RESUMO
Urinary tract infection (UTI) commonly afflicts people with diabetes. This augmented infection risk is partly due to deregulated insulin receptor (IR) signaling in the kidney collecting duct. The collecting duct is composed of intercalated cells (ICs) and principal cells (PCs). Evidence suggests that ICs contribute to UTI defenses. Here, we interrogate how IR deletion in ICs impacts antibacterial defenses against uropathogenic Escherichia coli. We also explore how IR deletion affects immune responses in neighboring PCs with intact IR expression. To accomplish this objective, we profile the transcriptomes of IC and PC populations enriched from kidneys of wild-type and IC-specific IR knock-out mice that have increased UTI susceptibility. Transcriptomic analysis demonstrates that IR deletion suppresses IC-integrated stress responses and innate immune defenses. To define how IR shapes these immune defenses, we employ murine and human kidney cultures. When challenged with bacteria, murine ICs and human kidney cells with deregulated IR signaling cannot engage central components of the integrated stress response-including activating transcriptional factor 4 (ATF4). Silencing ATF4 impairs NFkB activation and promotes infection. In turn, NFkB silencing augments infection and suppresses antimicrobial peptide expression. In diabetic mice and people with diabetes, collecting duct cells show reduced IR expression, impaired integrated stress response engagement, and compromised immunity. Collectively, these translational data illustrate how IR orchestrates collecting duct antibacterial responses and the communication between ICs and PCs.
Assuntos
Camundongos Knockout , Receptor de Insulina , Infecções Urinárias , Escherichia coli Uropatogênica , Animais , Humanos , Camundongos , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Imunidade Inata , Rim/metabolismo , Túbulos Renais Coletores/metabolismo , Camundongos Endogâmicos C57BL , Receptor de Insulina/metabolismo , Transdução de Sinais , Infecções Urinárias/microbiologia , Infecções Urinárias/metabolismo , Infecções Urinárias/imunologia , Escherichia coli Uropatogênica/imunologiaRESUMO
There is a critical gap in knowledge about how Gram-negative bacterial pathogens, using survival strategies developed for other niches, cause lethal bacteremia. Facultative anaerobic species of the Enterobacterales order are the most common cause of Gram-negative bacteremia, including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter hormaechei. Bacteremia often leads to sepsis, a life-threatening organ dysfunction resulting from unregulated immune responses to infection. Despite a lack of specialization for this host environment, Gram-negative pathogens cause nearly half of bacteremia cases annually. Based on our existing Tn-Seq fitness factor data from a murine model of bacteremia combined with comparative genomics of the five Enterobacterales species above, we prioritized 18 conserved fitness genes or operons for further characterization. Mutants were constructed for all genes in all five species. Each mutant was used to cochallenge C57BL/6 mice via tail vein injection along with each respective wild-type strain to determine competitive indices for each fitness gene. Five fitness factor genes, when mutated, attenuated mutants in four or five species in the spleen and liver (tatC, ruvA, gmhB, wzxE, arcA). Five additional fitness factor genes or operons were validated as outcompeted by wild-type in three, four, or five bacterial species in the spleen (xerC, prc, apaGH, atpG, aroC). Overall, 17 of 18 fitness factor mutants were attenuated in at least one species in the spleen or liver. Together, these findings allow for the development of a model of bacteremia pathogenesis that may include future targets of therapy against bloodstream infections.
