Daprodustat prevents cyclosporine-A–mediated anemia and peritubular capillary loss

Abstract

Chronic Cyclosporine-A treatment is associated with serious side effects, including kidney toxicity and anemia. Although pathophysiology of Cyclosporine-A-induced kidney injury remains incompletely understood, hypoxia is likely involved. Here, we investigated the effect of the hypoxia inducible factor activator daprodustat on Cyclosporine-A -induced kidney toxicity. As Cyclosporine-A profoundly alters protein phosphorylation by inhibiting the phosphatase calcineurin, special attention was directed towards the kidney phospho-proteome. Mice received Cyclosporine-A with or without daprodustat for up to eight weeks. In kidney homogenates, 1360 selected proteins were analyzed at expression and phosphorylation levels. Of these, Cyclosporine-A changed the expression of 79 and the phosphorylation of 86 proteins. However, when Cyclosporine-A treatment was combined with daprodustat, the expression of 95 proteins and phosphorylation of only six proteins was altered suggesting that daprodustat prevented most protein phosphorylation brought about by Cyclosporine-A. Although daprodustat showed only marginal effect on its own, angiogenesis-related pathways were among the most profoundly impacted by daprodustat when given on top of Cyclosporine-A. Additionally, Cyclosporine-A lowered the blood hemoglobin concentration and caused kidney capillary rarefaction, which daprodustat prevented. Thus, combined daprodustat/Cyclosporine-A treatment prevented deleterious Cyclosporine-A effects on microcirculation and hemoglobin, and the protective action of daprodustat involves suppression of broad protein phosphorylation changes caused by Cyclosporine-A. Chronic Cyclosporine-A treatment is associated with serious side effects, including kidney toxicity and anemia. Although pathophysiology of Cyclosporine-A-induced kidney injury remains incompletely understood, hypoxia is likely involved. Here, we investigated the effect of the hypoxia inducible factor activator daprodustat on Cyclosporine-A -induced kidney toxicity. As Cyclosporine-A profoundly alters protein phosphorylation by inhibiting the phosphatase calcineurin, special attention was directed towards the kidney phospho-proteome. Mice received Cyclosporine-A with or without daprodustat for up to eight weeks. In kidney homogenates, 1360 selected proteins were analyzed at expression and phosphorylation levels. Of these, Cyclosporine-A changed the expression of 79 and the phosphorylation of 86 proteins. However, when Cyclosporine-A treatment was combined with daprodustat, the expression of 95 proteins and phosphorylation of only six proteins was altered suggesting that daprodustat prevented most protein phosphorylation brought about by Cyclosporine-A. Although daprodustat showed only marginal effect on its own, angiogenesis-related pathways were among the most profoundly impacted by daprodustat when given on top of Cyclosporine-A. Additionally, Cyclosporine-A lowered the blood hemoglobin concentration and caused kidney capillary rarefaction, which daprodustat prevented. Thus, combined daprodustat/Cyclosporine-A treatment prevented deleterious Cyclosporine-A effects on microcirculation and hemoglobin, and the protective action of daprodustat involves suppression of broad protein phosphorylation changes caused by Cyclosporine-A. Translational StatementCyclosporine-A (CsA) treatment per se may cause anemia, which is amenable to intervention with the hypoxia-inducible factor activator daprodustat. The latter is currently under US Food and Drug Administration and European Medicines Agency review for treatment of renal anemia. Long-term daprodustat therapy offsets most of the CsA-induced protein phosphorylation changes that are key for immunosuppression, but probably also for adverse effects, like kidney toxicity. Early CsA-induced kidney toxicity presents with diminished kidney capillaries, which are restored by daprodustat. CsA may inhibit angiogenesis (e.g., through dephosphorylation of protein kinase cyclic adenosine