RESUMO
Chronic kidney disease (CKD) affects >10% of the world population, with increasing prevalence in middle age. The risk for CKD is dependent on the number of functioning nephrons through the life cycle, and 50% of nephrons are lost through normal aging, revealing their vulnerability to internal and external stressors. Factors responsible for CKD remain poorly understood, with limited availability of biomarkers or effective therapy to slow progression. This review draws on the disciplines of evolutionary medicine and bioenergetics to account for the heterogeneous nephron injury that characterizes progressive CKD following episodes of acute kidney injury with incomplete recovery. The evolution of symbiosis in eukaryotes led to the efficiencies of oxidative phosphorylation and the rise of metazoa. Adaptations to ancestral environments are the products of natural selection that have shaped the mammalian nephron with its vulnerabilities to ischemic, hypoxic, and toxic injury. Reproductive fitness rather than longevity has served as the driver of evolution, constrained by available energy and its allocation to homeostatic responses through the life cycle. Metabolic plasticity has evolved in parallel with robustness necessary to preserve complex developmental programs, and adaptations that optimize survival through reproductive years can become maladaptive with aging, reflecting antagonistic pleiotropy. Consequently, environmental stresses promote trade-offs and mismatches that result in cell fate decisions that ultimately lead to nephron loss. Elucidation of the bioenergetic adaptations by the nephron to ancestral and contemporary environments may lead to the development of new biomarkers of kidney disease and new therapies to reduce the global burden of progressive CKD.
Assuntos
Rim , Insuficiência Renal Crônica , Pessoa de Meia-Idade , Animais , Humanos , Rim/metabolismo , Néfrons/metabolismo , Insuficiência Renal Crônica/epidemiologia , Insuficiência Renal Crônica/metabolismo , Envelhecimento , Metabolismo Energético , MamíferosRESUMO
A kidney organoid is a three-dimensional (3D) cellular aggregate grown from stem cells in vitro that undergoes self-organization, recapitulating aspects of normal renal development to produce nephron structures that resemble the native kidney organ. These miniature kidney-like structures can also be derived from primary patient cells and thus provide simplified context to observe how mutations in kidney-disease-associated genes affect organogenesis and physiological function. In the past several years, advances in kidney organoid technologies have achieved the formation of renal organoids with enhanced numbers of specialized cell types, less heterogeneity, and more architectural complexity. Microfluidic bioreactor culture devices, single-cell transcriptomics, and bioinformatic analyses have accelerated the development of more sophisticated renal organoids and tailored them to become increasingly amenable to high-throughput experimentation. However, many significant challenges remain in realizing the use of kidney organoids for renal replacement therapies. This review presents an overview of the renal organoid field and selected highlights of recent cutting-edge kidney organoid research with a focus on embryonic development, modeling renal disease, and personalized drug screening.
Assuntos
Rim , Néfrons , Humanos , Avaliação Pré-Clínica de Medicamentos , Rim/metabolismo , Néfrons/metabolismo , Organoides/metabolismo , OrganogêneseRESUMO
A reduced nephron number at birth, due to critical gestational conditions, including maternal malnutrition, is associated with the risk of developing hypertension and chronic kidney disease in adulthood. No interventions are currently available to augment nephron number. We have recently shown that sirtuin 3 (SIRT3) has an important role in dictating proper nephron endowment. The present study explored whether SIRT3 stimulation, by means of supplementation with nicotinamide riboside (NR), a precursor of the SIRT3 co-substrate nicotinamide adenine dinucleotide (NAD+), was able to improve nephron number in a murine model of a low protein (LP) diet. Our findings show that reduced nephron number in newborn mice (day 1) born to mothers fed a LP diet was associated with impaired renal SIRT3 expression, which was restored through supplementation with NR. Glomerular podocyte density, as well as the rarefaction of renal capillaries, also improved through NR administration. In mechanistic terms, the restoration of SIRT3 expression through NR was mediated by the induction of proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α). Moreover, NR restored SIRT3 activity, as shown by the reduction of the acetylation of optic atrophy 1 (OPA1) and superoxide dismutase 2 (SOD2), which resulted in improved mitochondrial morphology and protection against oxidative damage in mice born to mothers fed the LP diet. Our results provide evidence that it is feasible to prevent nephron mass shortage at birth through SIRT3 boosting during nephrogenesis, thus providing a therapeutic option to possibly limit the long-term sequelae of reduced nephron number in adulthood.
