Nanotherapeutic kidney cell-specific targeting to ameliorate acute kidney injury

Abstract

Acute kidney injury (AKI) increases the risk of in-hospital death, adds to expense of care, and risk of early chronic kidney disease. AKI often follows an acute event such that timely treatment could ameliorate AKI and potentially reduce the risk of additional disease. Despite therapeutic success of dexamethasone in animal models, clinical trials have not demonstrated broad success. To improve the safety and efficacy of dexamethasone for AKI, we developed and characterized a novel, kidney-specific nanoparticle enabling specific within-kidney targeting to proximal tubular epithelial cells provided by the megalin ligand cilastatin. Cilastatin and dexamethasone were complexed to H-Dot nanoparticles, which were constructed from generally recognized as safe components. Cilastatin/Dexamethasone/H-Dot nanotherapeutics were found to be stable at plasma pH and demonstrated salutary release kinetics at urine pH. In vivo, they were specifically biodistributed to the kidney and bladder, with 75% recovery in the urine and with reduced systemic toxicity compared to native dexamethasone. Cilastatin complexation conferred proximal tubular epithelial cell specificity within the kidney in vivo and enabled dexamethasone delivery to the proximal tubular epithelial cell nucleus in vitro. The Cilastatin/Dexamethasone/H-Dot nanotherapeutic improved kidney function and reduced kidney cellular injury when administered to male C57BL/6 mice in two translational models of AKI (rhabdomyolysis and bilateral ischemia reperfusion). Thus, our design-based targeting and therapeutic loading of a kidney-specific nanoparticle resulted in preservation of the efficacy of dexamethasone, combined with reduced off-target disposition and toxic effects. Hence, our study illustrates a potential strategy to target AKI and other diseases of the kidney. Acute kidney injury (AKI) increases the risk of in-hospital death, adds to expense of care, and risk of early chronic kidney disease. AKI often follows an acute event such that timely treatment could ameliorate AKI and potentially reduce the risk of additional disease. Despite therapeutic success of dexamethasone in animal models, clinical trials have not demonstrated broad success. To improve the safety and efficacy of dexamethasone for AKI, we developed and characterized a novel, kidney-specific nanoparticle enabling specific within-kidney targeting to proximal tubular epithelial cells provided by the megalin ligand cilastatin. Cilastatin and dexamethasone were complexed to H-Dot nanoparticles, which were constructed from generally recognized as safe components. Cilastatin/Dexamethasone/H-Dot nanotherapeutics were found to be stable at plasma pH and demonstrated salutary release kinetics at urine pH. In vivo, they were specifically biodistributed to the kidney and bladder, with 75% recovery in the urine and with reduced systemic toxicity compared to native dexamethasone. Cilastatin complexation conferred proximal tubular epithelial cell specificity within the kidney in vivo and enabled dexamethasone delivery to the proximal tubular epithelial cell nucleus in vitro. The Cilastatin/Dexamethasone/H-Dot nanotherapeutic improved kidney function and reduced kidney cellular injury when administered to male C57BL/6 mice in two translational models of AKI (rhabdomyolysis and bilateral ischemia reperfusion). Thus, our design-based targeting and therapeutic loading of a kidney-specific nanoparticle resulted in preservation of the efficacy of dexamethasone, combined with reduced off-target disposition and toxic effects. Hence, our study illustrates a potential strategy to target AKI and other diseases of the kidney. Translational StatementAcute kidney injury complicates severe illness and can lead to death or chronic kidney disease. One challenge for treatment is targeting the kidney. Here we specifically target the injured cell population with a kidney-specific nanoparticle that contains a targeting moiety and dexamethasone as a treatment moiety. The nanoparticle has a generally recognized as safe backbone and is complexed to US Food and Drug Administration–approved targeting and therapeutic moieties, reducing barriers to translation. Kidney-specific delivery and reduction of toxicity increase utility in the complex environment of care around acute kidney injury. These data support the rodent and large animal translational study and scale-up pharmaceutical-grade production to support phase I trials. Acute kidney injury complicates severe illness and can lead to death or chronic kidney disease. One challenge for treatment is targeting the kidney. Here we specifically target the injured cell population with a kidney-specific nanoparticle that contains a targeting moiety and dexamethasone as a treatment moiety. The nanoparticle has a generally recognized as safe