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1.
Proc Natl Acad Sci U S A ; 119(40): e2206515119, 2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-36161923

RESUMO

Antimicrobial peptides (AMPs) are critical to the protection of the urinary tract of humans and other animals from pathogenic microbial invasion. AMPs rapidly destroy pathogens by disrupting microbial membranes and/or augmenting or inhibiting the host immune system through a variety of signaling pathways. We have previously demonstrated that alpha-defensins 1-3 (DEFA1A3) are AMPs expressed in the epithelial cells of the human kidney collecting duct in response to uropathogens. We also demonstrated that DNA copy number variations in the DEFA1A3 locus are associated with UTI and pyelonephritis risk. Because DEFA1A3 is not expressed in mice, we utilized human DEFA1A3 gene transgenic mice (DEFA4/4) to further elucidate the biological relevance of this locus in the murine urinary tract. We demonstrate that the kidney transcriptional and translational expression pattern is similar in humans and the human gene transgenic mouse upon uropathogenic Escherichia coli (UPEC) stimulus in vitro and in vivo. We also demonstrate transgenic human DEFA4/4 gene mice are protected from UTI and pyelonephritis under various UPEC challenges. This study serves as the foundation to start the exploration of manipulating the DEFA1A3 locus and alpha-defensins 1-3 expression as a potential therapeutic target for UTIs and other infectious diseases.


Assuntos
Infecções por Escherichia coli , Pielonefrite , Infecções Urinárias , Escherichia coli Uropatogênica , alfa-Defensinas , Animais , Variações do Número de Cópias de DNA , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Loci Gênicos , Humanos , Camundongos , Camundongos Transgênicos , Pielonefrite/genética , Pielonefrite/imunologia , Pielonefrite/microbiologia , Sistema Urinário/microbiologia , Infecções Urinárias/genética , Infecções Urinárias/imunologia , Infecções Urinárias/microbiologia , alfa-Defensinas/genética
2.
Pflugers Arch ; 476(4): 565-578, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38227050

RESUMO

Intercalated cells (ICs) in the kidney collecting duct have a versatile role in acid-base and electrolyte regulation along with the host immune defense. Located in the terminal kidney tubule segment, ICs are among the first kidney cells to encounter bacteria when bacteria ascend from the bladder into the kidney. ICs have developed several mechanisms to combat bacterial infections of the kidneys. For example, ICs produce antimicrobial peptides (AMPs), which have direct bactericidal activity, and in many cases are upregulated in response to infections. Some AMP genes with IC-specific kidney expression are multiallelic, and having more copies of the gene confers increased resistance to bacterial infections of the kidney and urinary tract. Similarly, studies in human children demonstrate that those with history of UTIs are more likely to have single-nucleotide polymorphisms in IC-expressed AMP genes that impair the AMP's bactericidal activity. In murine models, depleted or impaired ICs result in decreased clearance of bacterial load following transurethral challenge with uropathogenic E. coli. A 2021 study demonstrated that ICs even act as phagocytes and acidify bacteria within phagolysosomes. Several immune signaling pathways have been identified in ICs which may represent future therapeutic targets in managing kidney infections or inflammation. This review's objective is to highlight IC structure and function with an emphasis on current knowledge of IC's diverse innate immune capabilities.


Assuntos
Infecções Bacterianas , Túbulos Renais Coletores , Infecções Urinárias , Criança , Camundongos , Humanos , Animais , Escherichia coli , Rim/metabolismo , Infecções Urinárias/metabolismo , Infecções Urinárias/microbiologia , Túbulos Renais Coletores/metabolismo , Imunidade Inata , Infecções Bacterianas/metabolismo
3.
Pediatr Nephrol ; 39(11): 3217-3219, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-38842722

RESUMO

Tumor lysis syndrome (TLS) is a life-threatening metabolic disorder caused by massive tumor lysis. Allopurinol, a xanthine oxidase inhibitor, is initiated during chemotherapy to prevent hyperuricemia and subsequent acute kidney injury (AKI). We report two cases of xanthine nephrolithiasis during TLS in newly diagnosed hematologic malignancy patients receiving prophylactic allopurinol. Allopurinol use likely promoted xanthine crystallization, stone formation, and AKI.