Assuntos
Sirtuína 3 , Camundongos , Animais , Sirtuína 3/metabolismo , NAD , Dieta com Restrição de Proteínas , PPAR gama , Néfrons/metabolismo , Suplementos NutricionaisRESUMO
We measured the activities of epithelial Na channels (ENaC) and ROMK channels in the distal nephron of the mouse kidney and assessed their role in the process of K+ secretion under different physiological conditions. Under basal dietary conditions (0.5% K), ENaC activity, measured as amiloride-sensitive currents, was high in cells at the distal end of the distal convoluted tubule (DCT) and proximal end of the connecting tubule (CNT), a region we call the early CNT (CNTe). In more distal parts of the CNT (aldosterone-sensitive portion [CNTas]), these currents were minimal. This functional difference correlated with alterations in the intracellular location of ENaC, which was at or near the apical membrane in CNTe and more cytoplasmic in the CNTas. ROMK activity, measured as TPNQ-sensitive currents, was substantial in both segments. A mathematical model of the rat nephron suggested that K+ secretion by the CNTe predicted from these currents provides much of the urinary K+ required for K balance on this diet. In animals fed a K-deficient diet (0.1% K), both ENaC and ROMK currents in the CNTe decreased by â¼50%, predicting a 50% decline in K+ secretion. Enhanced reabsorption by a separate mechanism is required to avoid excessive urinary K+ losses. In animals fed a diet supplemented with 3% K, ENaC currents increased modestly in the CNTe but strongly in the CNTas, while ROMK currents tripled in both segments. The enhanced secretion of K+ by the CNTe and the recruitment of secretion by the CNTas account for the additional transport required for K balance. Therefore, adaptation to increased K+ intake involves the extension of robust K+ secretion to more distal parts of the nephron.
Assuntos
Canais Epiteliais de Sódio , Canais de Potássio Corretores do Fluxo de Internalização , Animais , Túbulos Renais Distais/metabolismo , Camundongos , Néfrons/metabolismo , Ratos , Sódio/metabolismoRESUMO
Several groups have developed in vitro expansion cultures for mouse metanephric nephron progenitor cells (NPCs) using cocktails of small molecules and growth factors including BMP7. However, the detailed mechanisms by which BMP7 acts in the NPC expansion remain to be elucidated. Here, by performing chemical screening for BMP substitutes, we identified a small molecule, TCS21311, that can replace BMP7 and revealed a novel inhibitory role of BMP7 in JAK3-STAT3 signaling in NPC expansion culture. Further, we found that TCS21311 facilitates the proliferation of mouse embryonic NPCs and human induced pluripotent stem cell-derived NPCs when added to the expansion culture. These results will contribute to understanding the mechanisms of action of BMP7 in NPC proliferation in vitro and in vivo and to the stable supply of NPCs for regenerative therapy, disease modeling and drug discovery for kidney diseases.
Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Inibidores de Janus Quinases/farmacologia , Néfrons/citologia , Néfrons/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 7/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Meios de Cultura , Avaliação Pré-Clínica de Medicamentos , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Janus Quinase 3/antagonistas & inibidores , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Néfrons/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas PequenasRESUMO
BACKGROUND: Regulation of sodium chloride transport in the aldosterone-sensitive distal nephron is essential for fluid homeostasis and BP control. The chloride-bicarbonate exchanger pendrin in ß-intercalated cells, along with sodium chloride cotransporter (NCC) in distal convoluted tubules, complementarily regulate sodium chloride handling, which is controlled by the renin-angiotensin-aldosterone system. METHODS: Using mice with mineralocorticoid receptor deletion in intercalated cells, we examined the mechanism and roles of pendrin upregulation via mineralocorticoid receptor in two different models of renin-angiotensin-aldosterone system activation. We also used aldosterone-treated NCC knockout mice to examine the role of pendrin regulation in salt-sensitive hypertension. RESULTS: Deletion of mineralocorticoid receptor in intercalated cells suppressed the increase in renal pendrin expression induced by either exogenous angiotensin II infusion or endogenous angiotensin II upregulation via salt restriction. When fed a low-salt diet, intercalated cell-specific mineralocorticoid receptor knockout mice with suppression of pendrin upregulation showed BP reduction that was attenuated by compensatory activation of NCC. In contrast, upregulation of pendrin induced by aldosterone excess combined with a high-salt diet was scarcely affected by deletion of mineralocorticoid receptor in intercalated cells, but depended instead on hypokalemic alkalosis through the activated mineralocorticoid receptor-epithelial sodium channel cascade in principal cells. In aldosterone-treated NCC knockout mice showing upregulation of pendrin, potassium supplementation corrected alkalosis and inhibited the pendrin upregulation, thereby lowering BP. CONCLUSIONS: In conjunction with NCC, the two pathways of pendrin upregulation, induced by angiotensin II through mineralocorticoid receptor activation in intercalated cells and by alkalosis through mineralocorticoid receptor activation in principal cells, play important roles in fluid homeostasis during salt depletion and salt-sensitive hypertension mediated by aldosterone excess.
