldenström Macroglobulinemia and Non-IgM-Type Lymphoplasmacytic Lymphoma Are Genetically Similar.

INTRODUCTION: Waldenström macroglobulinemia (WM) represents a subset of lymphoplasmacytic lymphoma (LPL) with the immunoglobulin (Ig)M paraprotein. MYD88 L265P and CXCR4 mutations are common mutations in WM patients, and mutations in ARID1A and KMT2D (MLL2) have also been reported. However, little information has been accumulated on genetic changes in LPL with other paraproteins like IgG. METHODS: We therefore aimed to evaluate genetic differences between WM and LPL with non-IgM paraprotein (non-IgM-type LPL) using targeted next-generation sequencing (NGS) in 20 Japanese patients (10 with WM, 10 with non-IgM-type LPL). RESULTS: Mutations were detected in ARID1A (10%), CXCR4 (20%), MYD88 (90%), and KMT2D (0%) for WM patients and in ARID1A (10%), CXCR4 (20%), MYD88 (70%), and KMT2D (10%) for non-IgM-type LPL patients. No significant differences were identified. No mutations were detected in NOTCH2, PRDM1, CD274 (PD-L1), PDCD1LG2 (PD-L2), RAG2, MYBBP1A, TP53, or CD79B. DISCUSSION: Mutant allele frequency in MYD88 L265P did not differ significantly between WM and non-IgM-type LPL. Most mutations detected by NGS were subclonal following MYD88 L265P, although one non-IgM-type LPL patient harbored only CXCR4 S338X mutation. Our NGS analyses reveal genetic characteristics in LPL patients and suggest genetic similarities between these two subsets of LPL, WM and non-IgM-type.

Sub-lethal doses of chemotherapeutic agents induce senescence in T cells and upregulation of PD-1 expression.

Cellular senescence is a stable cell cycle arrest, usually in response to internal and/or external stress, including telomere dysfunction, abnormal cellular growth, and DNA damage. Several chemotherapeutic drugs, such as melphalan (MEL) and doxorubicin (DXR), induce cellular senescence in cancer cells. However, it is not clear whether these drugs induce senescence in immune cells. We evaluated the induction of cellular senescence in T cells were derived from human peripheral blood mononuclear cells (PBMNCs) in healthy donors using sub-lethal doses of chemotherapeutic agents. The PBMNCs were kept overnight in RPMI 1640 medium with 2% phytohemagglutinin and 10% fetal bovine serum and then cultured in RPMI 1640 with 20 ng/mL IL-2 and sub-lethal doses of chemotherapeutic drugs (2 µM MEL and 50 nM DXR) for 48 h. Sub-lethal doses of chemotherapeutic agents induced phenotypes associated with senescence, such as the formation of γH2AX nuclear foci, cell proliferation arrest, and induction of senescence-associated beta-galactosidase (SA-ß-Gal) activity, (control vs. MEL, DXR; median mean fluorescence intensity (MFI) 1883 (1130-2163) vs. 2233 (1385-2254), 2406.5 (1377-3119), respectively) in T cells. IL6 and SPP1 mRNA, which are senescence-associated secretory phenotype (SASP) factors, were significantly upregulated by sublethal doses of MEL and DXR compared to the control (P = 0.043 and 0.018, respectively). Moreover, sub-lethal doses of chemotherapeutic agents significantly enhanced the expression of programmed death 1 (PD-1) on CD3 + CD4 + and CD3 + CD8 + T cells compared to the control (CD4 + T cells; P = 0.043, 0.043, and 0.043, respectively, CD8 + T cells; P = 0.043, 0.043, and 0.043, respectively). Our results suggest that sub-lethal doses of chemotherapeutic agents induce senescence in T cells and tumor immunosuppression by upregulating PD-1 expression on T cells.

Myeloma Microenvironmental TIMP1 Induces the Invasive Phenotype in Fibroblasts to Modulate Disease Progression.