monophosphate–dependent type II regulatory subunit α [PRKAR2A]). In kidney biopsies, reduced phospho-PRKAR2A may represent an early sign of CsA toxicity. Cyclosporine-A (CsA) treatment per se may cause anemia, which is amenable to intervention with the hypoxia-inducible factor activator daprodustat. The latter is currently under US Food and Drug Administration and European Medicines Agency review for treatment of renal anemia. Long-term daprodustat therapy offsets most of the CsA-induced protein phosphorylation changes that are key for immunosuppression, but probably also for adverse effects, like kidney toxicity. Early CsA-induced kidney toxicity presents with diminished kidney capillaries, which are restored by daprodustat. CsA may inhibit angiogenesis (e.g., through dephosphorylation of protein kinase cyclic adenosine monophosphate–dependent type II regulatory subunit α [PRKAR2A]). In kidney biopsies, reduced phospho-PRKAR2A may represent an early sign of CsA toxicity. The immunosuppressant cyclosporine-A (CsA) acts through inhibition of the phosphatase calcineurin. CsA is standard of care in organ transplantation and in a variety of autoimmune diseases.1Azzi J.R. Sayegh M.H. Mallat S.G. Calcineurin inhibitors: 40 years later, can't live without.J Immunol. 2013; 191: 5785-5791Crossref PubMed Scopus (230) Google Scholar, 2Issa N. Kukla A. Ibrahim H.N. Calcineurin inhibitor nephrotoxicity: a review and perspective of the evidence.Am J Nephrol. 2013; 37: 602-612Crossref PubMed Scopus (172) Google Scholar, 3Bauer A.C. Franco R.F. Manfro R.C. Immunosuppression in kidney transplantation: state of the art and current protocols.Curr Pharm Des. 2020; 26: 3440-3450Crossref PubMed Scopus (16) Google Scholar Although CsA’s benefits are substantial, CsA may exert short- and long-term adverse effects on the kidney. Long-term CsA treatment causes renal vascular constriction, occlusive afferent arteriolopathy, nephron ischemia, and striped tubular-interstitial fibrosis.4Myers B.D. Newton L. Cyclosporine-induced chronic nephropathy: an obliterative microvascular renal injury.J Am Soc Nephrol. 1991; 2: S45-52Crossref PubMed Google Scholar,5Young B.A. Burdmann E.A. Johnson R.J. et al.Cyclosporine A induced arteriolopathy in a rat model of chronic cyclosporine nephropathy.Kidney Int. 1995; 48: 431-438Abstract Full Text PDF PubMed Scopus (97) Google Scholar Therefore, loss of kidney function may outweigh the benefits of CsA.6Naesens M. Kuypers D.R. Sarwal M. Calcineurin inhibitor nephrotoxicity.Clin J Am Soc Nephrol. 2009; 4: 481-508Crossref PubMed Scopus (1068) Google Scholar Although pathophysiology of CsA-induced kidney injury remains incompletely understood, hypoxia of nephron segments likely is involved.7Heyman S.N. Abassi Z. Rosenberger C. et al.Cyclosporine A induces endothelin-converting enzyme-1: studies in vivo and in vitro.Acta Physiol (Oxf). 2018; 223e13033Crossref PubMed Scopus (8) Google Scholar Mice receiving CsA repetitively over days experience episodic kidney hypoxia.8Fahling M. Mathia S. Scheidl J. et al.Cyclosporin a induces renal episodic hypoxia.Acta Physiol (Oxf). 2017; 219: 625-639Crossref PubMed Scopus (23) Google Scholar Interestingly, some groups report anemia under CsA treatment,9Bardet V. Junior A.P. Coste J. et al.Impaired erythropoietin production in liver transplant recipients: the role of calcineurin inhibitors.Liver Transpl. 2006; 12: 1649-1654Crossref PubMed Scopus (16) Google Scholar,10Lei D.M. Piao S.G. Jin Y.S. et al.Expression of erythropoietin and its receptor in kidneys from normal and cyclosporine-treated rats.Transplant Proc. 2014; 46: 521-528Crossref PubMed Scopus (6) Google Scholar as well as relatively low erythropoietin (EPO) blood levels.11Sinkeler S.J. Zelle D.M. Homan van der Heide J.J. et al.Endogenous plasma erythropoietin, cardiovascular mortality and all-cause mortality in renal transplant recipients.Am J Transplant. 