Assuntos
Hipertensão/etiologia , Néfrons/metabolismo , Néfrons/patologia , Receptores de Mineralocorticoides/fisiologia , Simportadores de Cloreto de Sódio/fisiologia , Transportadores de Sulfato/metabolismo , Aldosterona , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Sistema Renina-Angiotensina/fisiologiaRESUMO
In a recent issue of Nephron, Abu-Amer et al.[
Assuntos
Cardiotônicos/efeitos adversos , Digoxina/efeitos adversos , Magnésio/urina , Humanos , Rim/metabolismo , Túbulos Renais/metabolismo , Magnésio/sangue , Magnésio/metabolismo , Néfrons/metabolismoRESUMO
Most vertebrates, including cartilaginous fishes, maintain their plasma SO4 (2-) concentration ([SO4 (2-)]) within a narrow range of 0.2-1 mM. As seawater has a [SO4 (2-)] about 40 times higher than that of the plasma, SO4 (2-) excretion is the major role of kidneys in marine teleost fishes. It has been suggested that cartilaginous fishes also excrete excess SO4 (2-) via the kidney. However, little is known about the underlying mechanisms for SO4 (2-) transport in cartilaginous fish, largely due to the extraordinarily elaborate four-loop configuration of the nephron, which consists of at least 10 morphologically distinguishable segments. In the present study, we determined cDNA sequences from the kidney of holocephalan elephant fish (Callorhinchus milii) that encoded solute carrier family 26 member 1 (Slc26a1) and member 6 (Slc26a6), which are SO4 (2-) transporters that are expressed in mammalian and teleost kidneys. Elephant fish Slc26a1 (cmSlc26a1) and cmSlc26a6 mRNAs were coexpressed in the proximal II (PII) segment of the nephron, which comprises the second loop in the sinus zone. Functional analyses using Xenopus oocytes and the results of immunohistochemistry revealed that cmSlc26a1 is a basolaterally located electroneutral SO4 (2-) transporter, while cmSlc26a6 is an apically located, electrogenic Cl(-)/SO4 (2-) exchanger. In addition, we found that both cmSlc26a1 and cmSlc26a6 were abundantly expressed in the kidney of embryos; SO4 (2-) was concentrated in a bladder-like structure of elephant fish embryos. Our results demonstrated that the PII segment of the nephron contributes to the secretion of excess SO4 (2-) by the kidney of elephant fish. Possible mechanisms for SO4 (2-) secretion in the PII segment are discussed.
Assuntos
Peixe Elétrico/metabolismo , Túbulos Renais Proximais/metabolismo , Rim/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sulfatos/metabolismo , Sequência de Aminoácidos , Animais , DNA Complementar/genética , Embrião não Mamífero/metabolismo , Proteínas de Peixes/metabolismo , Néfrons/metabolismo , Oócitos/metabolismo , Distribuição Tecidual , XenopusRESUMO
A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells. Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport. Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein. A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3' untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.