Assuntos
Alopurinol , Síndrome de Lise Tumoral , Humanos , Alopurinol/efeitos adversos , Síndrome de Lise Tumoral/etiologia , Síndrome de Lise Tumoral/diagnóstico , Masculino , Feminino , Criança , Xantina , Nefrolitíase/induzido quimicamente , Adolescente , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/diagnóstico , Hiperuricemia/tratamento farmacológico , Hiperuricemia/diagnóstico , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/uso terapêutico
4.
Dev Dyn ; 252(4): 463-482, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36335435

RESUMO

BACKGROUND: The interstitial extracellular matrix (ECM) is comprised of proteins and glycosaminoglycans and provides structural and biochemical information during development. Our previous work revealed the presence of transient ECM-based structures in the interstitial matrix of developing kidneys. Stromal cells are the main contributors to interstitial ECM synthesis, and the transcription factor Forkhead Box D1 (Foxd1) is critical for stromal cell function. To investigate the role of Foxd1 in interstitial ECM patterning, we combined 3D imaging and proteomics to explore how the matrix changes in the murine developing kidney when Foxd1 is knocked out. RESULTS: We found that COL26A1, FBN2, EMILIN1, and TNC, interstitial ECM proteins that are transiently upregulated during development, had a similar distribution perinatally but then diverged in patterning in the adult. Abnormally clustered cortical vertical fibers and fused glomeruli were observed when Foxd1 was knocked out. The changes in the interstitial ECM of Foxd1 knockout kidneys corresponded to disrupted Foxd1+ cell patterning but did not precede branching dysmorphogenesis. CONCLUSIONS: The transient ECM networks affected by Foxd1 knockout may provide support for later-stage nephrogenic structures.


Assuntos
Fatores de Transcrição Forkhead , Rim , Animais , Camundongos , Matriz Extracelular/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Rim/metabolismo
5.
Am J Physiol Renal Physiol ; 325(6): F770-F778, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37823193

RESUMO

Kidney intercalated cells (ICs) maintain acid-base homeostasis and recent studies have demonstrated that they function in the kidney's innate defense. To study kidney innate immune function, ICs have been enriched using vacuolar ATPase (V-ATPase) B1 subunit (Atp6v1b1)-Cre (B1-Cre) mice. Although Atp6v1b1 is considered kidney specific, it is expressed in multiple organ systems, both in mice and humans, raising the possibility of off-target effects when using the Cre-lox system. We have recently shown using single-cell RNA sequencing that the gene that codes for the V-ATPase G3 subunit (mouse gene: Atp6v1g3; human gene: ATP6V1G3; protein abbreviation: G3) mRNA is selectively enriched in human kidney ICs. In this study, we generated Atp6v1g3-Cre (G3-Cre) reporter mice using CRISPR/CAS technology and crossed them with Tdtomatoflox/flox mice. The resultant G3-Cre+Tdt+ progeny was evaluated for kidney specificity in multiple tissues and found to be highly specific to kidney cells with minimal or no expression in other organs evaluated compared with B1-Cre mice. Tdt+ cells were flow sorted and were enriched for IC marker genes on RT-PCR analysis. Next, we crossed these mice to ihCD59 mice to generate an IC depletion mouse model (G3-Cre+ihCD59+/+). ICs were depleted in these mice using intermedilysin, which resulted in lower blood pH, suggestive of a distal renal tubular acidosis phenotype. The G3-Cre mice were healthy, bred normally, and produce regular-sized litter. Thus, this new "IC reporter" mice can be a useful tool to study ICs.NEW & NOTEWORTHY This study details the development, validation, and experimental use of a new mouse model to study the collecting duct and intercalated cells. Kidney intercalated cells are a cell type increasingly recognized to be important in several human diseases including kidney infections, acid-base disorders, and acute kidney injury.


Assuntos
Acidose Tubular Renal , Túbulos Renais Coletores , ATPases Vacuolares Próton-Translocadoras , Camundongos , Humanos , Animais , Rim/metabolismo , Integrases/genética , Integrases/metabolismo , Acidose Tubular Renal/genética , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Túbulos Renais Coletores/metabolismo
6.
Pediatr Nephrol ; 38(12): 4051-4059, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37418011