RESUMO
With the emergence of antibiotic-resistant bacteria, innovative approaches are needed for the treatment of urinary tract infections. Boosting antimicrobial peptide expression may provide an alternative to antibiotics. Here, we developed reporter cell lines and performed a high-throughput screen of clinically used drugs to identify compounds that boost ribonuclease 4 and 7 expression (RNase 4 and 7), peptides that have antimicrobial activity against antibiotic-resistant uropathogens. This screen identified histone deacetylase (HDAC) inhibitors as effective RNase 4 and RNase 7 inducers. Validation studies in primary human kidney and bladder cells confirmed pan-HDAC inhibitors as well as the HDAC class I inhibitor, MS-275, induce RNase 4 and RNase 7 to protect human kidney and bladder cells from uropathogenic Escherichia coli. When we administered MS-275 to mice, RNase 4 and 7 expression increased and mice were protected from acute transurethral E. coli challenge. In support of this mechanism, MS-275 treatment increased acetylated histone H3 binding to the RNASE4 and RNASE7 promoters. Overexpression and knockdown of HDAC class I proteins identified HDAC3 as a primary regulator of RNase 4 and 7. These results demonstrate the protective effects of enhancing RNase 4 and RNase 7, opening the door to repurposing medications as antibiotic conserving therapeutics for urinary tract infection.
Assuntos
Inibidores de Histona Desacetilases , Infecções Urinárias , Humanos , Camundongos , Animais , Inibidores de Histona Desacetilases/farmacologia , Escherichia coli/metabolismo , Reposicionamento de Medicamentos , Ribonucleases/metabolismo , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia , AntibacterianosRESUMO
Antimicrobial peptides are essential host defense mechanisms that prevent urinary tract infections. Recent studies have demonstrated that peptides in the ribonuclease A superfamily have antimicrobial activity against uropathogens and protect the urinary tract from uropathogenic Escherichia coli (UPEC). Little is known about the antibacterial function or expression of ribonuclease 4 (RNase 4) in the human urinary tract. Here, we show that full-length recombinant RNase 4 peptide and synthetic amino-terminal RNase 4 peptide fragment have antibacterial activity against UPEC and multidrug-resistant (MDR)-UPEC. RNASE4 transcript expression was detected in human kidney and bladder tissue using quantitative real-time PCR. Immunostaining or in situ hybridization localized RNase 4 expression to proximal tubules, principal and intercalated cells in the kidney's collecting duct, and the bladder urothelium. Urinary RNase 4 concentrations were quantified in healthy controls and females with a history of urinary tract infection. Compared with controls, urinary RNase 4 concentrations were significantly lower in females with a history of urinary tract infection. When RNase 4 was neutralized in human urine or silenced in vitro using siRNA, urinary UPEC replication or attachment to and invasion of urothelial and kidney medullary cells increased. These data show that RNase 4 has antibacterial activity against UPEC, is expressed in the human urinary tract, and can contribute to host defense against urinary tract infections.NEW & NOTEWORTHY Ribonuclease 4 (RNase 4) is a newly identified host defense peptide in the human kidney and bladder. RNase 4 kills uropathogenic Escherichia coli (UPEC) and multidrug-resistant UPEC. RNase 4 prevents invasive UPEC infection and suppressed RNase 4 expression may be a risk factor for more severe or recurrent urinary tract infection.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Rim/enzimologia , Ribonucleases/metabolismo , Bexiga Urinária/enzimologia , Adolescente , Peptídeos Catiônicos Antimicrobianos , Criança , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Feminino , Inativação Gênica , História Antiga , História Medieval , Humanos , Rim/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Ribonucleases/genética , Ribonucleases/urina , Bexiga Urinária/metabolismo , Escherichia coli Uropatogênica , Urotélio/citologiaRESUMO
BACKGROUND: Evidence suggests that antimicrobial peptides, components of the innate immune response, protect the kidneys and bladder from bacterial challenge. We previously identified ribonuclease 7 (RNase 7) as a human antimicrobial peptide that has bactericidal activity against uropathogenic Escherichia coli (UPEC). Functional studies assessing RNase 7's contributions to urinary tract defense are limited. METHODS: To investigate RNase 7's role in preventing urinary tract infection (UTI), we quantified urinary RNase 7 concentrations in 29 girls and adolescents with a UTI history and 29 healthy female human controls. To assess RNase 7's antimicrobial activity in vitro in human urothelial cells, we used siRNA to silence urothelial RNase 7 production and retroviral constructs to stably overexpress RNase 7; we then evaluated UPEC's ability to bind and invade these cells. For RNase 7 in vivo studies, we developed humanized RNase 7 transgenic mice, subjected them to experimental UTI, and enumerated UPEC burden in the urine, bladder, and kidneys. RESULTS: Compared with controls, study participants with a UTI history had 1.5-fold lower urinary RNase 7 concentrations. When RNase 7 was silenced in vitro, the percentage of UPEC binding or invading human urothelial cells increased; when cells overexpressed RNase 7, UPEC attachment and invasion decreased. In the transgenic mice, we detected RNase 7 expression in the kidney's intercalated cells and bladder urothelium. RNase 7 humanized mice exhibited marked protection from UPEC. CONCLUSIONS: These findings provide evidence that RNase 7 has a role in kidney and bladder host defense against UPEC and establish a foundation for investigating RNase 7 as a UTI prognostic marker or nonantibiotic-based therapy.