Assuntos
Aldosterona/metabolismo , Córtex Renal/metabolismo , Túbulos Renais Coletores/metabolismo , MicroRNAs/metabolismo , Sódio/metabolismo , Aldosterona/genética , Animais , Anquirinas/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Canais Epiteliais de Sódio/metabolismo , Córtex Renal/citologia , Túbulos Renais Coletores/citologia , Luciferases/genética , Camundongos Endogâmicos C57BL , Néfrons/citologia , Néfrons/metabolismo , RNA Interferente Pequeno/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The zebrafish pronephros provides a conserved model to study kidney development, in particular to delineate the poorly understood processes of how nephron segment pattern and cell type choice are established. Zebrafish nephrons are divided into distinct epithelial regions that include a series of proximal and distal tubule segments, which are comprised of intercalated transporting epithelial cells and multiciliated cells (MCC). Previous studies have shown that retinoic acid (RA) regionalizes the renal progenitor field into proximal and distal domains and that Notch signaling later represses MCC differentiation, but further understanding of these pathways has remained unknown. The transcription factor mecom (mds1/evi1 complex) is broadly expressed in renal progenitors, and then subsequently marks the distal tubule. Here, we show that mecom is necessary to form the distal tubule and to restrict both proximal tubule formation and MCC fate choice. We found that mecom and RA have opposing roles in patterning discrete proximal and distal segments. Further, we discovered that RA is required for MCC formation, and that one mechanism by which RA promotes MCC fate choice is to inhibit mecom. Next, we determined the epistatic relationship between mecom and Notch signaling, which limits MCC fate choice by lateral inhibition. Abrogation of Notch signaling with the γ-secretase inhibitor DAPT revealed that Notch and mecom did not have additive effects in blocking MCC formation, suggesting that they function in the same pathway. Ectopic expression of the Notch signaling effector, Notch intracellular domain (NICD), rescued the expansion of MCCs in mecom morphants, indicating that mecom acts upstream to induce Notch signaling. These findings suggest a model in which mecom and RA arbitrate proximodistal segment domains, while MCC fate is modulated by a complex interplay in which RA inhibition of mecom, and mecom promotion of Notch, titrates MCC number. Taken together, our studies have revealed several essential and novel mechanisms that control pronephros development in the zebrafish.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Néfrons/embriologia , Receptores Notch/metabolismo , Tretinoína/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Diferenciação Celular , Linhagem da Célula , Epistasia Genética , Genômica , Rim/embriologia , Proteína do Locus do Complexo MDS1 e EVI1 , Néfrons/metabolismo , Organogênese/fisiologia , Pronefro/metabolismo , Estrutura Terciária de Proteína , RNA Complementar/metabolismo , Transdução de Sinais , Fatores de Tempo , Peixe-Zebra/genéticaRESUMO
Oxidative stress causes the generation of reactive oxygen species (ROS) that lead to nephrotoxicity. An aminoglycoside, gentamicin, has pronounced nephrotoxic effect in humans and animals and this study was planned to observe the nephro-protective effect of antioxidants, vitamin C and Nigella sativa oil. Serum creatinine, blood urea nitrogen, and antioxidant activity were measured as indicators of nephrotoxicity for all the groups of rabbits. Results showed that vitamin C and Nigella sativa oil both had nephro-protective effect as they lowered the values of nephrotoxicity indicators (serum creatinine, blood urea nitrogen, and antioxidant activity) as compared to gentamicin control group values. When these two antioxidants were given as combination, they proved to have synergistic nephro-protective effect.
Assuntos
Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Gentamicinas , Nefropatias/prevenção & controle , Néfrons/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Óleos de Plantas/farmacologia , Animais , Biomarcadores/sangue , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Citoproteção , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Masculino , Néfrons/metabolismo , Coelhos , Fatores de TempoRESUMO
Dietary potassium (K(+)) restriction and hypokalemia have been reported to change the abundance of most renal Na(+) and K(+) transporters and aquaporin-2 isoform, but results have not been consistent. The aim of this study was to reexamine Na(+), K(+) and H(2)O transporters' pool size regulation in response to removing K(+) from a diet containing 0.74% NaCl, as well as from a diet containing 2% NaCl (as found in American diets) to blunt reducing total diet electrolytes. Sprague-Dawley rats (n = 5-6) were fed for 6 days with one of these diets: 2% KCl, 0.74% NaCl (2K1Na, control chow) compared with 0.03% KCl, 0.74% NaCl (0K1Na); or 2% KCl, 2%NaCl (2K2Na) compared with 0.03% KCl, 2% NaCl (0K2Na, Na(+) replete). In both 0K1Na and 0K2Na there were significant decreases in: 1) plasma [K(+)] (<2.5 mM); 2) urinary K(+) excretion (<5% of control); 3) urine osmolality and plasma [aldosterone], as well as 4) an increase in urine volume and medullary hypertrophy. The 0K2Na group had the lowest [aldosterone] (172.0 ± 17.4 pg/ml) and lower blood pressure (93.2 ± 4.9 vs. 112.0 ± 3.1 mmHg in 2K2Na). Transporter pool size regulation was determined by quantitative immunoblotting of renal cortex and medulla homogenates. The only differences measured in both 0K1Na and 0K2Na groups were a 20-30% decrease in cortical ß-ENaC, 30-40% increases in kidney-specific Ste20/SPS1-related proline/alanine-rich kinase, and a 40% increase in medullary sodium pump abundance. The following proteins were not significantly changed in both the 0 K groups: Na(+)/H(+) exchanger isoform 3; Na(+)-K(+)-Cl(-) cotransporter; Na(+)-Cl(-) cotransporter, oxidative stress response kinase-1; renal outer medullary K(+) channel; autosomal recessive hypercholesterolemia; c-Src, aquaporin 2 isoform; or renin. Thus, despite profound hypokalemia and renal K(+) conservation, we did not confirm many of the changes that were previously reported. We predict that changes in transporter distribution and activity are likely more important for conserving K(+) than changes in total abundance.