RESUMO

BACKGROUND: The objective was to determine the extent that eGFR formulas correspond to measured plasma iohexol clearance (iGFR) in children with normal or near normal kidney function, particularly how different eGFR formulas yield discordant results. METHODS: iGFR from 2 (iGFR-2pt) and 4 (iGFR-4pt) time points along with creatinine and/or cystatin C-based eGFR were measured in children with mild CKD, stages 1-2. eGFR was calculated using 6 equations: 3 under 25 (U25) formulas from the Chronic Kidney Disease in Children (CKiD) study, the full age-combined cystatin C (cysC) and creatinine spectrum (FAS-combined), the European Kidney Function Consortium (EKFC-creatinine) equation, and the Chronic Kidney Disease Epidemiology Collaboration (CKD-epi) cysC-based equation. RESULTS: Twenty-nine children were included, of which 22 had discordant creatinine vs. cystatin C-based eGFR by ≥ 15mL/min/1.73 m2. Overall, the FAS-combined had the least bias, while the U25 most accurately identified children with an eGFR < 90 mL/min/1.73 m2. When Cr-eGFR was ≥ 15 mL/min higher than CysC-eGFR, the U25 creatinine eGFR was closest to iGFR-4pt. When CysC eGFR was higher, the U25-combined was closest to iGFR-4pt. CONCLUSION: The formulas that most closely approximated the measured GFR varied depending on the pattern of discordant eGFR results. Based on the results, we recommend using the CKiD U25-combined formula to screen for children with a low GFR. Either the CKiD U25-combined or FAS-combined would be recommended for changes in eGFR longitudinally. However, because all formulas were discordant from the iGFR-4pt in over a third of participants, further refinement of pediatric eGFR formulas is needed at the normal/near-normal range. A higher resolution version of the Graphical abstract is available as Supplementary information.


Assuntos
Cistatina C , Insuficiência Renal Crônica , Humanos , Criança , Taxa de Filtração Glomerular , Creatinina , Testes de Função Renal
7.
Clin Infect Dis ; 75(9): 1511-1519, 2022 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-35349633

RESUMO

BACKGROUND: Mortality in severe malaria remains high in children treated with intravenous artesunate. Acute kidney injury (AKI) is a common complication of severe malaria, but the interactions between AKI and other complications on the risk of mortality in severe malaria are not well characterized. METHODS: Between 2014 and 2017, 600 children aged 6-48 months to 4 years hospitalized with severe malaria were enrolled in a prospective clinical cohort study evaluating clinical predictors of mortality in children with severe malaria. RESULTS: The mean age of children in this cohort was 2.1 years (standard deviation, 0.9 years) and 338 children (56.3%) were male. Mortality was 7.3%, and 52.3% of deaths occurred within 12 hours of admission. Coma, acidosis, impaired perfusion, AKI, elevated blood urea nitrogen (BUN), and hyperkalemia were associated with increased mortality (all P < .001). AKI interacted with each risk factor to increase mortality (P < .001 for interaction). Children with clinical indications for dialysis (14.4% of all children) had an increased risk of death compared with those with no indications for dialysis (odds ratio, 6.56; 95% confidence interval, 3.41-12.59). CONCLUSIONS: AKI interacts with coma, acidosis, or impaired perfusion to significantly increase the risk of death in severe malaria. Among children with AKI, those who have hyperkalemia or elevated BUN have a higher risk of death. A better understanding of the causes of these complications of severe malaria, and development and implementation of measures to prevent and treat them, such as dialysis, are needed to reduce mortality in severe malaria.


Assuntos
Acidose , Injúria Renal Aguda , Hiperpotassemia , Malária , Criança , Masculino , Humanos , Pré-Escolar , Feminino , Coma/complicações , Estudos Prospectivos , Estudos de Coortes , Hiperpotassemia/complicações , Injúria Renal Aguda/terapia , Malária/complicações , Acidose/complicações , Fatores de Risco , Perfusão
8.
Pediatr Nephrol ; 37(2): 385-391, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34272611