Tissue inhibitors of metalloproteinases (TIMPs) are endogenous matrix metalloproteinase inhibitors. TIMP1 is produced by cancer cells and has pleiotropic activities. However, its role and source in multiple myeloma (MM) are unclear. Here, we evaluated TIMP1 protein and mRNA levels in bone marrow (BM) plasma cells and assessed the effects of TIMP1 expression on fibroblast invasive capacity using three-dimensional spheroid cell invasion assays. TIMP1 mRNA and protein levels were elevated when patients progressed from monoclonal gammopathy of undetermined significance or smouldering myeloma to MM. Furthermore, TIMP1 levels decreased at complete response and TIMP1 protein levels increased with higher international staging. TIMP1 mRNA levels were markedly higher in extramedullary plasmacytoma and MM with t(4;14). Overall survival and post-progression survival were significantly lower in MM patients with high TIMP1 protein. Recombinant TIMP1 did not directly affect MM cells but enhanced the invasive capacity of fibroblasts; this effect was suppressed by treatment with anti-TIMP1 antibodies. Fibroblasts supported myeloma cell invasion and expansion in extracellular matrix. Overall, these results suggested that MM-derived TIMP1 induces the invasive phenotype in fibroblasts and is involved in disease progression. Further studies are required to elucidate the specific roles of TIMP1 in MM and facilitate the development of novel therapies targeting the TIMP1 pathway.

DNA damage-induced cellular senescence is regulated by 53BP1 accumulation in the nuclear foci and phase separation.

Cellular senescence is linked to a wide range of age-related diseases and can be triggered by a variety of stresses, including DNA damage. A variety of genotoxic stressors, such as anti-cancer drugs, cause DNA double-strand breaks (DSBs), which trigger the accumulation of the tumour suppressor protein p53 in the nucleus. Cellular stresses stabilize and activate the p53 signalling pathway, which regulates various cellular processes, such as apoptosis, DNA repair, and senescence. Although p53 signalling is a well-known tumour suppressor pathway, it remains unclear how it is regulated during cellular senescence. Here, we show that p53-binding protein 1 (53BP1) accumulation in the nuclear foci is required for DNA damage-induced cellular senescence via p53 activation. In human immortalized fibroblast, shRNA-mediated 53BP1 depletion decreased not only the expression of p53-target genes but also the cellular senescence induced by adriamycin treatment. Furthermore, we confirmed that DSBs trigger the hyperaccumulation of 53BP1 in the nuclear foci, which plays a key role in the regulation of cellular senescence. To prevent the accumulation of 53BP1 in the nuclear foci, we used phase separation inhibitors, and siRNA against RNF168, which accumulates at DSB loci and forms complexes with 53BP1. This blocks the formation of 53BP1 nuclear foci and DNA damage-induced cellular senescence by activating the p53 signaling pathway. In conclusion,   we demonstrated that increased accumulation of 53BP1 in the nuclear foci following DNA damage activates p53 and governs cellular senescence via a liquid-liquid phase separation mechanism.

Clinical significance of human endogenous retrovirus K (HERV-K) in multiple myeloma progression.

Human endogenous retroviruses (HERVs) are retrotransposons that infect human germline cells and occupy 5-8% of the human genome. Their expression, though inhibited by mutation, deletion, and epigenetic mechanisms under normal conditions, is associated with diseases including cancer. This study aimed to clarify the association between HERVs and multiple myeloma (MM) progression. We found that HERV-K envelope (env) and long-term repeat (LTR) expression was statistically significantly higher within plasma cells in MM than in monoclonal gammopathy of undetermined significance or controls. HERV-K env knockdown increased proliferation in the MM.1S cell line and decreased the expression of the tumor suppressor genes TP53 and CDKN1A. TP53 and CDKN1A were highly expressed in MM, and their expression was correlated with HERV-K expression. HERV-K knockdown reduced apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3F, 3G, and 3H expression by 10-20% in MM.1S cells. The anti-retroviral agents nevirapine and nelfinavir suppressed proliferation and increased HERV-K expression in MM cell lines. Our results suggest that HERV-K is involved in MM progression, but its role is likely to go beyond promoting cell proliferation. Clarifying the role of HERV-K in MM will lead to the discovery of novel treatment strategies and supply new insights into MM pathogenesis.

DNA-double strand breaks enhance the expression of major histocompatibility complex class II through the ATM-NF-κΒ-IRF1-CIITA pathway.