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Néfrons/metabolismo , Deficiência de Potássio/metabolismo , Potássio na Dieta/farmacologia , Cloreto de Sódio na Dieta/farmacologia , Trocadores de Sódio-Hidrogênio/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Animais , Masculino , Néfrons/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Suboptimal kidney development resulting from a genetic deficit in nephron number can have lifelong consequences that may lead to cardiorenal complications upon exposure to secondary insults in later life. To determine whether the inherited reduced renal reserve compromises the ability to handle osmotic stress in the adult animal, we challenged the heterozygous 3H1 Brachyrrhine (Br/+) mouse, which displays heritable renal hypoplasia associated with reduced embryonic six2 expression, to a solution of 2% NaCl for 5 days or to fluid restriction for 48 h. Blood chemistry, fluid intake, and physiological parameters, including renal measurements, were determined. Systemic hypertonicity by prolonged salt loading led to significant increases in plasma osmolality and plasma Na(+), along with polydipsia and polyuria, with a significant urine-concentrating defect that was resistant to DDAVP treatment in the adult Br/+ mouse compared with wild-type littermates. The Br/+ mouse also developed a significant increase in blood urea nitrogen at baseline that was further elevated when 2% NaCl was given. Fluid restriction for 48 h further enhanced plasma osmolality and plasma Na(+) responses, although the Br/+ mouse was evidently able to produce a small amount of concentrated urine at this time. Hypothalamic c-Fos expression was appropriately activated in the Br/+ mouse in response to both osmotic challenges, indicating an intact central neuroendocrine pathway that was not affected by the lack of congenital six2 expression. Collectively, our results demonstrate impaired osmoregulatory mechanisms consistent with chronic renal failure in the Br/+ mouse and indicate that six2 haploinsufficiency has a direct effect on postnatal fluid and electrolyte handling associated with fluid imbalance.
Assuntos
Falência Renal Crônica/metabolismo , Néfrons/metabolismo , Fatores de Transcrição/deficiência , Equilíbrio Hidroeletrolítico , Análise de Variância , Animais , Antidiuréticos/administração & dosagem , Nitrogênio da Ureia Sanguínea , Desamino Arginina Vasopressina/administração & dosagem , Ingestão de Líquidos , Regulação da Expressão Gênica no Desenvolvimento , Haploinsuficiência , Proteínas de Homeodomínio/genética , Hipotálamo/metabolismo , Hipotálamo/fisiopatologia , Capacidade de Concentração Renal , Falência Renal Crônica/genética , Falência Renal Crônica/fisiopatologia , Camundongos , Camundongos Mutantes , Néfrons/anormalidades , Néfrons/efeitos dos fármacos , Néfrons/fisiopatologia , Organogênese , Concentração Osmolar , Poliúria/genética , Poliúria/metabolismo , Poliúria/fisiopatologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Solução Salina Hipertônica/administração & dosagem , Solução Salina Hipertônica/metabolismo , Sódio/sangue , Cloreto de Sódio na Dieta/administração & dosagem , Cloreto de Sódio na Dieta/metabolismo , Fatores de Transcrição/genética , Equilíbrio Hidroeletrolítico/efeitos dos fármacos , Equilíbrio Hidroeletrolítico/genéticaRESUMO
The mode of cellular toxicity of aqueous extract of Fadogia agrestis stem in male rats was investigated. Rats were grouped into four: A, B, C and D where A (the control) received orally 1 mL of distilled water; B, C and D (test groups) received orally 18, 50 and 100 mg/kg body weight of the extract, respectively, for 28 days. Infrared spectroscopy indicated the presence of hydroxyl (OH) and primary amine (CONH). Clinical toxicity symptoms such as respiratory distress, epistasis, salivation, hypo- and hyperactivity were not observed at any period of the experiment. No mortality was also recorded. Extract administration significantly reduced (p < .05) the activities of alkaline phosphatase, lactate dehydrogenase and gamma glutamyl transferase in the liver and kidney with corresponding increases in the serum. Serum malondialdehyde also increased significantly in all the extract-treated groups. The liver and kidney body weight ratios of the extract-treated animals compared well (P > .05) with their controls throughout the experimental period. The extract did not cause any swelling, atrophy or hypertrophy of the organs. The other evidence in this study suggests disruption of the ordered lipid bilayer of the plasma membranes of the hepatocytes and nephrons. This might have resulted from peroxidation of the polyunsaturated fatty acids on the membranes of the hepatocytes and nephrons made possible by the functional groups or the product of metabolism of the extract. This may be responsible for the compromise of the integrity of the plasma membranes of the hepatocytes and nephrons.