RESUMO

BACKGROUND: Emergency departments (EDs) often rely on urinalysis (UA) to rapidly identify urinary tract infections (UTIs) in children. However, the suboptimal test characteristics of UA can lead to false-positive results. Novel urinary biomarkers may increase the diagnostic precision of UA. In this study, we compared the concentrations of 6 pre-selected proteins: BH3 interacting domain death agonist (BID), B-cell lymphoma 6 protein, ras GTPase-activating protein 1, cathepsin S (CTSS), 3-hydroxyanthranilate 3,4-dioxygenase, and transgelin-2. METHODS: In a pediatric ED, we prospectively enrolled 167 children with UA and urine culture collected. Pyuria was defined as either ≥ 5 white blood cells per high-power field on microscopy or positive leukocyte esterase (LE). The urine culture was considered positive if it yielded ≥ 50,000 colony-forming units per milliliter of any single urinary pathogen. Urine protein levels were measured by enzyme-linked immunosorbent assay and normalized to urine creatinine. RESULTS: BID was significantly higher in the UTI group compared to the culture-negative pyuria group with a mean ratio of 1.42 (95% confidence interval (CI), 1.15, 1.76) when uncorrected for creatinine concentration. When corrected for creatinine concentration, CTSS was significantly elevated in the UTI group compared to the culture-negative pyuria group with a mean ratio of 2.11 (95% CI, 1.39, 3.21). CONCLUSIONS: BID and CTSS concentrations were elevated in the urine of children with UTI compared to those with culture-negative pyuria. These proteins deserve further research into their utility to serve as novel biomarkers for UTI.


Assuntos
Piúria , Infecções Urinárias , Biomarcadores , Criança , Humanos , Contagem de Leucócitos , Piúria/diagnóstico , Urinálise/métodos , Infecções Urinárias/diagnóstico , Infecções Urinárias/urina , Urina
9.
Clin Nephrol ; 97(2): 93-102, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34779389

RESUMO

Hospital readmissions experienced by kidney transplant recipients may be secondary to a range of conditions, including infections and rejection episodes. The objective was to identify trends in patients with kidney transplant complications, in regard to hospital discharges, ED visits, and charges over the years available from 1993 to 2015. Using the Healthcare Cost and Utilization Project database, trends were identified in hospitalizations, emergency department visits, and charges from 1993 to 2015 for complications following kidney transplantation. Hospital discharges have significantly increased over time and at a faster rate than the increase in number of kidney transplants performed, while emergency department visits numbers and rates remain unchanged. The type of kidney transplant complications experienced were analyzed by incidence and proportion of total charges. Rejection made up the largest proportion of hospitalizations and of total cost in patients suffering from kidney transplant complications. Improved immunosuppression regimens have resulted in longer allograft survival. We speculate that the overlap between infection and rejection is compounding and contributing to graft injury and thus, it is important to try to prevent and/or properly identify those episodes as well in order to improve graft survival.


Assuntos
Transplante de Rim , Rejeição de Enxerto/epidemiologia , Sobrevivência de Enxerto , Humanos , Terapia de Imunossupressão , Transplante de Rim/efeitos adversos , Readmissão do Paciente , Transplantados
10.
Am J Physiol Renal Physiol ; 318(2): F468-F474, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31841391

RESUMO

Acute pyelonephritis is frequently associated with metabolic acidosis. We previously reported that metabolic acidosis stimulates expression of hypoxia-inducible factor (HIF)-1α-induced target genes such as stromal derived factor-1 and cathelicidin, an antimicrobial peptide. Since the collecting duct (CD) plays a pivotal role in regulating acid-base homeostasis and is the first nephron segment encountered by an ascending microbial infection, we examined the contribution of HIF-1α to innate immune responses elicited by acid loading of an M-1 immortalized mouse CD cell line. Acid loading of confluent M-1 cells was achieved by culture in pH 6.8 medium supplemented with 5-(N-ethyl-N-isopropyl)-amiloride to block Na+/H+ exchange activity for 24 h. Acid loading induced antimicrobial peptide [cathelicidin and ß-defensin (Defb2 and Defb26)] mRNA expression and M-1 cell resistance to uropathogenic Escherichia coli infection to an extent similar to that obtained by inhibition of HIF prolyl hydroxylases, which promote HIF-1α protein degradation. The effect of acid loading on M-1 cell resistance to uropathogenic E. coli infection was reduced by inhibition of HIF-1α (PX-478), and, in combination with prolyl hydroxylase inhibitors, acidosis did not confer additional resistance. Thus, metabolic stress of acidosis triggers HIF-1α-dependent innate immune responses in CD (M-1) cells. Whether pharmacological stabilization of HIF prevents or ameliorates pyelonephritis in vivo warrants further investigation.


