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1.
PNAS Nexus ; 3(5): pgae187, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38807632

RESUMO

Chronic and genetic kidney diseases such as autosomal dominant polycystic kidney disease (ADPKD) have few therapeutic options, and clinical trials testing small molecule drugs have been unfavorable due to low kidney bioavailability and adverse side effects. Although nanoparticles can be designed to deliver drugs directly to the diseased site, there are no kidney-targeted nanomedicines clinically available, and most FDA-approved nanoparticles are administered intravenously which is not ideal for chronic diseases. To meet these challenges of chronic diseases, we developed a biomaterials-based strategy using chitosan particles (CP) for oral delivery of therapeutic, kidney-targeting peptide amphiphile micelles (KMs). We hypothesized that encapsuling KMs into CP would enhance the bioavailability of KMs upon oral administration given the high stability of chitosan in acidic conditions and mucoadhesive properties enabling absorption within the intestines. To test this, we evaluated the mechanism of KM access to the kidneys via intravital imaging and investigated the KM biodistribution in a porcine model. Next, we loaded KMs carrying the ADPKD drug metformin into CP (KM-CP-met) and measured in vitro therapeutic effect. Upon oral administration in vivo, KM-CP-met showed significantly greater bioavailability and accumulation in the kidneys as compared to KM only or free drug. As such, KM-CP-met treatment in ADPKD mice (Pkd1fl/fl;Pax8-rtTA;Tet-O-Cre which develops the disease over 120 days and mimics the slow development of ADPKD) showed enhanced therapeutic efficacy without affecting safety despite repeated treatment. Herein, we demonstrate the potential of KM-CP as a nanomedicine strategy for oral delivery for the long-term treatment of chronic kidney diseases.

2.
Biomacromolecules ; 25(5): 2749-2761, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38652072

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is a complex disorder characterized by uncontrolled renal cyst growth, leading to kidney function decline. The multifaceted nature of ADPKD suggests that single-pathway interventions using individual small molecule drugs may not be optimally effective. As such, a strategy encompassing combination therapy that addresses multiple ADPKD-associated signaling pathways could offer synergistic therapeutic results. However, severe off-targeting side effects of small molecule drugs pose a major hurdle to their clinical transition. To address this, we identified four drug candidates from ADPKD clinical trials, bardoxolone methyl (Bar), octreotide (Oct), salsalate (Sal), and pravastatin (Pra), and incorporated them into peptide amphiphile micelles containing the RGD peptide (GRGDSP), which binds to the basolateral surface of renal tubules via integrin receptors on the extracellular matrix. We hypothesized that encapsulating drug combinations into RGD micelles would enable targeting to the basolateral side of renal tubules, which is the site of disease, via renal secretion, leading to superior therapeutic benefits compared to free drugs. To test this, we first evaluated the synergistic effect of drug combinations using the 20% inhibitory concentration for each drug (IC20) on renal proximal tubule cells derived from Pkd1flox/-:TSLargeT mice. Next, we synthesized and characterized the RGD micelles encapsulated with drug combinations and measured their in vitro therapeutic effects via a 3D PKD growth model. Upon both IV and IP injections in vivo, RGD micelles showed a significantly higher accumulation in the kidneys compared to NT micelles, and the renal access of RGD micelles was significantly reduced after the inhibition of renal secretion. Specifically, both Bar+Oct and Bar+Sal in the RGD micelle treatment showed enhanced therapeutic efficacy in ADPKD mice (Pkd1fl/fl;Pax8-rtTA;Tet-O-Cre) with a significantly lower KW/BW ratio and cyst index as compared to PBS and free drug-treated controls, while other combinations did not show a significant difference. Hence, we demonstrate that renal targeting through basolateral targeting micelles enhances the therapeutic potential of combination therapy in genetic kidney disease.


Assuntos
Sistemas de Liberação de Medicamentos , Micelas , Animais , Camundongos , Sistemas de Liberação de Medicamentos/métodos , Humanos , Rim Policístico Autossômico Dominante/tratamento farmacológico , Rim Policístico Autossômico Dominante/patologia , Oligopeptídeos/química , Doenças Renais Policísticas/tratamento farmacológico , Doenças Renais Policísticas/patologia
3.
Front Mol Biosci ; 9: 1001941, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36504724