Assuntos
Membrana Celular/efeitos dos fármacos , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Extratos Vegetais/toxicidade , Rubiaceae/química , Fosfatase Alcalina/sangue , Fosfatase Alcalina/metabolismo , Animais , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Ácidos Graxos Insaturados/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Rim/enzimologia , Rim/metabolismo , Rim/patologia , L-Lactato Desidrogenase/sangue , L-Lactato Desidrogenase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Masculino , Malondialdeído/sangue , Néfrons/efeitos dos fármacos , Néfrons/enzimologia , Néfrons/metabolismo , Néfrons/patologia , Oxirredução , Caules de Planta/química , Ratos , gama-Glutamiltransferase/sangue , gama-Glutamiltransferase/metabolismoRESUMO
A novel member of the organic anion transporter (OAT) family, Oat5 (Slc22a19), has been reported to transport a naturally occurring mycotoxin, ochratoxin A (OTA). However, neither its endogenous substrate and driving force nor physiological functions have been determined. Herein, we report the functional characterization of rat Oat5 (rOat5), as well as its intrarenal distribution and membrane localization. When expressed in Xenopus laevis oocytes, rOat5 mediated the transport of sulfate conjugates of steroids such as estrone-3-sulfate (E(1)S; K(m) = 18.9 +/- 3.9 microM) and dehydroepiandrosterone sulfate (K(m) = 2.3 +/- 0.2 microM) in a sodium-independent manner, in addition to OTA. The rOat5-mediated E(1)S transport was strongly inhibited by four-carbon (C4) dicarboxylate succinate and longer dicarboxylates (C7-C9). The uptake of [(3)H]E(1)S via rOat5 was significantly trans-stimulated by succinate, and the efflux of [(14)C]succinate was significantly trans-stimulated by E(1)S. A similar trans-stimulatory effect of preloaded succinate on E(1)S uptake was also detected in cells stably expressing rOat5 (S(2) rOat5). rOat5 interacted with chemically heterogenous anionic compounds. The rOat5-mediated E(1)S transport was inhibited by several sulfate conjugates, such as 4-methylumbelliferyl sulfate and beta-estradiol sulfate, but not by glucuronide conjugates. An immunohistochemical study showed that rOat5 was localized at the apical membrane of renal proximal tubules in the corticomedullary region. rOat5 mRNA was expressed in the late segments (S(2) and S(3)) of proximal tubules. These results indicate that rOat5 is renal organic anion/dicarboxylates exchanger and, under physiological conditions, may function as an apical reabsorptive pathway for organic anions in proximal tubules driven by an outward gradient of dicarboxylates.
Assuntos
Transportadores de Ácidos Dicarboxílicos/metabolismo , Túbulos Renais Proximais/metabolismo , Animais , Western Blotting , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , DNA Complementar/biossíntese , DNA Complementar/genética , Sulfato de Desidroepiandrosterona/metabolismo , Estrona/análogos & derivados , Estrona/metabolismo , Etiquetas de Sequências Expressas , Imuno-Histoquímica , Cinética , Membranas/metabolismo , Néfrons/citologia , Néfrons/metabolismo , Oócitos/metabolismo , RNA Complementar/biossíntese , RNA Complementar/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Succinatos/metabolismo , Transfecção , Xenopus laevisRESUMO
Lipid-mediated injury plays an important role in the pathogenesis of many renal diseases including diabetic nephropathy. Liver X receptor-alpha (LXRalpha) is an intracellular sterol sensor that regulates expression of genes controlling cholesterol absorption, excretion, catabolism, and cellular efflux. The present study was aimed at examining the role of LXRalpha in cholesterol metabolism in glomerular mesangial cells. A 1,561-bp fragment of full-length rabbit LXR cDNA was cloned. The deduced protein sequence exhibited 92.4 and 89.2% identity to human and mouse LXRalpha, respectively. Tissue distribution studies showed that rabbit LXRalpha was expressed in the liver, spleen, and kidney. In situ hybridization and RT-PCR assays further indicated that LXRalpha mRNA was widely expressed in the kidney and present in every nephron segment including the glomeruli. To determine intrarenal regulation of LXRalpha, rabbits were treated with thiazolidinedione (TZD) peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists, which have been previously shown to enhance LXRalpha expression via PPARgamma and increase cholesterol efflux in macrophages. The results showed that glomerular LXRalpha expression was markedly induced by TZDs. In cultured rabbit mesangial cells, LXRalpha mRNA and protein were detected by RT-PCR and immunoblotting. Treatment of mesangial cells with a specific LXRalpha agonist, TO-901317, significantly increased basal and apolipoprotein AI-mediated cholesterol efflux and markedly enhanced the promoter activity of an LXRalpha target gene, ATP-binding cassette transporter A1 (ABCA1). In conclusion, LXRalpha is expressed in renal glomeruli and functionally present in mesangial cells where its activation mediates cholesterol efflux via ABCA1. These data suggest that LXRalpha may be a potential therapeutic target for treating lipid-related renal glomerular disease.