Major histocompatibility complex class II (MHC II) is important for the adaptive immune response because MHC II presents processed antigens to a cluster of differentiation 4 (CD4)-positive T-cells. Conventional doses of chemotherapeutic agents induce tumor cell death by causing DNA double-strand breaks (DSBs). However, cellular responses caused by sub-lethal doses of chemotherapeutic agents are poorly understood. In this study, using low doses of chemotherapeutic agents, we showed that DSBs enhanced the expression of MHC II on cells that originate from antigen-presenting cells (APCs). These agents induced the MHC class II transactivator (CIITA), the master regulator of MHC II, and interferon regulatory factor 1 (IRF1), a transcription factor for CIITA. Short hairpin RNA against IRF1 suppressed chemotherapeutic agent-induced CIITA expression, whereas exogenous expression of IRF1 induced CIITA. Inhibition of ataxia-telangiectasia mutated (ATM), a DSB-activated kinase, suppressed induction of IRF1, CIITA, and MHC II. Similar results were observed by inhibiting NF-κB, a downstream target of ATM. These results suggest that DSBs induce MHC II activity via the ATM-NF-κB-IRF1-CIITA pathway in cells that intrinsically present antigens. Additionally, chemotherapeutic agents induced T-cell regulatory molecules. Our findings suggest that chemotherapeutic agents enhance the antigen presentation activity of APCs for T-cell activation.

MYC Causes Multiple Myeloma Progression via Attenuating TP53-Induced MicroRNA-34 Expression.

MicroRNAs (miRNAs and miRs) are small (19-25 base pairs) non-coding RNAs with the ability to modulate gene expression. Previously, we showed that the miR-34 family is downregulated in multiple myeloma (MM) as the cancer progressed. In this study, we aimed to clarify the mechanism of miRNA dysregulation in MM. We focused particularly on the interaction between MYC and the TP53-miR34 axis because there is a discrepancy between increased TP53 and decreased miR-34 expressions in MM. Using the nutlin-3 or Tet-on systems, we caused wild-type (WT) p53 protein accumulation in human MM cell lines (HMCLs) and observed upregulated miR-34 expression. Next, we found that treatment with an Myc inhibitor alone did not affect miR-34 expression levels, but when it was coupled with p53 accumulation, miR-34 expression increased. In contrast, forced MYC activation by the MYC-ER system reduced nutlin-3-induced miR-34 expression. We also observed that TP53 and MYC were negatively correlated with mature miR-34 expressions in the plasma cells of patients with MM. Our results suggest that MYC participates in the suppression of p53-dependent miRNA expressions. Because miRNA expression suppresses tumors, its inhibition leads to MM development and malignant transformation.

IDO2 rs10109853 polymorphism affects the susceptibility to multiple myeloma.

Single-nucleotide polymorphisms (SNPs) of the IDO1 and IDO2 genes have been associated with some diseases. Here, we investigated the association of IDO1 and IDO2 SNPs with the susceptibility to multiple myeloma (MM) and their relationships with MM clinical features. We obtained genomic DNA from 100 patients with MM and 149 healthy race-matched controls and determined IDO1 promoter - 1849G/T (rs3824259) and IDO2 R248W (rs10109853) genotypes by using the polymerase chain reaction-restriction fragment length polymorphism method. The patients with MM had a significantly higher frequency of the IDO2 R248W RR genotype (high-activity type) (59.0% vs. 43.6%, odds ratio = 1.86, 95% confidence interval = 1.11-3.11, P = 0.017) compared with those in healthy controls. Patients with the IDO2 R248W RR genotype (high-activity type) were significantly younger and had a significantly lower frequency of International Staging System (ISS) stage III condition than those with the RW and WW genotypes (median 63 years vs. 69 years, P = 0.025; 15 [25.4%] vs. 50 [48.8%]). In addition, the IDO2 R248W RR genotype was significantly associated with a higher level of hemoglobin at diagnosis (mean ± standard deviation, 10.7 ± 2.36 vs. 9.27 ± 2.40 g/dL; P = 0.0032). Neither polymorphism significantly affected overall survival. Our study indicates that IDO2 R248W may be associated with the susceptibility to MM and severity of anemia.

PARP1 V762A polymorphism affects the prognosis of myelodysplastic syndromes.