RESUMO

ADPKD has few therapeutic options. Tolvaptan slows disease but has side effects limiting its tolerability. Bempedoic acid (BA), an ATP citrate-lyase (ACLY) inhibitor FDA-approved for hypercholesterolemia, catalyzes a key step in fatty acid/sterol synthesis important for cell proliferation. BA is activated by very long-chain acyl-CoA synthetase (FATP2) expressed primarily in kidney and liver. BA also activates AMPK. We hypothesized that BA could be a novel ADPKD therapy by inhibiting cyst growth, proliferation, injury, and metabolic dysregulation via ACLY inhibition and AMPK activation. Pkd1-null kidney cell lines derived from mouse proximal tubule (PT) and collecting duct (IMCD) were grown in 2D or 3D Matrigel cultures and treated ± BA, ± SB-204990 (another ACLY inhibitor) or with Acly shRNA before cyst analysis, immunoblotting or mitochondrial assays using MitoSox and MitoTracker staining. Pkd1 fl/fl ; Pax8-rtTA; Tet-O-Cre C57BL/6J mice were induced with doxycycline injection on postnatal days 10 and 11 (P10-P11) and then treated ± BA (30 mg/kg/d) ± tolvaptan (30-100 mg/kg/d) by gavage from P12-21. Disease severity was determined by % total-kidney-weight-to-bodyweight (%TKW/BW) and BUN levels at euthanasia (P22). Kidney and liver homogenates were immunoblotted for expression of key biomarkers. ACLY expression and activity were upregulated in Pkd1-null PT and IMCD-derived cells vs. controls. Relative to controls, both BA and SB-204990 inhibited cystic growth in Pkd1-null kidney cells, as did Acly knockdown. BA inhibited mitochondrial superoxide production and promoted mitochondrial elongation, suggesting improved mitochondrial function. In ADPKD mice, BA reduced %TKW/BW and BUN to a similar extent as tolvaptan vs. untreated controls. Addition of BA to tolvaptan caused a further reduction in %TKW/BW and BUN vs. tolvaptan alone. BA generally reduced ACLY and stimulated AMPK activity in kidneys and livers vs. controls. BA also inhibited mTOR and ERK signaling and reduced kidney injury markers. In liver, BA treatment, both alone and together with tolvaptan, increased mitochondrial biogenesis while inhibiting apoptosis. We conclude that BA and ACLY inhibition inhibited cyst growth in vitro, and BA decreased ADPKD severity in vivo. Combining BA with tolvaptan further improved various ADPKD disease parameters. Repurposing BA may be a promising new ADPKD therapy, having beneficial effects alone and along with tolvaptan.

4.
Am J Physiol Renal Physiol ; 322(1): F27-F41, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34806449

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in the polycystin 1 (PKD1) or polycystin 2 genes, presents with progressive development of kidney cysts and eventual end-stage kidney disease with limited treatment options. Previous work has shown that metformin reduces cyst growth in rapid ADPKD mouse models via inhibition of cystic fibrosis transmembrane conductance regulator-mediated fluid secretion, mammalian target of rapamycin, and cAMP pathways. The present study importantly tested the effectiveness of metformin as a therapy for ADPKD in a more clinically relevant Pkd1RC/RC mouse model, homozygous for the R3277C knockin point mutation in the Pkd1 gene. This mutation causes ADPKD in humans. Pkd1RC/RC male and female mice, which have a slow progression to end-stage kidney disease, received metformin (300 mg/kg/day in drinking water vs. water alone) from 3 to 9 or 12 mo of age. As previously reported, Pkd1RC/RC females had a more severe disease phenotype as compared with males. Metformin treatment reduced the ratio of total kidney weight-to-body weight relative to age-matched and sex-matched untreated controls at both 9 and 12 mo and reduced the cystic index in females at 9 mo. Metformin also increased glomerular filtration rate, lowered systolic blood pressure, improved anemia, and lowered blood urea nitrogen levels relative to controls in both sexes. Moreover, metformin reduced gene expression of key inflammatory markers and both gene and protein expression of kidney injury marker-1 and cyclin-dependent kinase-1 versus untreated controls. Altogether, these findings suggest several beneficial effects of metformin in this highly relevant slowly progressive ADPKD mouse model, which may help inform new ADPKD therapies in patients.NEW & NOTEWORTHY Metformin treatment improved ADPKD disease severity in a relevant, slowly progressive ADPKD mouse model that recapitulates a PKD-associated PKD1 mutation. Relative to controls, metformin reduced kidney weight/body weight, cystic index and BUN levels, while improving GFR, blood pressure and anemia. Metformin also reduced key inflammatory and injury markers, along with cell proliferation markers. These findings suggest several beneficial effects of metformin in this ADPKD mouse model, which may help inform new ADPKD therapies in patients.