Assuntos
Infecções por Escherichia coli/enzimologia , Rim/enzimologia , Ribonucleases/genética , Bexiga Urinária/enzimologia , Infecções Urinárias/enzimologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica , Adolescente , Animais , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Criança , Pré-Escolar , Feminino , Inativação Gênica , Humanos , Imunidade Inata , Lactente , Rim/microbiologia , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , Prognóstico , Bexiga Urinária/microbiologia , Urotélio/metabolismo , Urotélio/patologia , Adulto JovemRESUMO
Diabetes mellitus is a systemic disease associated with a deficiency of insulin production or action. Diabetic patients have an increased susceptibility to infection with the urinary tract being the most common site. Recent studies suggest that Ribonuclease 7 (RNase 7) is a potent antimicrobial peptide that plays an important role in protecting the urinary tract from bacterial insult. Because the impact of diabetes on RNase 7 expression and function are unknown, we investigated the effects of insulin on RNase 7 using human urine specimens. The urinary RNase 7 concentrations were measured in healthy control patients and insulin-deficient type 1 diabetics before and after starting insulin therapy. Compared with controls, diabetic patients had suppressed urinary RNase 7 concentrations, which increased with insulin. Using primary human urothelial cells, the mechanisms by which insulin stimulates RNase 7 synthesis were next explored. Insulin induced RNase 7 production via the phosphatidylinositide 3-kinase signaling pathway (PI3K/AKT) to shield urothelial cells from uropathogenic E. coli. In contrast, uropathogenic E. coli suppressed PI3K/AKT activity and RNase 7 production. Thus, insulin and PI3K/AKT signaling are essential for RNase 7 expression and increased infection risks in diabetic patients may be secondary to suppressed RNase 7 production. Our data may provide unique insight into novel urinary tract infection therapeutic strategies in at-risk populations.
Assuntos
Diabetes Mellitus Tipo 1/complicações , Infecções por Escherichia coli/metabolismo , Insulina/metabolismo , Ribonucleases/metabolismo , Infecções Urinárias/metabolismo , Sistema Urinário/metabolismo , Adolescente , Antígenos CD/metabolismo , Linhagem Celular Tumoral , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/urina , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/etiologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/urina , Feminino , Humanos , Insulina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/metabolismo , Ribonucleases/urina , Transdução de Sinais , Sistema Urinário/microbiologia , Infecções Urinárias/etiologia , Infecções Urinárias/microbiologia , Infecções Urinárias/urinaRESUMO
Recent evidence suggests antimicrobial peptides protect the urinary tract from infection. Ribonuclease 7 (RNase 7), a member of the RNase A superfamily, is a potent epithelial-derived protein that maintains human urinary tract sterility. RNase 7 expression is restricted to primates, limiting evaluation of its antimicrobial activity in vivo. Here we identified ribonuclease 6 (RNase 6) as the RNase A superfamily member present in humans and mice that is most conserved at the amino acid level relative to RNase 7. Like RNase 7, recombinant human and murine RNase 6 has potent antimicrobial activity against uropathogens. Quantitative real-time PCR and immunoblot analysis indicate that RNase 6 mRNA and protein are upregulated in the human and murine urinary tract during infection. Immunostaining located RNase 6 to resident and infiltrating monocytes, macrophages, and neutrophils. Uropathogenic E. coli induces RNase 6 peptide expression in human CD14(+) monocytes and murine bone marrow-derived macrophages. Thus, RNase 6 is an inducible, myeloid-derived protein with markedly different expression from the epithelial-derived RNase 7 but with equally potent antimicrobial activity. Our studies suggest RNase 6 serves as an evolutionarily conserved antimicrobial peptide that participates in the maintenance of urinary tract sterility.