OBJECTIVE: Myelodysplastic syndromes (MDS), caused by various genetic mutations in hematopoietic stem cells, are associated with highly variable outcomes. Poly (ADP-ribose) polymerase-1 (PARP1) plays an important role in DNA damage repair and contributes to the progression of several types of cancer. Here, we investigated the impact of PARP1 V762A polymorphism on the susceptibility to and prognosis of MDS. METHODS: Samples collected from 105 MDS patients and 202 race-matched healthy controls were subjected to polymerase chain reaction-restriction fragment length polymorphism for genotyping. RESULTS: The allele and genotype frequencies of PARP1 V762A did not differ between MDS patients and the control group. However, MDS patients with the PARP1 V762A non-AA genotype, which is associated with high gene activity, had shorter overall survival rates (P = .01) than those with the AA genotype. Multivariate analysis of overall survival also revealed PARP1 V762A non-AA genotype as a poor prognostic factor (P = .02). When patients were analyzed according to treatment history, the PARP1 V762A non-AA genotype was only associated with poor survival in patients who had received treatment (P = .02). CONCLUSION: PARP1 V762A polymorphism may be an independent prognostic factor for MDS, and a predictive biomarker for MDS treatment.

PDCD1 and PDCD1LG1 polymorphisms affect the susceptibility to multiple myeloma.

Single-nucleotide polymorphisms (SNPs) of the programmed cell death protein-1 (PDCD1), programmed cell death protein-1 ligand-1 (PDCD1LG1), and cytotoxic T lymphocyte-associated antigen-4 (CTLA4) genes are implicated in the pathogenesis of some cancers. We investigated the role of PDCD1, PDCD1LG1, and CTLA4 SNPs in MM pathogenesis and the susceptibility to and clinical features of multiple myeloma (MM). We obtained genomic DNA from 124 patients with MM and 211 healthy controls and detected PDCD1 (rs36084323, rs41386349, and rs2227982), PDCD1LG1 (rs2297136 and rs4143815), and CTLA4 (rs733618, rs11571316, rs231775, and rs3087243) genotypes using the polymerase chain reaction-restriction fragment length polymorphism method or the TaqMan allelic discrimination real-time PCR method. The patients with MM had a significantly higher frequency of the PDCD1 GCC/GCC haplotype (rs36084323/rs41386349/rs2227982) compared with the healthy controls. PDCD1 rs2227982 CC genotype was associated significantly with a higher frequency of bone lesions. Patients with PDCD1LG1 rs2297136 TT and TC types (high-expression types) showed lower albumin level than those with CC genotype. In addition, the PDCD1LG1 rs4143815 CC and CG types (high-expression types) were associated significantly with higher frequency of patients who were treated with thalidomide and/or bortezomib. However, there was no statistical significance between CTLA4 polymorphisms and clinical variables of patients with MM. There were no significant differences between all the polymorphisms and OS. Our study indicates that the PDCD1 haplotype is associated with a susceptibility to MM. The PDCD1 rs2227982 and PDCD1LG1 rs2297136 affect the clinical features of multiple myeloma patients.

Association between OGG1 S326C CC genotype and elevated relapse risk in acute myeloid leukemia.

Recent studies have shown that tumors of relapsed acute myeloid leukemia (AML) present additional genetic mutations compared to the primary tumors. The base excision repair (BER) pathway corrects oxidatively damaged mutagenic bases and plays an important role in maintaining genetic stability. The purpose of the present study was to investigate the relationship between BER functional polymorphisms and AML relapse. We focused on five major polymorphisms: OGG1 S326C, MUTYH Q324H, APE1 D148E, XRCC1 R194W, and XRCC1 R399Q. Ninety-four adults with AML who achieved first complete remission were recruited. Genotyping was performed with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The OGG1 S326C CC genotype (associated with lower OGG1 activity) was observed more frequently in patients with AML relapse [28.9 vs. 8.9%, odds ratio (OR) = 4.10, 95% confidence interval (CI) = 1.35-12.70, P = 0.01]. Patients with the CC genotype exhibited shorter relapse-free survival (RFS). Moreover, the TCGA database suggested that low OGG1 expression in AML cells is associated with a higher frequency of mutations. The present findings suggest that the OGG1 S326C polymorphism increased the probability of AML relapse and may be useful as a prognostic factor for AML relapse risk.