Assuntos
Falência Renal Crônica/prevenção & controle , Rim/efeitos dos fármacos , Metformina/farmacologia , Rim Policístico Autossômico Dominante/tratamento farmacológico , Fármacos Renais/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Feminino , Predisposição Genética para Doença , Taxa de Filtração Glomerular/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Falência Renal Crônica/metabolismo , Falência Renal Crônica/patologia , Falência Renal Crônica/fisiopatologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Rim Policístico Autossômico Dominante/fisiopatologia , Canais de Cátion TRPP/genética , Fatores de Tempo
5.
J Control Release ; 329: 1198-1209, 2021 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-33127449

RESUMO

Nanoparticle drug delivery has many advantages over small molecule therapeutics, including reducing off-target side effects and increasing drug potency. However, many nanoparticles are administered parenterally, which is challenging for chronic diseases such as polycystic kidney disease (PKD), the most common hereditary disease worldwide in which patients need continuous treatment over decades. To address this clinical need, we present the development of nanoparticles synthesized from chitosan, a widely available polymer chosen for its ability to improve oral bioavailability. Specifically, we optimized the synthesis parameters of chitosan nanoparticles and demonstrate mucoadhesion and permeation across an intestinal barrier model in vitro. Furthermore, when administered orally to mice, ex vivo imaging of rhodamine-loaded chitosan nanoparticles showed significantly higher accumulation in the intestines compared to the free model drug, as well as 1.3 times higher serum area under the curve (AUC), demonstrating controlled release and improved serum delivery over 24 h. To test its utility for chronic diseases such as PKD, we loaded the candidate PKD drug, metformin, into chitosan nanoparticles, and upon oral administration to a PKD murine model (Pkd1fl/fl;Pax8-rtTA;Tet-O cre), a lower cyst burden was observed compared to free metformin, and was well tolerated upon repeated dosages. Blood urea nitrogen (BUN) and creatinine levels were similar to untreated mice, demonstrating kidney and biocompatibility health. Our study builds upon previous chitosan-based drug delivery approaches, and demonstrates a novel, oral nanoformulation for PKD.


Assuntos
Quitosana , Metformina , Nanopartículas , Doenças Renais Policísticas , Administração Oral , Animais , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Humanos , Camundongos
6.
Neuron ; 52(4): 691-703, 2006 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-17114052

RESUMO

Phospholipase Cbeta (PLCbeta) isozymes represent a family of molecules that link G protein-coupled receptors (GPCRs) to an intracellular signaling network. Here, we investigated the function of PLCbeta isozymes in sensory neurons by using mutant mice deficient for specific PLCbeta family members. Expression analysis indicated that PLCbeta3, one of the four isoforms, is predominantly expressed in a subpopulation of C-fiber nociceptors. A subset of these neurons expressed the histamine H1 receptor. Ca(2+) imaging studies revealed that PLCbeta3 specifically mediates histamine-induced calcium responses through the histamine H1 receptor in cultured sensory neurons. In line with this, we found that PLCbeta3(-/-) mice showed significant defects in scratching behavior induced by histamine; histamine-trifluoromethyl-toluidine (HTMT), a selective H1 agonist; and compound 48/80, a mast cell activator. These results demonstrate that PLCbeta3 is required to mediate "itch" sensation in response to histamine acting on the histamine H1 receptor in C-fiber nociceptive neurons.


Assuntos
Gânglios Espinais/metabolismo , Isoenzimas/metabolismo , Fibras Nervosas Amielínicas/metabolismo , Neurônios Aferentes/metabolismo , Nociceptores/metabolismo , Receptores Histamínicos H1/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Gânglios Espinais/efeitos dos fármacos , Histamina/metabolismo , Histamina/farmacologia , Agonistas dos Receptores Histamínicos/farmacologia , Isoenzimas/genética , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Nervosas Amielínicas/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Fosfolipase C beta , Prurido/induzido quimicamente , Prurido/metabolismo , Prurido/fisiopatologia , Ratos , Ratos Wistar , Receptores Histamínicos H1/efeitos dos fármacos , Reflexo/efeitos dos fármacos , Reflexo/fisiologia , Neuropatia Ciática/metabolismo , Neuropatia Ciática/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/genética
7.
Proc Natl Acad Sci U S A ; 100(11): 6604-9, 2003 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-12738888