Assuntos
Endorribonucleases/fisiologia , Ribonucleases/fisiologia , Sistema Urinário/enzimologia , Sistema Urinário/microbiologia , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade MicrobianaRESUMO
Recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Previously, we have shown that ribonuclease 7 (RNase 7) is a potent antimicrobial peptide that has a broad-spectrum antimicrobial activity against uropathogenic bacteria. The urothelium of the lower urinary tract and intercalated cells of the kidney produce RNase 7, but regulation of its antimicrobial activity has not been well defined. Here, we characterize the expression of an endogenous inhibitor, ribonuclease inhibitor (RI), in the urinary tract and evaluate its effect on the antimicrobial activity of RNase 7. Using RNA isolated from non-infected human bladder and kidney tissue, quantitative real-time polymerase chain reaction showed that RNH1, the gene encoding RI, is constitutively expressed throughout the urinary tract. With pyelonephritis, RNH1 expression and RI peptide production significantly decrease. Immunostaining localized RI production to the umbrella cells of the bladder and intercalated cells of the renal collecting tubule. In vitro assays showed that RI bound to RNase 7 and suppressed its antimicrobial activity by blocking its ability to bind the cell wall of uropathogenic bacteria. Thus, these results demonstrate a new immunomodulatory role for RI and identified a unique regulatory pathway that may affect how RNase 7 maintains urinary tract sterility.
Assuntos
Proteínas de Transporte/metabolismo , Rim/enzimologia , Pielonefrite/enzimologia , Ribonucleases/antagonistas & inibidores , Bexiga Urinária/enzimologia , Urotélio/enzimologia , Adolescente , Adulto , Idoso , Proteínas de Transporte/genética , Proteínas de Transporte/urina , Estudos de Casos e Controles , Parede Celular/enzimologia , Criança , Pré-Escolar , Enterococcus faecalis/enzimologia , Enterococcus faecalis/patogenicidade , Escherichia coli/enzimologia , Escherichia coli/patogenicidade , Feminino , Regulação Enzimológica da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Rim/microbiologia , Elastase de Leucócito/metabolismo , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Proteólise , Pielonefrite/genética , Pielonefrite/microbiologia , Pielonefrite/urina , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo , Ribonucleases/urina , Fatores de Tempo , Bexiga Urinária/microbiologia , Urotélio/microbiologiaRESUMO
Urinary tract infections (UTIs) commonly afflict people with diabetes. To better understand the mechanisms that predispose diabetics to UTIs, we employ diabetic mouse models and altered insulin signaling to show that insulin receptor (IR) shapes UTI defenses. Our findings are validated in human biosamples. We report that diabetic mice have suppressed IR expression and are more susceptible to UTIs caused by uropathogenic Escherichia coli (UPEC). Systemic IR inhibition increases UPEC susceptibility, while IR activation reduces UTIs. Localized IR deletion in bladder urothelium promotes UTI by increasing barrier permeability and suppressing antimicrobial peptides. Mechanistically, IR deletion reduces nuclear factor κB (NF-κB)-dependent programming that co-regulates urothelial tight junction integrity and antimicrobial peptides. Exfoliated urothelial cells or urine samples from diabetic youths show suppressed expression of IR, barrier genes, and antimicrobial peptides. These observations demonstrate that urothelial insulin signaling has a role in UTI prevention and link IR to urothelial barrier maintenance and antimicrobial peptide expression.