Short-term administration of recombinant human erythropoietin decreases B cell number in human peripheral blood.

OBJECTIVES: There are conflicting results on the influence of recombinant human erythropoietin (rHuEPO) administration to lymphocytes, especially to B cells. METHODS: We analyzed peripheral white blood cell (WBC) subsets in patients who received one bolus administration of rHuEPO. 119 autologous blood donors were enrolled in this study. Fourty-nine out of them were treated with rHuEPO. Blood samples were obtained before the first phlebotomy and one week later before the second one. By flow cytometry, we measured the numbers of WBC, lymphocytes, dendritic cells, CD4+ T cells, CD8+ T cells, natural killer (NK) cells, B cells, monocytes, and neutrophils, further details of B cell subsets. RESULTS: In the EPO-treatment group, absolute numbers of lymphocytes, especially CD8+ T cells, NK cells, and B cells, significantly decreased after rHuEPO administration. In B cell subsets, absolute numbers of naïve B cells and IgD-CD27- B cells significantly decreased. Other B cell subsets, such as transitional B cells, memory B cells, and marginal zone B cells, also showed a decreasing trend. CONCLUSION: These findings suggest that a single administration of rHuEPO can influence human immune system via reduction of B cell number in peripheral blood.

PDCD1 and CTLA4 polymorphisms affect the susceptibility to, and clinical features of, chronic immune thrombocytopenia.

Programmed death-1 (PD-1, PDCD1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4, CTLA4) play central roles in immune checkpoint pathways. Single nucleotide polymorphisms (SNPs) of PDCD1 and CTLA4 have been reported to be associated with susceptibility to some autoimmune diseases. However, the potential association between SNPs in these immune checkpoint genes and risk of chronic immune thrombocytopenia (cITP) remain controversial and obscure. The aims of this study were to clarify the influence of PDCD1 and CTLA4 SNPs on the risk of developing cITP and its clinical features. We obtained genomic DNA from 119 patients with cITP and 223 healthy controls; their genotypes were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Patients with cITP had a significantly higher frequency of the PDCD1 +7209 TT genotype compared with healthy controls. The CTLA4 -1577 GG genotype and CT60 GG genotype showed higher frequencies of platelet count <5 × 109 /l at diagnosis, minimum platelet count <5 × 109 /l, and bleeding symptoms. Moreover, the PDCD1 -606 AA genotype and +63379 TT genotype were significantly associated with a lower number of patients who achieved a complete response to prednisolone treatment. Our results suggest that the immune checkpoint polymorphisms may affect the susceptibility to the clinical features of cITP, and treatment response of the affected patients.

IL17A and IL23R gene polymorphisms affect the clinical features and prognosis of patients with multiple myeloma.

Single nucleotide polymorphisms (SNPs) in interleukin 17 (IL17A) and IL-23 receptor (IL23R) are involved in the pathogenesis of many cancers and autoimmune diseases. We investigated the influence of IL17A and IL23R SNPs on the risk of developing multiple myeloma (MM) and its clinical features. We obtained genomic DNA from 120 patients with MM and 201 healthy controls and detected IL17A -197 G/A (rs2275913) and IL23R H3Q (rs1884444) genotypes using the polymerase chain reaction-restriction fragment length polymorphism method. There were no significant differences in the genotype and allele frequencies of IL17A -197 G/A and IL23R H3Q between the controls and patients with MM. Compared with the GG and GA genotypes, the IL17A AA genotype was significantly associated with lower hemoglobin levels. The IL23R HH genotype was significantly associated with higher frequency of bone lesions and plasmacytoma than the HQ and QQ genotypes. We observed significant differences in overall survival (OS) between patients treated with thalidomide and/or bortezomib and those treated conventionally. Therefore, we also examined the effect of IL17A and IL23R polymorphisms on the clinical variables and OS in patients treated with thalidomide and/or bortezomib. We observed that the IL23R HH genotype was significantly associated with poor survival compared with the QH and HH genotypes in these patients. Our findings indicate that IL17A -197 G/A and IL23R H3Q are not associated with susceptibility to MM. However, IL-17 and IL-23R polymorphisms may affect severity, bone lesions, and extra-medullary disease in patients with MM. Moreover, IL23R polymorphisms may contribute to poor prognosis in patients with MM treated with thalidomide and/or bortezomib.