Assuntos
Acidose/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Infecções por Escherichia coli/prevenção & controle , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Túbulos Renais Coletores/metabolismo , Infecções Urinárias/prevenção & controle , Escherichia coli Uropatogênica/patogenicidade , Acidose/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Linhagem Celular , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Túbulos Renais Coletores/imunologia , Túbulos Renais Coletores/microbiologia , Camundongos , Prolil Hidroxilases/metabolismo , Estabilidade Proteica , Transdução de Sinais , Regulação para Cima , Infecções Urinárias/imunologia , Infecções Urinárias/metabolismo , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/imunologia , beta-Defensinas/metabolismo , Catelicidinas
11.
Am J Physiol Renal Physiol ; 318(6): F1441-F1453, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32390512

RESUMO

Carbonic anhydrase II knockout (Car2-/-) mice have depleted numbers of renal intercalated cells, which are increasingly recognized to be innate immune effectors. We compared pyelonephritis susceptibility following reciprocal renal transplantations between Car2-/- and wild-type mice. We examined the effect of pharmacological CA suppression using acetazolamide in an experimental murine model of urinary tract infection. Car2-/- versus wild-type mice were compared for differences in renal innate immunity. In our transplant scheme, mice lacking CA-II in the kidney had increased pyelonephritis risk. Mice treated with acetazolamide had lower kidney bacterial burdens at 6 h postinfection, which appeared to be due to tubular flow from diuresis because comparable results were obtained when furosemide was substituted for acetazolamide. Isolated Car2-/- kidney cells enriched for intercalated cells demonstrated altered intercalated cell innate immune gene expression, notably increased calgizzarin and insulin receptor expression. Intercalated cell number and function along with renal tubular flow are determinants of pyelonephritis risk.


Assuntos
Acetazolamida/farmacologia , Anidrase Carbônica II/deficiência , Inibidores da Anidrase Carbônica/farmacologia , Infecções por Escherichia coli/prevenção & controle , Rim/efeitos dos fármacos , Pielonefrite/prevenção & controle , Infecções Urinárias/prevenção & controle , Acidose/enzimologia , Acidose/genética , Animais , Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica II/genética , Modelos Animais de Doenças , Infecções por Escherichia coli/enzimologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Imunidade Inata , Rim/enzimologia , Rim/imunologia , Rim/microbiologia , Transplante de Rim , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pielonefrite/enzimologia , Pielonefrite/genética , Pielonefrite/microbiologia , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Proteínas S100/genética , Proteínas S100/metabolismo , Infecções Urinárias/enzimologia , Infecções Urinárias/genética , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/patogenicidade
12.
BMC Nephrol ; 21(1): 417, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32993548

RESUMO

BACKGROUND: Acute kidney injury (AKI) is increasingly recognized as a consequential clinical complication in children with severe malaria. However, approaches to estimate baseline creatinine (bSCr) are not standardized in this unique patient population. Prior to wide-spread utilization, bSCr estimation methods need to be evaluated in many populations, particularly in children from low-income countries. METHODS: We evaluated six methods to estimate bSCr in Ugandan children aged 6 months to 12 years of age in two cohorts of children with severe malaria (n = 1078) and healthy community children (n = 289). Using isotope dilution mass spectrometry (IDMS)-traceable creatinine measures from community children, we evaluated the bias, accuracy and precision of estimating bSCr using height-dependent and height-independent estimated glomerular filtration (eGFR) equations to back-calculate bSCr or estimating bSCr directly using published or population-specific norms. RESULTS: We compared methods to estimate bSCr in healthy community children against the IDMS-traceable SCr measure. The Pottel-age based equation, assuming a normal GFR of 120 mL/min per 1.73m2, was the more accurate method with minimal bias when compared to the Schwartz height-based equation. Using the different bSCr estimates, we demonstrated the prevalence of KDIGO-defined AKI in children with severe malaria ranged from 15.6-43.4%. The lowest estimate was derived using population upper levels of normal and the highest estimate was derived using the mean GFR of the community children (137 mL/min per 1.73m2) to back-calculate the bSCr. Irrespective of approach, AKI was strongly associated with mortality with a step-wise increase in mortality across AKI stages (p < 0.0001 for all). AKI defined using the Pottel-age based equation to estimate bSCr showed the strongest relationship with mortality with a risk ratio of 5.13 (95% CI 3.03-8.68) adjusting for child age and sex. CONCLUSIONS: We recommend using height-independent age-based approaches to estimate bSCr in hospitalized children in sub-Saharan Africa due to challenges in accurate height measurements and undernutrition which may impact bSCr estimates. In this population the Pottel-age based GFR estimating equation obtained comparable bSCr estimates to population-based estimates in healthy children.