Assuntos
Colesterol/metabolismo , Mesângio Glomerular/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Região 3'-Flanqueadora/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Células Cultivadas , DNA Complementar/biossíntese , DNA Complementar/genética , Proteínas de Ligação a DNA , Genes Reporter/genética , Vetores Genéticos , Mesângio Glomerular/citologia , Homeostase/fisiologia , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Rim/metabolismo , Receptores X do Fígado , Luciferases/genética , Masculino , Dados de Sequência Molecular , Néfrons/metabolismo , Receptores Nucleares Órfãos , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The chloride channel ClC-Kb is expressed in the basolateral cell membrane of the distal nephron and participates in renal NaCl reabsorption. Loss-of-function mutations of ClC-Kb lead to classic Bartter syndrome, a rare salt-wasting disorder. Recently, we identified the ClC-Kb(T481S) polymorphism, which confers a strong gain-of-function effect on the ClC-Kb chloride channel. The present study has been performed to explore the prevalence of the mutation and its functional significance in renal salt handling and blood pressure regulation. As evident from electrophysiological analysis with the 2-electrode voltage-clamp technique, heterologous expression of ClC-Kb(T481S) in Xenopus oocytes gave rise to a current that was 7-fold larger than the current produced by wild-type ClC-Kb. The prevalence of the mutant allele was significantly higher in an African population from Ghana (22%) than in whites (12%). As tested in 1 white population, carriers of ClC-Kb(T481S) were associated with significantly higher systolic (by approximately 6.0 mm Hg) and diastolic (by approximately 4.2 mm Hg) blood pressures and significantly higher prevalence (45% versus 25%) of hypertensive (> or =140/90 mm Hg) blood pressure levels. Individuals carrying ClC-Kb(T481S) had significantly higher plasma Na+ concentrations and significantly decreased glomerular filtration rate. In conclusion, the mutation ClC-Kb(T481S) of the renal epithelial Cl- channel ClC-Kb strongly activates ClC-Kb chloride channel function in vitro and may predispose to the development of essential hypertension in vivo.
Assuntos
Substituição de Aminoácidos , Proteínas de Transporte de Ânions/genética , Canais de Cloreto/genética , Hipertensão/genética , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Mutação Puntual , Adulto , Animais , Proteínas de Transporte de Ânions/metabolismo , População Negra/genética , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Alemanha , Gana , Humanos , Transporte de Íons/genética , Masculino , Proteínas de Membrana/metabolismo , Natriurese/genética , Néfrons/metabolismo , Oócitos , Técnicas de Patch-Clamp , Potássio/metabolismo , RNA Complementar/genética , Sódio/metabolismo , População Branca/genética , Xenopus laevisRESUMO
The great advantage of the tetracycline-inducible system lies in its ability to address a large variety of biological questions in a time-dependent and tissue-specific manner. This study describes a transgenic mouse line, rTA(LAP)-1, which produces the reverse tetracycline transactivator under control of the liver activator protein (LAP) promoter. Two reporter lines with luciferase and LacZ reporter genes were used to demonstrate predominant expression in the kidney and liver when doxycycline was added to the drinking water. In the kidney, transgene expression was found primarily in cortical proximal tubules. No luciferase and beta-galactosidase activity was detected in mice without doxycycline in the drinking water, which attests to the tight control of this system. One of the advantages of the tet system lies in its reversibility, and indeed, a virtually complete remission of transgene activity in both the kidney and liver was observed when doxycycline was withdrawn. Also examined was transactivator activity during development by exposing the mothers producing the reverse transactivator to doxycycline before mating. Transgene activity was detected in newborn kidneys and liver, indicating that sufficient amounts of doxycycline had crossed the placental barrier. During nephron development, the LAP promoter appeared to be only active in the more mature proximal tubules. Finally, the rTA(LAP)-1 line was used to inducibly express the human PKD2 cDNA in proximal tubules of transgenic mice, but no cystic changes were detected, even after 6 mo of induction.