The cytokine polymorphisms affecting Th1/Th2 increase the susceptibility to, and severity of, chronic ITP.

BACKGROUND: T-helper cell type 1 (Th1) polarization in chronic immune thrombocytopenia (cITP) has been reported at the protein and mRNA levels. We evaluated the impact of Th1/Th2 cytokine and cytokine receptor functional polymorphisms on both susceptibility to, and severity of, cITP. We analysed IFN-γ + 874 T/A, IFN-γR -611G/A, IL-4 -590C/T, and IL-4Rα Q576R polymorphisms in 126 cITP patients (male/female: 34/92; median age: 47.7 years) and 202 healthy control donors. Genotyping was determined by PCR and direct sequencing. The Th1/Th2 ratio was detected in peripheral blood mononuclear cells via flow cytometry. RESULTS: cITP patients had a higher frequency of the IL-4Rα 576 non-QQ genotype compared to healthy subjects (P = 0.04). cITP patients with the IFN-γ +874 non-AA genotype (high expression type) showed more severe thrombocytopenia than those with the AA genotype (P < 0.05). cITP patients had a significantly higher Th1/Th2 ratio than control patients (P < 0.01); this ratio was inversely correlated with platelet counts. Furthermore, patients with both IFN-γ +874 non-AA genotype (high expression type) and IFN-γR -611 non-AA genotype (high-function type) had a significantly higher Th1/Th2 ratio (P < 0.05). CONCLUSIONS: The cytokine polymorphisms affecting Th1/Th2 increase the susceptibility to, and severity of, chronic ITP.

Polymorphism of IL-10 receptor ß affects the prognosis of multiple myeloma patients treated with thalidomide and/or bortezomib.

Interleukin-10 (IL-10) and IL-10 receptor (IL-10R) single nucleotide polymorphisms have been implicated in the pathogenesis of many cancers. We investigated the influence of IL-10 -592C/A, IL-10RA I224V, and IL-10RB K47E on the risk of developing multiple myeloma (MM) and the clinical features of MM. We extracted the genomic DNA from 128 MM patients and 202 healthy controls and used polymerase chain reaction-restriction fragment length polymorphism method to detect IL-10 promoter -592C/A (rs1800872), IL-10RA (rs2228055), and IL-10RB K47E (rs2834167) genotypes. Overall survival (OS) was defined as the interval from the date of diagnosis to the date of death or last clinical appointment. No statistically significant difference was observed in the genotype and allele frequencies of IL-10 -592C/A, IL-10RA I224V, and IL-10RB K47E between MM patients and healthy controls. IL-10RA II genotype was significantly associated with a hemoglobin level lower than that of IV and VV genotypes (mean ± standard deviation, 9.21 ± 2.46 vs 10.3 ± 2.33 g/dL; P = .021). IL-10 -592 AA genotype was significantly associated with OS better than that of CA and CC genotypes (median OS, 74.5 vs 46.3 months; P = .047). We observed significant differences in survival between patients treated with thalidomide and/or bortezomib and those treated with conventional treatments (median OS, 74.5 vs 38.2 months; P = .021). Therefore, we also examined the effect of IL-10 and IL-10R polymorphisms on the clinical variables and OS of patients treated with thalidomide and/or bortezomib. In addition, IL-10RB EE genotype was significantly associated with poorer survival than KK and KE genotypes (median OS, 46.3 vs 78.8 months; P = .015). Our findings indicate that IL-10 and IL-10R gene polymorphisms may not contribute to the susceptibility to MM but may be associated with the severity and prognosis of MM. In particular, IL-10RB K47E polymorphism may contribute to the poor prognosis of MM patients treated with thalidomide and/or bortezomib.

Podocyte-associated talin1 is critical for glomerular filtration barrier maintenance.