Assuntos
Injúria Renal Aguda/sangue , Análise Química do Sangue/métodos , Creatinina/sangue , Taxa de Filtração Glomerular , Malária/complicações , Injúria Renal Aguda/complicações , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/epidemiologia , Análise de Variância , Biomarcadores/sangue , Peso Corporal , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Malária/sangue , Malária/mortalidade , Masculino , Modelos Estatísticos , Prevalência , Estudos Prospectivos , Magreza , Uganda/epidemiologia
13.
Am J Physiol Renal Physiol ; 315(4): F812-F823, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28468965

RESUMO

The urinary tract is usually culture negative despite its close proximity to microbial flora. The precise mechanism by which the kidneys and urinary tract defends against infection is not well understood. The initial kidney cells to encounter ascending pathogens are the collecting tubule cells that consist of principal cells (PCs) that express aquaporin 2 (AQP2) and intercalated cells (ICs) that express vacuolar H+-ATPase (V-ATPase, B1 subunit). We have previously shown that ICs are involved with the human renal innate immune defense. Here we generated two reporter mice, VATPase B1-cre+tdT+ mice to fluorescently label ICs and AQP2-cre+tdT+ mice to fluorescently label PCs, and then performed flow sorting to enrich PCs and ICs for analysis. Isolated ICs and PCs along with proximal tubular cells were used to measure antimicrobial peptide (AMP) mRNA expression. ICs and PCs were significantly enriched for AMPs. Isolated ICs responded to uropathogenic Escherichia coli (UPEC) challenge in vitro and had higher RNase4 gene expression than control while both ICs and PCs responded to UPEC challenge in vivo by upregulating Defb1 mRNA expression. To our knowledge, this is the first report of isolating murine collecting tubule cells and performing targeted analysis for multiple classes of AMPs.


Assuntos
Aquaporina 2/imunologia , Células Epiteliais/metabolismo , Túbulos Renais Coletores/imunologia , Reação em Cadeia da Polimerase , Animais , Aquaporina 2/genética , Imunidade Inata/imunologia , Rim/imunologia , Rim/metabolismo , Camundongos Transgênicos , Reação em Cadeia da Polimerase/métodos , Regulação para Cima/imunologia , ATPases Vacuolares Próton-Translocadoras/imunologia , ATPases Vacuolares Próton-Translocadoras/metabolismo
14.
Kidney Int ; 93(1): 204-213, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28893421

RESUMO

The incidence of nephrolithiasis continues to rise. Previously, we showed that a monogenic cause could be detected in 11.4% of individuals with adult-onset nephrolithiasis or nephrocalcinosis and in 16.7-20.8% of individuals with onset before 18 years of age, using gene panel sequencing of 30 genes known to cause nephrolithiasis/nephrocalcinosis. To overcome the limitations of panel sequencing, we utilized whole exome sequencing in 51 families, who presented before age 25 years with at least one renal stone or with a renal ultrasound finding of nephrocalcinosis to identify the underlying molecular genetic cause of disease. In 15 of 51 families, we detected a monogenic causative mutation by whole exome sequencing. A mutation in seven recessive genes (AGXT, ATP6V1B1, CLDN16, CLDN19, GRHPR, SLC3A1, SLC12A1), in one dominant gene (SLC9A3R1), and in one gene (SLC34A1) with both recessive and dominant inheritance was detected. Seven of the 19 different mutations were not previously described as disease-causing. In one family, a causative mutation in one of 117 genes that may represent phenocopies of nephrolithiasis-causing genes was detected. In nine of 15 families, the genetic diagnosis may have specific implications for stone management and prevention. Several factors that correlated with the higher detection rate in our cohort were younger age at onset of nephrolithiasis/nephrocalcinosis, presence of multiple affected members in a family, and presence of consanguinity. Thus, we established whole exome sequencing as an efficient approach toward a molecular genetic diagnosis in individuals with nephrolithiasis/nephrocalcinosis who manifest before age 25 years.