RESUMO

RGS (regulator of G protein signaling) proteins containing the G protein gamma-like (GGL) domain (RGS6, RGS7, RGS9, and RGS11) interact with the fifth member of the G protein beta-subunit family, Gbeta5. This interaction is necessary for the stability of both the RGS protein and for Gbeta5. Consistent with this notion, we have found that elevation of RGS9-1 mRNA levels by transgene expression does not increase RGS9-1 protein level in the retina, suggesting that Gbeta5 levels may be limiting. To examine further the interactions of Gbeta5 and the GGL domain-containing RGS proteins, we inactivated the Gbeta5 gene. We found that the levels of GGL domain-containing RGS proteins in retinas and in striatum are eliminated or reduced drastically, whereas the levels of Ggamma2 and RGS4 proteins remain normal in the absence of Gbeta5. The homozygous Gbeta5 knockout (Gbeta5-/-) mice derived from heterozygous knockout mating are runty and exhibit a high preweaning mortality rate. We concluded that complex formation between GGL domain-containing RGS proteins and the Gbeta5 protein is necessary to maintain their mutual stability in vivo. Furthermore, in the absence of Gbeta5 and all four RGS proteins that form protein complexes with Gbeta5, the animals that survive into adulthood are viable and have no gross defects in brain or retinal morphology.


Assuntos
Proteínas RGS/genética , Animais , Sequência de Bases , Primers do DNA , Heterozigoto , Homozigoto , Camundongos , Camundongos Transgênicos , Proteínas RGS/fisiologia , RNA Mensageiro/genética
8.
Proc Natl Acad Sci U S A ; 99(14): 9352-7, 2002 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-12077299

RESUMO

The G(12) subfamily of heterotrimeric G-proteins consists of two members, G(12) and G(13). Gene-targeting studies have revealed a role for G(13) in blood vessel development. Mice lacking the alpha subunit of G(13) die around embryonic day 10 as the result of an angiogenic defect. On the other hand, the physiological role of G(12) is still unclear. To address this issue, we generated G alpha(12)-deficient mice. In contrast to the G alpha(13)-deficient mice, G alpha(12)-deficient mice are viable, fertile, and do not show apparent abnormalities. However, G alpha(12) does not seem to be entirely redundant, because in the offspring generated from G alpha(12)+/- G alpha(13)+/- intercrosses, at least one intact G alpha(12) allele is required for the survival of animals with only one G alpha(13) allele. In addition, G alpha(12) and G alpha(13) showed a difference in mediating cell migratory response to lysophosphatidic acid in embryonic fibroblast cells. Furthermore, mice lacking both G alpha(12) and G alpha(q) die in utero at about embryonic day 13. These data indicate that the G alpha(12)-mediated signaling pathway functionally interacts not only with the G alpha(13)- but also with the G alpha(q/11)-mediated signaling systems.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Transdução de Sinais/fisiologia , Animais , Movimento Celular/fisiologia , Células Cultivadas , Cruzamentos Genéticos , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário e Fetal/genética , Desenvolvimento Embrionário e Fetal/fisiologia , Feminino , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Proteínas Heterotriméricas de Ligação ao GTP/deficiência , Proteínas Heterotriméricas de Ligação ao GTP/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
9.
J Immunol ; 168(7): 3543-9, 2002 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-11907117

RESUMO

The complexity and magnitude of interactions leading to the selective infiltration of eosinophils in response to inhaled allergens are formidable obstacles to a larger understanding of the pulmonary pathology associated with allergic asthma. This study uses knockout mice to demonstrate a novel function for the heterotrimeric G protein, G(q), in the regulation of pulmonary eosinophil recruitment. In the absence of G(q) signaling, eosinophils failed to accumulate in the lungs following allergen challenge. These studies demonstrate that the inhibition of eosinophil accumulation in the airways is attributed to the failure of hemopoietically derived cells to elaborate GM-CSF in the airways. The data suggest that activation of a G(q)-coupled receptor(s) on resident leukocytes in the lung elicits expression of GM-CSF, which, in turn, is required for allergen-induced pulmonary eosinophilia, identifying a novel pathway of eosinophil-associated effector functions leading to pulmonary pathology in diseases such as asthma.


Assuntos
Alérgenos/administração & dosagem , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Eosinofilia Pulmonar/imunologia , Aerossóis , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Fatores Quimiotáticos de Eosinófilos/farmacologia , Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Citocinas/biossíntese , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Proteínas Heterotriméricas de Ligação ao GTP/deficiência , Proteínas Heterotriméricas de Ligação ao GTP/genética , Injeções Intraperitoneais , Intubação Intratraqueal , Contagem de Leucócitos , Ativação Linfocitária/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Eosinofilia Pulmonar/etiologia , Eosinofilia Pulmonar/genética , Eosinofilia Pulmonar/patologia , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th2/imunologia , Células Th2/metabolismo
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