Podocytes are specialized actin-rich epithelial cells that line the kidney glomerular filtration barrier. The interface between the podocyte and the glomerular basement membrane requires integrins, and defects in either α3 or ß1 integrin, or the α3ß1 ligand laminin result in nephrotic syndrome in murine models. The large cytoskeletal protein talin1 is not only pivotal for integrin activation, but also directly links integrins to the actin cytoskeleton. Here, we found that mice lacking talin1 specifically in podocytes display severe proteinuria, foot process effacement, and kidney failure. Loss of talin1 in podocytes caused only a modest reduction in ß1 integrin activation, podocyte cell adhesion, and cell spreading; however, the actin cytoskeleton of podocytes was profoundly altered by the loss of talin1. Evaluation of murine models of glomerular injury and patients with nephrotic syndrome revealed that calpain-induced talin1 cleavage in podocytes might promote pathogenesis of nephrotic syndrome. Furthermore, pharmacologic inhibition of calpain activity following glomerular injury substantially reduced talin1 cleavage, albuminuria, and foot process effacement. Collectively, these findings indicate that podocyte talin1 is critical for maintaining the integrity of the glomerular filtration barrier and provide insight into the pathogenesis of nephrotic syndrome.

Altered renal proximal tubular endocytosis and histology in mice lacking myosin-VI.

Myosin VI (Myo6) is an actin-based molecular motor involved in clathrin-mediated endocytosis that is highly expressed in the renal proximal tubule brush border. We investigated the renal physiological consequences of loss of Myo6 function by performing renal clearance and physiological measurements on Myo6 functional null Snell's waltzer (sv/sv) and control heterozygous (+/sv) mice. Sv/sv mice showed reduced body weight and elevated blood pressure compared with controls; no differences were observed for glomerular flow rate, urine volume, blood acid-base parameters, and plasma concentrations and urinary excretions of Na(+) and K(+). To assess the integrity of endocytosis-mediated protein absorption by the kidney, urinary albumin excretion was measured, and the proximal tubular uptake of intravenously injected endocytic marker horseradish peroxidase (HRP) was examined. Albumin excretion was increased nearly 4-fold in sv/sv mice relative to controls. Conversely, HRP uptake was reduced and delayed in proximal tubule cells of the sv/sv kidney observed by electron microscopy at 5 and 30 min after injection. Consistent with impaired endocytosis, we also observed defects indicating alterations along the endocytic pathway in sv/sv proximal tubule cells: (1) decreased membrane association of the clathrin adaptor subunit, adaptin beta, and Disabled-2 (Dab2) after sedimentation of renal homogenates and (2) reduced apical vacuole number. In addition, proximal tubular dilation and fibrosis, likely secondary effects of the loss of Myo6, were observed in sv/sv kidneys. These results indicate that Myo6 plays a key role in endocytosis-mediated protein absorption in the mouse kidney proximal tubule.

Shear-induced reorganization of renal proximal tubule cell actin cytoskeleton and apical junctional complexes.

In this study, we demonstrate that fluid shear stress (FSS)-induced actin cytoskeletal reorganization and junctional formation in renal epithelial cells are nearly completely opposite the corresponding changes in vascular endothelial cells (ECs) [Thi MM et al. (2004) Proc Natl Acad Sci USA 101:16483-16488]. Mouse proximal tubule cells (PTCs) were subjected to 5 h of FSS (1 dyn/cm(2)) to investigate the dynamic responses of the cytoskeletal distribution of filamentous actin (F-actin), ZO-1, E-cadherin, vinculin, and paxillin to FSS. Immunofluorescence analysis revealed that FSS caused basal stress fiber disruption, more densely distributed peripheral actin bands (DPABs), and the formation of both tight junctions (TJs) and adherens junctions (AJs). A dramatic reinforcement of vinculin staining was found at the cell borders as well as the cell interior. These responses were abrogated by the actin-disrupting drug, cytochalasin D. To interpret these results, we propose a "junctional buttressing" model for PTCs in which FSS enables the DPABs, TJs, and AJs to become more tightly connected. In contrast, in the "bumper-car" model for ECs, all junctional connections were severely disrupted by FSS. This "junctional buttressing" model explains why a FSS of only 1/10 of that used in the EC study can cause a similarly dramatic, cytoskeletal response in these tall, cuboidal epithelial cells; and why junctional buttressing between adjacent cells may benefit renal epithelium in maximizing flow-activated, brush border-dependent, transcellular salt and water reabsorption.