Assuntos
Sequenciamento do Exoma , Mutação , Nefrocalcinose/genética , Nefrolitíase/genética , Adolescente , Idade de Início , Criança , Pré-Escolar , Progressão da Doença , Feminino , Estudos de Associação Genética , Marcadores Genéticos , Predisposição Genética para Doença , Hereditariedade , Humanos , Lactente , Masculino , Nefrocalcinose/diagnóstico por imagem , Nefrocalcinose/epidemiologia , Nefrolitíase/diagnóstico por imagem , Nefrolitíase/epidemiologia , Linhagem , Fenótipo , Valor Preditivo dos Testes , Prognóstico , Fatores de Risco , Tomografia Computadorizada por Raios X , Ultrassonografia , Adulto Jovem
15.
Am J Kidney Dis ; 71(3): 441-445, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29198386

RESUMO

Alport syndrome is a rare hereditary disorder caused by rare variants in 1 of 3 genes encoding for type IV collagen. Rare variants in COL4A5 on chromosome Xq22 cause X-linked Alport syndrome, which accounts for ∼80% of the cases. Alport syndrome has a variable clinical presentation, including progressive kidney failure, hearing loss, and ocular defects. Exome sequencing performed in 2 affected related males with an undefined X-linked glomerulopathy characterized by global and segmental glomerulosclerosis, mesangial hypercellularity, and vague basement membrane immune complex deposition revealed a COL4A5 sequence variant, a substitution of a thymine by a guanine at nucleotide 665 (c.T665G; rs281874761) of the coding DNA predicted to lead to a cysteine to phenylalanine substitution at amino acid 222, which was not seen in databases cataloguing natural human genetic variation, including dbSNP138, 1000 Genomes Project release version 01-11-2004, Exome Sequencing Project 21-06-2014, or ExAC 01-11-2014. Review of the literature identified 2 additional families with the same COL4A5 variant leading to similar atypical histopathologic features, suggesting a unique pathologic mechanism initiated by this specific rare variant. Homology modeling suggests that the substitution alters the structural and dynamic properties of the type IV collagen trimer. Genetic analysis comparing members of the 3 families indicated a distant relationship with a shared haplotype, implying a founder effect.


Assuntos
Colágeno Tipo IV/genética , Predisposição Genética para Doença , Nefrite Hereditária/genética , Nefrite Hereditária/patologia , Linhagem , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Biópsia por Agulha , Análise Mutacional de DNA , Seguimentos , Efeito Fundador , Testes Genéticos/métodos , Variação Genética , Humanos , Imuno-Histoquímica , Masculino , Nefrite Hereditária/diagnóstico , Nefrite Hereditária/tratamento farmacológico , Medição de Risco , Índice de Gravidade de Doença , Esteroides/uso terapêutico , Adulto Jovem
16.
Am J Physiol Renal Physiol ; 312(1): F43-F53, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27760770

RESUMO

Acquired renal scarring occurs in a subset of patients following febrile urinary tract infections and is associated with hypertension, proteinuria, and chronic kidney disease. Limited knowledge of histopathology, immune cell recruitment, and gene expression changes during pyelonephritis restricts the development of therapies to limit renal scarring. Here, we address this knowledge gap using immunocompetent mice with vesicoureteral reflux. Transurethral inoculation of uropathogenic Escherichia coli in C3H/HeOuJ mice leads to renal mucosal injury, tubulointerstitial nephritis, and cortical fibrosis. The extent of fibrosis correlates most significantly with inflammation at 7 and 28 days postinfection. The recruitment of neutrophils and inflammatory macrophages to infected kidneys is proportional to renal bacterial burden. Transcriptome analysis reveals molecular signatures associated with renal ischemia-reperfusion injury, immune cell chemotaxis, and leukocyte activation. This murine model recapitulates the cardinal histopathological features observed in humans with acquired renal scarring following pyelonephritis. The integration of histopathology, quantification of cellular immune influx, and unbiased transcriptional profiling begins to define potential mechanisms of tissue injury during pyelonephritis in the context of an intact immune response. The clear relationship between inflammatory cell recruitment and fibrosis supports the hypothesis that acquired renal scarring arises as a consequence of excessive host inflammation and suggests that immunomodulatory therapies should be investigated to reduce renal scarring in patients with pyelonephritis.