backbone and is complexed to US Food and Drug Administration–approved targeting and therapeutic moieties, reducing barriers to translation. Kidney-specific delivery and reduction of toxicity increase utility in the complex environment of care around acute kidney injury. These data support the rodent and large animal translational study and scale-up pharmaceutical-grade production to support phase I trials. Acute kidney injury (AKI) is a lethal syndrome caused by traumatic injury, surgery, coronavirus disease 2019, critical illness, and therapeutic drugs. In these settings, the time of kidney injury is often known, and timely treatment could ameliorate AKI and prevent additional disease. AKI increases hospital length of stay, risk of in-hospital death, and expense of care.1Dasta J.F. Kane-Gill S. Review of the literature on the costs associated with acute kidney injury.J Pharm Pract. 2019; 32: 292-302Crossref PubMed Scopus (35) Google Scholar,2Silver S.A. Long J. Zheng Y. et al.Cost of acute kidney injury in hospitalized patients.J Hosp Med. 2017; 12: 70-76Crossref PubMed Scopus (154) Google Scholar The risk of transition to chronic kidney disease in patients with AKI older than 66 years is 50%,3United States Renal Data System 2020 USRDS Annual Data Report: epidemiology of kidney disease in the United States.National Institute of Diabetes and Digestive and Kidney Diseases. National Institutes of Health, 2000Google Scholar and there is a considerable increase in the risk of distant organ disease, as well.4Hebert J.F. Funahashi Y. Hutchens M.P. Harm! foul! How acute kidney injury SHReDDs patient futures.Curr Opin Nephrol Hypertens. 2023; 32: 165-171Crossref PubMed Scopus (1) Google Scholar Despite success in animal models, clinical trials have failed to demonstrate a safe therapeutic agent. A major reason for such failures is pleiotropic extrarenal effects of systemically delivered agents. For example, the anti-inflammatory glucocorticoid dexamethasone (Dex) is protective in mice subjected to renal ischemia-reperfusion (a model of early graft dysfunction after renal transplantation) and experimental rhabdomyolysis (a model of traumatic crush syndrome).5Kumar S. Allen D.A. Kieswich J.E. et al.Dexamethasone ameliorates renal ischemia-reperfusion injury.J Am Soc Nephrol. 2009; 20: 2412-2425Crossref PubMed Scopus (101) Google Scholar, 6Zager R.A. Johnson A.C.M. Acute kidney injury induces dramatic p21 upregulation via a novel, glucocorticoid-activated, pathway.Am J Physiol Renal Physiol. 2019; 316: F674-F681Crossref PubMed Scopus (14) Google Scholar, 7Moonen L. Geryl H. D’Haese P.C. et al.Short-term dexamethasone treatment transiently, but not permanently, attenuates fibrosis after acute-to-chronic kidney injury.BMC Nephrol. 2018; 19: 343Crossref PubMed Scopus (11) Google Scholar Dex has been administered to patients undergoing cardiac surgery, a setting with a high risk of AKI and in which severe side effects of Dex (immunosuppression, hyperglycemia, and delirium) can be managed in the intensive care unit. In the largest and best-conducted clinical study, Dex reduced the risk of severe AKI, especially in high-risk patients.8Jacob K.A. Leaf D.E. Dieleman J.M. et al.Intraoperative high-dose dexamethasone and severe AKI after cardiac surgery.J Am Soc Nephrol. 2015; 26: 2947-2951Crossref PubMed Scopus (74) Google Scholar However, severe side effects of Dex, including delirium, hyperglycemia, hypertension, muscle catabolism, and immunosuppression,9Dexamethasone. Package insert. Acrotech Biopharma, 2019Google Scholar prevent its wide use for AKI. Although Dex has pleiotropic anti-inflammatory and immune suppressant properties, Dex protects cultured proximal tubular epithelial cell (PTEC) survival, morphology, and mitochondrial function in injury models, suggesting a specific effect on PTEC.6Zager R.A. Johnson A.C.M. Acute kidney injury induces dramatic p21 upregulation via a novel, glucocorticoid-activated, pathway.Am J Physiol Renal Physiol. 2019; 316: F674-F681Crossref PubMed Scopus (14) Google Scholar,10Schirris T.J.J. Jansen J. Mihajlovic M. et al.Mild intracellular acidification by dexamethasone attenuates mitochondrial dysfunction in a human inflammatory proximal tubule epithelial cell model.Sci Rep. 2017; 7: 10623Crossref PubMed Scopus (4) Google Scholar,11Zager R.A. Johnson A.C. Becker K. Acute unilateral ischemic renal injury induces progressive renal inflammation, lipid accumulation, histone modification, and “end-stage” kidney disease.Am J Physiol Renal Physiol. 2011; 301: F1334-F1345Crossref PubMed Scopus (134) Google Scholar AKI-related tubular cell cycle arrest mediates recovery, repair, and development of AKI–chronic kidney disease transition.12Lin X. Jin H. Chai Y. et al.Cellular senescence and acute kidney injury.Pediatr Nephrol. 