Assuntos
Receptor de Insulina , Transdução de Sinais , Bexiga Urinária , Infecções Urinárias , Urotélio , Receptor de Insulina/metabolismo , Infecções Urinárias/microbiologia , Infecções Urinárias/metabolismo , Infecções Urinárias/patologia , Animais , Urotélio/metabolismo , Urotélio/patologia , Urotélio/microbiologia , Humanos , Bexiga Urinária/microbiologia , Bexiga Urinária/patologia , Bexiga Urinária/metabolismo , Camundongos , Escherichia coli Uropatogênica/patogenicidade , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Feminino , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Insulina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , MasculinoRESUMO
The mechanisms that maintain sterility in the urinary tract are incompletely understood; however, recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Ribonuclease 7 (RNase 7), a potent antimicrobial peptide contributing to urinary tract sterility, is expressed by intercalated cells in the renal collecting tubules and is present in the urine at levels sufficient to kill bacteria at baseline. Here, we characterize the expression and function of RNase 7 in the human urinary tract during infection. Both quantitative real-time PCR and enzyme-linked immunosorbant assays demonstrated increases in RNASE7 expression in the kidney along with kidney and urinary RNase 7 peptide concentrations with infection. While immunostaining localized RNase 7 production to the intercalated cells of the collecting tubule during sterility, its expression during pyelonephritis was found to increase throughout the nephron but not in glomeruli or the interstitium. Recombinant RNase 7 exhibited antimicrobial activity against uropathogens at low micromolar concentrations by disrupting the microbial membrane as determined by atomic force microscopy. Thus, RNase 7 expression is increased in the urinary tract with infection and has antibacterial activity against uropathogens at micromolar concentrations.
Assuntos
Rim/enzimologia , Pielonefrite/enzimologia , Ribonucleases/metabolismo , Infecções Urinárias/enzimologia , Bactérias/enzimologia , Estudos de Casos e Controles , Membrana Celular/enzimologia , Ensaio de Imunoadsorção Enzimática , Interações Hospedeiro-Patógeno , Humanos , Rim/microbiologia , Cinética , Viabilidade Microbiana , Microscopia de Força Atômica , Pielonefrite/genética , Pielonefrite/microbiologia , Pielonefrite/urina , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Ribonucleases/genética , Ribonucleases/urina , Regulação para Cima , Infecções Urinárias/genética , Infecções Urinárias/microbiologia , Infecções Urinárias/urinaRESUMO
Obesity-associated type 2 diabetes (DM) leads to adipose tissue dysfunction. Lumican is a proteoglycan implicated in obesity, insulin resistance (IR), and adipocyte dysfunction. Using human visceral adipose tissue (VAT) from subjects with and without DM, we studied lumican effects on adipocyte function. Lumican was increased in VAT and adipocytes in DM. Lumican knockdown in adipocytes decreased lipolysis and improved adipogenesis and insulin sensitivity in VAT adipocytes in DM, while treatment with human recombinant lumican increased lipolysis and impaired insulin-sensitivity in an ERK-dependent manner. We demonstrate that lumican impairs adipocyte metabolism, partially via ERK signalling, and is a potential target for developing adipose tissue-targeted therapeutics in DM.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Humanos , Lumicana/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Adipócitos/metabolismo , Lipólise , Obesidade/complicações , Obesidade/metabolismo , Tecido Adiposo/metabolismoRESUMO