Assuntos
Cicatriz/metabolismo , Escherichia coli/isolamento & purificação , Inflamação/microbiologia , Rim/microbiologia , Pielonefrite/microbiologia , Refluxo Vesicoureteral/imunologia , Animais , Modelos Animais de Doenças , Feminino , Fibrose/imunologia , Fibrose/microbiologia , Inflamação/imunologia , Inflamação/patologia , Rim/patologia , Camundongos , Camundongos Endogâmicos C3H , Nefrite Intersticial/imunologia , Nefrite Intersticial/microbiologia , Nefrite Intersticial/patologia , Pielonefrite/imunologia , Traumatismo por Reperfusão/microbiologia , Traumatismo por Reperfusão/patologia , Refluxo Vesicoureteral/microbiologia
17.
J Am Soc Nephrol ; 27(10): 3175-3186, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26940096

RESUMO

The contribution of genetic variation to urinary tract infection (UTI) risk in children with vesicoureteral reflux is largely unknown. The innate immune system, which includes antimicrobial peptides, such as the α-defensins, encoded by DEFA1A3, is important in preventing UTIs but has not been investigated in the vesicoureteral reflux population. We used quantitative real-time PCR to determine DEFA1A3 DNA copy numbers in 298 individuals with confirmed UTIs and vesicoureteral reflux from the Randomized Intervention for Children with Vesicoureteral Reflux (RIVUR) Study and 295 controls, and we correlated copy numbers with outcomes. Outcomes studied included reflux grade, UTIs during the study on placebo or antibiotics, bowel and bladder dysfunction, and renal scarring. Overall, 29% of patients and 16% of controls had less than or equal to five copies of DEFA1A3 (odds ratio, 2.09; 95% confidence interval, 1.40 to 3.11; P<0.001). For each additional copy of DEFA1A3, the odds of recurrent UTI in patients receiving antibiotic prophylaxis decreased by 47% when adjusting for vesicoureteral reflux grade and bowel and bladder dysfunction. In patients receiving placebo, DEFA1A3 copy number did not associate with risk of recurrent UTI. Notably, we found that DEFA1A3 is expressed in renal epithelium and not restricted to myeloid-derived cells, such as neutrophils. In conclusion, low DEFA1A3 copy number associated with recurrent UTIs in subjects in the RIVUR Study randomized to prophylactic antibiotics, providing evidence that copy number polymorphisms in an antimicrobial peptide associate with UTI risk.


Assuntos
Peptídeos Cíclicos/genética , Polimorfismo Genético , Infecções Urinárias/genética , Refluxo Vesicoureteral/genética , alfa-Defensinas/fisiologia , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Fatores de Risco , Infecções Urinárias/etiologia , Refluxo Vesicoureteral/complicações , alfa-Defensinas/genética
18.
Kidney Int ; 90(3): 568-79, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27401534

RESUMO

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/urina
20.
Pediatr Res ; 79(6): 934-9, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26885759

RESUMO

BACKGROUND: Pediatricians frequently use urinalysis to diagnose urinary tract infection (UTI) while awaiting urine culture results, but sensitivity and specificity of urinalysis are limited. This study evaluated the diagnostic accuracy of the antimicrobial peptides (AMPs) human α-defensin 5 (HD5) and human neutrophil peptides (HNP) 1-3 as novel UTI biomarkers in children. METHODS: We prospectively enrolled 199 pediatric Emergency Department or Urgent Care patients evaluated for a UTI. Urine concentrations of HD5 and HNP1-3 were measured by enzyme-linked immunosorbent assay. Urine culture was the reference standard. Sensitivities and specificities of leukocyte esterase (LE), HD5, HNP1-3, and test combinations were compared. RESULTS: For predicting positive urine culture, the areas under the receiver-operating characteristic curves for HD5 and HNP1-3 were 0.86 (95% confidence interval (CI): 0.81-0.92) and 0.88 (95% CI: 0.82-0.93), respectively. Compared to LE ≥ trace, the combination test "LE and HD5" increased specificity by 6% (95% CI: 3-10%) without decreasing sensitivity. In the subgroup whose urine was collected by a clean-catch method, combination tests "LE and HD5" and "HD5 and HNP1-3" increased specificity by > 10% compared to LE alone. CONCLUSION: Urine AMP profiles are a promising novel strategy as an adjunct to urinalysis to aid UTI diagnosis in children.


Assuntos
Infecções Urinárias/urina , alfa-Defensinas/urina , Adolescente , Biomarcadores/urina , Criança , Pré-Escolar , Registros Eletrônicos de Saúde , Serviço Hospitalar de Emergência , Feminino , Humanos , Lactente , Masculino , Pediatria , Estudos Prospectivos , Curva ROC , Reprodutibilidade dos Testes , Tamanho da Amostra , Sensibilidade e Especificidade , Urinálise
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