2022; 37: 3009-3018Crossref Scopus (19) Google Scholar,13De Chiara L. Conte C. Antonelli G. et al.Tubular cell cycle response upon AKI: revising old and new paradigms to identify novel targets for CKD prevention.Int J Mol Sci. 2021; 22: 11093Crossref Scopus (18) Google Scholar Because it is proven in humans, control of the pharmacokinetics and pharmacodynamics of Dex to prevent toxicity its use as a development of on the of a generally as safe with which and H. J. K. et for and PubMed Scopus Google Scholar Because of and these have specificity for the renal in to salutary targeting and of with or other off-target H. J. K. et for and PubMed Scopus Google H. M. J. et for 2019; PubMed Scopus Google Scholar we that H-Dot kidney could be to target within the kidney to tubular epithelial cells and Dex to these AKI and To target we a megalin Y. S. et with Am Soc Nephrol. 2017; PubMed Scopus Google Scholar has a toxicity and is by the US Food and Drug in with the Food and Drug Scholar In that at high have in injury K. K. et ameliorates AKI in Am Soc Nephrol. 2021; 32: PubMed Scopus Google Scholar, M. et and in of tubular in 2022; Scopus Google Scholar, S. et effects of 2015; PubMed Scopus Google Scholar, et effect of renal injury in and in vivo its Scopus Google Scholar, M. M. et of on in patients undergoing J Mol Sci. 2021; 22: Scopus (11) Google Scholar To our we developed and characterized its pharmacokinetics and of human tubular epithelial cells to the of and efficacy in vivo in translational AKI provided in is and the backbone to the is to The is and a H-Dot Dex is complexed to the H-Dot and of in the and complexation of nanotherapeutic provided in The of as the Data The ligand with the at and the model with the The release of and were human for of were provided by the The renal and by and as of cells from and human 2019; PubMed Scopus (4) Google Scholar cells were by cell as for A of for is provided in A of for cell is provided in were and by for kidney injury early or For in were cultured on A of and for is provided in administered to on were in et in utility and 2021; 22: PubMed Scopus Google Scholar and were and To H-Dot for and at and were In vivo were on male mice as by and at the of and at a mice and the plasma of the H-Dot the and were by The Dex and urine the mice were with native Dex nanoparticle or as a and subjected to the J.F. 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Johnson A.C.M. Acute kidney injury induces dramatic p21 upregulation via a novel, glucocorticoid-activated, pathway.Am J Physiol Renal Physiol. 2019; 316: F674-F681Crossref PubMed Scopus (14) Google Scholar we in and in The in Although caused cell and glucocorticoid increase in and it not p21 in or in vivo these that has a glucocorticoid effect in and effect in AKI. Drug treatment of kidney disease the function which can reducing efficacy or the function of the kidney on the of at a effect within the the kidney has and cell Kidney disease can be organ and cell specificity to the kidney and to a cell within the kidney To these we design-based development of a proximal tubule for AKI. on a with renal we and characterized a nanotherapeutic that renal to target the injured population of cells in the kidney. have been considerable to delivery to the kidney and and nanoparticles, and J. et as for renal PubMed Google Scholar, M. G. et of with a human renal Google Scholar, L. C. et of by cells in vivo and in PubMed Scopus Google Scholar, K. H. S. et targeting a Google Scholar, Y. Y. Y. et as a novel renal targeting PubMed Scopus Google Scholar, J. et specific to the cell 26: PubMed Scopus Google Scholar, X. et as a novel renal targeting Drug PubMed Scopus Google Scholar, Zheng J. et targeting of to proximal renal tubular epithelial 2009; PubMed Scopus Google Scholar, S. G. et targeting to the in the targeting of to proximal tubular Drug PubMed Scopus Google Scholar, J.E. et kidney by of 2011; PubMed Scopus Google Scholar, S. et of a to renal proximal tubular J 7: Google Scholar, X. et via a for kidney PubMed Scopus Google Scholar, M. et delivery to proximal tubule PubMed Scopus Google Scholar, et for J Physiol Renal Physiol. 2017; PubMed Scopus Google Scholar, Y. J. et delivery of to renal attenuates renal fibrosis and systemic PubMed Scopus Google Scholar have been renal and extrarenal and and acute and chronic safety X. delivery Pharm PubMed Google M. et in delivery for the PubMed Scopus Google Scholar have targeting and improved pharmacokinetics than or X. delivery Pharm PubMed Google these to translation. we a generally as safe backbone and an moiety to the of To AKI, we Dex has efficacy for AKI but toxicity use at the S. Allen D.A. Kieswich J.E. et al.Dexamethasone ameliorates renal ischemia-reperfusion injury.J Am Soc Nephrol. 2009; 20: 2412-2425Crossref PubMed Scopus (101) Google L. Geryl H. D’Haese P.C. et al.Short-term dexamethasone treatment transiently, but not permanently, attenuates fibrosis after acute-to-chronic kidney injury.BMC Nephrol. 