The state-of-the-art cultured podocyte is conditionally immortalized by expression of a temperature-sensitive mutant of the SV40 large-T antigen. These cultures proliferate at 33°C and differentiate at 37°C into arborized cells that more closely resemble in vivo podocytes. However, the degree of resemblance remains controversial. In this study, several parameters were measured in podocyte cell lines derived from mouse (JR, KE), human (MS), and rat (HK). In all lines, the quantities of NEPH1 and podocin proteins and NEPH1 and SYNPO mRNAs were comparable to glomeruli, while synaptopodin and nephrin proteins and NPHS1 and NPHS2 mRNAs were <5% of glomerular levels. Expression of Wilms' tumor-1 (WT1) mRNA in mouse lines was comparable to glomeruli, but rat and human lines expressed little WT1. Undifferentiated human and mouse lines had similar proliferation rates that decreased after differentiation, while the rate in rat cells remained constant. The motility of different lines varied as measured by both general motility and wound-healing assays. The toxicity of puromycin aminonucleoside was MS â¼ JR >> KE, and of doxorubicin was JR â¼ KE > MS, while HK cells were almost unaffected. Process formation was largely a result of contractile action after formation of lamellipodia. These findings demonstrate dramatic differences in marker expression, response to toxins, and motility between lines of podocytes from different species and even between similarly-derived mouse lines.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Podócitos/citologia , Podócitos/metabolismo , Animais , Biomarcadores/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/fisiologia , Células Cultivadas , Doxorrubicina/farmacologia , Humanos , Masculino , Camundongos , Modelos Animais , Podócitos/efeitos dos fármacos , Puromicina Aminonucleosídeo/farmacologia , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Proteínas WT1/metabolismoRESUMO
Ribonuclease 7 (RNase 7) is an antimicrobial peptide that prevents urinary tract infections (UTI); however, it is yet unknown how RNASE7 genetic variations affect its antimicrobial activity and its mitigation of UTI risk. This study determined whether the RNASE7 SNP rs1263872 is more prevalent in children with UTI and defined how rs1263872 affects RNase 7's antimicrobial activity against uropathogenic E. coli (UPEC). We performed genotyping for rs1263872 in 2 national UTI cohorts, including children enrolled in the Randomized Intervention for Children with Vesicoureteral Reflux trial or the Careful Urinary Tract Infection Evaluation study. Genotypes from these cohorts were compared with those of female controls with no UTI. To assess whether rs1263872 affects RNase 7's antimicrobial activity, we generated RNase 7 peptides and genetically modified urothelial cultures encoding wild-type RNase 7 and its variant. Compared with controls, girls in both UTI cohorts had an increased prevalence of the RNASE7 variant. Compared with the missense variant, wild-type RNase 7 peptide showed greater bactericidal activity against UPEC. Wild-type RNase 7 overexpression in human urothelial cultures reduced UPEC invasive infection compared with mutant overexpression. These results show that children with UTI have an increased prevalence of RNASE7 rs1263872, which may increase UTI susceptibility by suppressing RNase 7's antibacterial activity.
Assuntos
Peptídeos Antimicrobianos/genética , Polimorfismo de Nucleotídeo Único , Ribonucleases/genética , Infecções Urinárias/etiologia , Criança , Pré-Escolar , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Lactente , Ribonucleases/fisiologia , Infecções Urinárias/genéticaRESUMO
People with diabetes mellitus have increased infection risk. With diabetes, urinary tract infection (UTI) is more common and has worse outcomes. Here, we investigate how diabetes and insulin resistance impact the kidney's innate defenses and urine sterility. We report that type 2 diabetic mice have increased UTI risk. Moreover, insulin-resistant prediabetic mice have increased UTI susceptibility, independent of hyperglycemia or glucosuria. To identify how insulin resistance affects renal antimicrobial defenses, we genetically deleted the insulin receptor in the kidney's collecting tubules and intercalated cells. Intercalated cells, located within collecting tubules, contribute to epithelial defenses by acidifying the urine and secreting antimicrobial peptides (AMPs) into the urinary stream. Collecting duct and intercalated cell-specific insulin receptor deletion did not impact urine acidification, suppressed downstream insulin-mediated targets and AMP expression, and increased UTI susceptibility. Specifically, insulin receptor-mediated signaling regulates AMPs, including lipocalin 2 and ribonuclease 4, via phosphatidylinositol-3-kinase signaling. These data suggest that insulin signaling plays a critical role in renal antibacterial defenses.