Assuntos
Técnicas Genéticas , Rim/metabolismo , Biossíntese de Proteínas , Tetraciclina/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Western Blotting , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular , DNA Complementar/metabolismo , Doxiciclina/metabolismo , Doxiciclina/farmacologia , Feminino , Genótipo , Células HeLa , Humanos , Imuno-Histoquímica , Cinética , Óperon Lac , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Néfrons/metabolismo , Regiões Promotoras Genéticas , Fatores de Tempo , Ativação Transcricional , Transfecção , Transgenes , beta-Galactosidase/metabolismoRESUMO
The aim of this study was to assess the influence of some anaesthesia methods on renal function in patients with no clinical and laboratory symptoms of nephropathy. The activity of various parts of the nephron was evaluated on the basis of the urinary excretion of albumins (marker of renal glomeruli function), beta-2-microglobulins (marker of proximal tubule function) and Tamm-Horsfall protein (marker of distal tubule function). 45 patients were divided into three groups, basing on the kind of anaesthesia: "O"--general, "P"--spinal and "N"--local. The patients who were routinely found to have no signs of nephropathy showed no dysfunction of renal glomeruli and distal or proximal tubule as determined by the urinary excretion of albumins, Tamm-Horsfall protein and beta-2-microglobulins prior to the operation. Different anaesthesia techniques had no effect on the urinary excretion of beta-2-microglobulins. In contrast to local and spinal anaesthesia, general anaesthesia significantly increased the urinary excretion of albumins. General, spinal as well as local anaesthesia significantly decreased the urinary excretion of Tamm-Horsfall protein. The decrease in the excretion of this glycoprotein suggests that all types of anaesthesia techniques may influence the function of the distal part of nephron.
Assuntos
Anestesia/efeitos adversos , Anestesia/métodos , Anestésicos/efeitos adversos , Néfrons/efeitos dos fármacos , Néfrons/metabolismo , Albuminas/metabolismo , Anestesia Geral/efeitos adversos , Anestesia Local/efeitos adversos , Raquianestesia/efeitos adversos , Anestésicos Gerais/efeitos adversos , Anestésicos Locais/efeitos adversos , Biomarcadores/análise , Feminino , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Pessoa de Meia-Idade , Mucoproteínas/metabolismo , Uromodulina , Microglobulina beta-2/metabolismoRESUMO
Sodium and potassium-exchanging adenosine triphosphatase (Na,K-ATPase) in the kidney is associated with the gamma subunit (gamma, FXYD2), a single-span membrane protein that modulates ATPase properties. Rat and human gamma occur in two splice variants, gamma(a) and gamma(b), with different N termini. Here we investigated their structural heterogeneity and functional effects on Na,K-ATPase properties. Both forms were post-translationally modified during in vitro translation with microsomes, indicating that there are four possible forms of gamma. Site-directed mutagenesis revealed Thr(2) and Ser(5) as potential sites for post-translational modification. Similar modification can occur in cells, with consequences for Na,K-ATPase properties. We showed previously that stable transfection of gamma(a) into NRK-52E cells resulted in reduction of apparent affinities for Na(+) and K(+). Individual clones differed in gamma post-translational modification, however, and the effect on Na(+) affinity was absent in clones with full modification. Here, transfection of gamma(b) also resulted in clones with or without post-translational modification. Both groups showed a reduction in Na(+) affinity, but modification was required for the effect on K(+) affinity. There were minor increases in ATP affinity. The physiological importance of the reduction in Na(+) affinity was shown by the slower growth of gamma(a), gamma(b), and gamma(b') transfectants in culture. The differential influence of the four structural variants of gamma on affinities of the Na,K-ATPase for Na(+) and K(+), together with our previous finding of different distributions of gamma(a) and gamma(b) along the rat nephron, suggests a highly specific mode of regulation of sodium pump properties in kidney.