2018; 19: 343Crossref PubMed Scopus (11) Google K.A. Leaf D.E. Dieleman J.M. et al.Intraoperative high-dose dexamethasone and severe AKI after cardiac surgery.J Am Soc Nephrol. 2015; 26: 2947-2951Crossref PubMed Scopus (74) Google K. S. et lethal can be by a of high-dose dexamethasone a Acute PubMed Scopus Google Scholar, J. Y. et of dexamethasone protects mice renal injury by J Google Scholar, J. L. et dexamethasone attenuates renal injury by Sci. PubMed Scopus Google Scholar, J. J. Y. et in renal ischemia injury and could be by of the pathway.Am J Google Scholar, S. C. of in renal epithelial PubMed Scopus (18) Google toxicity is a that targets the kidney be treatment of to target Dex to specific has been with and delivery for and S. J. et for delivery in inflammatory Med. 2015; Scopus Google Scholar, Y. J. et cell for delivery of dexamethasone to 2021; Scopus Google Scholar, J.M. J. et dexamethasone as a novel in Scopus Google Scholar, et al.Dexamethasone for PubMed Scopus Google Scholar, L. Y. et for inflammatory of and PubMed Scopus Google Scholar proximal tubule cells by to the H-Dot with specificity for renal tubular epithelial cells and renal its development as a renal in the M. et renal ischemia 2022; Scopus Google Scholar as a renal megalin ligand as Y. S. et with Am Soc Nephrol. 2017; PubMed Scopus Google have specificity and other as of in K. K. et ameliorates AKI in Am Soc Nephrol. 2021; 32: PubMed Scopus Google S. et effects of 2015; PubMed Scopus Google Scholar, et effect of renal injury in and in vivo its Scopus Google Scholar, M. M. et of on in patients undergoing J Mol Sci. 2021; 22: Scopus (11) Google Scholar demonstrate that in the or with H-Dot kidney targeting of the kidney delivery of Dex and reduced the acute off-target Thus, targeting and targeting moiety to salutary pharmacodynamics and reduced demonstrate that ameliorates AKI in translational The of clinical efficacy pleiotropic anti-inflammatory effects of Dex injury to cultured to Dex has effects on cell and cell cycle in epithelial its use in K. on and in Scopus Google Scholar and arrest of cell cycle critical to AKI, in the early recovery as cell cycle arrest fibrosis than L. et cell cycle arrest in mediates kidney fibrosis after Med. PubMed Scopus Google Scholar A in AKI is characterized by cell cycle which is by the and of and the of or Dex induces epithelial cell and S. et al.Dexamethasone mitochondrial and in the kidney Physiol 2021; Scopus Google Scholar and mediates cell and Y. L. et dexamethasone and in the Rep. 2021; Scopus (1) Google Scholar and and is protective in AKI models, reducing renal inflammation, and et of cell death in and in vivo on Am Soc Nephrol. PubMed Scopus Google Scholar, J. et induces epithelial cell cycle arrest and ameliorates acute kidney J Physiol Renal Physiol. PubMed Scopus Google Scholar, J. of in kidney tubule cells the of acute renal PubMed Google Scholar, L. G. et a of and death in renal tubular epithelial J Physiol Renal Physiol. PubMed Google Scholar, J. of renal cells from toxicity by cell cycle J Physiol Renal Physiol. PubMed Google Scholar, J. L. J.M. et effect of the of on the of ischemic acute renal PubMed Scopus Google Scholar et a dramatic upregulation of p21 in AKI, which by systemic native Dex and not by the of R.A. Johnson A.C. Becker K. Acute unilateral ischemic renal injury induces progressive renal inflammation, lipid accumulation, histone modification, and “end-stage” kidney disease.Am J Physiol Renal Physiol. 2011; 301: F1334-F1345Crossref PubMed Scopus (134) Google p21 has been to have a protective effect in J. of in kidney tubule cells the of acute renal PubMed Google Scholar, L. G. et a of and death in renal tubular epithelial J Physiol Renal Physiol. PubMed Google Scholar, J. of renal cells from toxicity by cell cycle J Physiol Renal Physiol. PubMed Google Scholar, J. L. J.M. et effect of the of on the of ischemic acute renal PubMed Scopus Google Scholar we Although we found that glucocorticoid effect in cultured we not effect on p21 in or in vivo, suggesting that other the from kidney injury. 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J. et PubMed Scopus Google Scholar and of clinical and in acute kidney Scopus (1) Google Scholar it be to in data from human kidney cells demonstrate that the targeting and the of in human these data provided to support additional study including in large animal models the clinical In we a design-based development of a nanotherapeutic for AKI. of and in the injured cell population of the kidney resulted in the preservation of the efficacy of Dex, combined with reduced off-target and toxic effects. from not to and has other other or from the to the and demonstrated in in the at data in support of the of in the at by the US of to the of to and the of and and to and is the of that with and the use of at the The not the of the US of or the US

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