Assuntos
Infecções Bacterianas/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Túbulos Renais Coletores/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais , Infecções Urinárias/metabolismo , Animais , Infecções Bacterianas/genética , Infecções Bacterianas/microbiologia , Infecções Bacterianas/patologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/microbiologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/microbiologia , Diabetes Mellitus Tipo 2/patologia , Túbulos Renais Coletores/microbiologia , Túbulos Renais Coletores/patologia , Camundongos , Camundongos Mutantes , Receptor de Insulina/genética , Infecções Urinárias/genética , Infecções Urinárias/patologia , alfa-Defensinas/genética , alfa-Defensinas/metabolismoRESUMO
Arsenite, cadmium, and mercury are among the most abundant toxic metals (TM) in the environment. Although the most common renal manifestation of TM toxicity is proximal tubular dysfunction, significant glomerular injury can also occur. We hypothesized that glomerular injury following TM exposure results from TM-induced apoptosis of podocytes. To test this hypothesis we examined the extent of apoptosis and the apoptotic pathways induced in cultured murine podocytes incubated for three days with arsenite, cadmium, or mercury, and with equimolar combinations of all three metals. Apoptosis was detected by DNA laddering, and the number of apoptotic nuclei determined by Tunel assay. Treatment for three days with each TM resulted in DNA laddering and induced a dose-dependent increase in apoptotic nuclei. In contrast, treatment with equimolar combinations of TM induced significantly fewer apoptotic nuclei than individual TM treatments. Apoptosis induced by each TM was associated with a significant (approximately 400%) increase in caspase 8 activity, but no change in caspase 9 activity, and Western analyses revealed a marked up-regulation of Fas (approximately 500%) and FADD (approximately 300%) with no change in expression of Bax, Bcl-2, or Bcl-xL. Similar to the apoptotic response, combinations of TM induced less caspase 8 activity and Fas/FADD expression than individual TM treatments. Collectively, these results demonstrate that (1) TM induced apoptosis in cultured murine podocytes via the extrinsic Fas-FADD caspase 8 pathway, rather than the mitochondrial apoptotic pathway, and (2) combination TM exposure induced less apoptosis than individual TM, indicating an antagonistic rather than an additive or synergistic toxicity.
Assuntos
Apoptose , Arsenitos/toxicidade , Cádmio/toxicidade , Mercúrio/toxicidade , Podócitos/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Caspase 8 , Caspase 9 , Caspases/metabolismo , Linhagem Celular , Fragmentação do DNA , Interações Medicamentosas , Proteína de Domínio de Morte Associada a Fas , Marcação In Situ das Extremidades Cortadas , Camundongos , Podócitos/metabolismo , Receptores do Fator de Necrose Tumoral/biossíntese , Receptor fasRESUMO
Cadmium, mercury, and arsenite are among the most abundant toxic metals (TM) in our environment, and chronic TM exposure leads to injury to the kidney's glomerular filtration barrier. The small heat shock protein hsp25, highly expressed in glomerular podocytes, is induced during development of experimental nephrotic syndrome, and hsp25 overexpression can protect cultured podocytes from injury. Because little is known about the effect of multiple TM on podocytes, we measured the response of cultured podocytes to prolonged exposures to single and multiple TM. Podocyte viability declined by approximately 50% after 3 days of treatment with 20 microM cadmium, mercury, or arsenite, and 40 microM of any of these metals was lethal. The toxicity of equimolar concentrations of two or all three metals in combination was significantly altered compared to individual metal treatments. Single TM treatments induced only modest increases in the amounts of hsp25, alphaB-crystallin, and inducible hsp70. Toxic metal combinations induced greater stress protein accumulation, especially arsenite + cadmium or arsenite + cadmium + mercury treatments, the TM mixtures with the lowest toxicity. All TM treatments caused a rapid and sustained increase in hsp25 phosphorylation. The intracellular accumulation of cadmium was greater and that of mercury was less in cells treated with TM combinations than in cells treated with a single TM. Our results showed that multiple TM effects on podocyte viability were neither additive nor synergistic and that induction of heat shock proteins correlated with increased resistance to TM injury, suggesting that induction of small heat shock proteins may play an important role in preventing TM-induced podocyte injury.