2012; 12: 485-491Crossref PubMed Scopus (19) Google Scholar Although the mechanisms of CsA-induced anemia are unclear, it can be assumed that anemia contributes to CsA nephrotoxicity, by limiting oxygen delivery. Hence, anemia and kidney insufficiency may create a vicious cycle. Small, highly selective inhibitors of prolyl-hydroxylase domain, like daprodustat (GSK-1278863), emerge as erythropoiesis-stimulating agents, based on their ability to upregulate hypoxia-inducible factor (HIF), and hence, EPO. Herein, we test daprodustat’s potential to alleviate both CsA-induced anemia and kidney toxicity. Moreover, in mice treated for up to 8 weeks with CsA, daprodustat, or daprodustat/CsA, phospho-proteomics and subsequent factor analysis assess the fingerprint of early CsA-induced kidney toxicity, as well as potential treatment options. Remarkably, through mechanisms yet unclear, daprodustat virtually offsets CsA-induced changes in protein phosphorylation. Daprodustat prevents CsA-induced kidney capillary rarefaction and anemia. Animal experiments were approved by local authorities (Landesamt für Gesundheit und Soziales: G0227-17) and performed according to the guidelines of the American Physiological Society. Mice (C57BL/6N, male, 10 weeks old) were obtained from Janvier and had free access to chow and water. Seven days before treatment, mice received a low-salt diet (≤0.03% sodium) and distilled water until termination. Daily doses of CsA (80 mg/kg), daprodustat (10 mg/kg), or a combination of both were given by oral gavage for 4 or 8 weeks. Control mice were treated with the vehicle methylcellulose. Blood samples were collected from the submandibular vein.12Golde W.T. Gollobin P. Rodriguez L.L. A rapid, simple, and humane method for submandibular bleeding of mice using a lancet.Lab Anim (NY). 2005; 34: 39-43Crossref PubMed Scopus (333) Google Scholar Kidneys were either fixed in 4% paraformaldehyde (24 hours, room temperature), dehydrated, and embedded in paraffin for histologic analysis or snap frozen in liquid nitrogen for molecular analyses. Plasma CsA and plasma creatinine were measured by Labor Berlin–Charité Vivantes GmbH. Hemoglobin (Hb) concentration was measured using an ABL800 Flex blood gas analyzer (Radiometer GmbH). Liquid nitrogen-frozen kidney samples were ground to fine powder. RNA was extracted using RNA-Bee (Biozol Diagnostica Vertrieb GmbH), according to the manufacturer’s instructions. RNA quantity and quality were estimated using a NanoDrop 2000 (Thermo Fisher Scientific Inc.). cDNA synthesis was performed using random primers and Superscript II reverse transcriptase (Thermo Fisher Scientific Inc.). Quantitative polymerase chain reaction was performed using the CFX Connect cycler (Bio-Rad Laboratories, Inc.) and SYBR Green Master Mix (Thermo Fisher Scientific Inc.; number 4367659). Primers were designed using the primer-BLAST program (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index) and obtained from TIB Molbiol Syntheselabor GmbH. All primers used are listed in Supplementary Table S1. The polymerase chain reaction protocol consisted of a 10-minute holding step at 95 °C and 40 cycles of 20 seconds at 95 °C and 40 seconds at 60 °C. Samples were measured in triplicate. The triplicate’s arithmetic means were normalized to the housekeeping gene β-actin by employing the ΔCt-method. Liquid nitrogen-frozen kidney samples were ground to fine powder and resolved in lysis buffer (50 mM Tris, pH 6.8, 4 M urea, 1% sodium dodecylsulfate, and 12.5 mM dithiothreitol). Protein concentration was measured with a NanoDrop 2000 (Thermo Fisher Scientific Inc.). Proteins were separated by sodium dodecylsulfate– polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. Membranes were blocked for 1 hour at room temperature with 5% skimmed milk in triethanolamine-buffered saline with Tween 20. Membranes were probed with primary antibodies (protein kinase cyclic adenosine monophosphate–dependent type II regulatory subunit α [PRKAR2A]: number 612243, BD Biosciences; phosphorylated PRKAR2A: number sc-377575, Santa Cruz Biotechnology; HIF-1α: number 10006421, Cayman Chemical) at 4 °C overnight. 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Z. M. Chronic in the a and PubMed Scopus Google Scholar Herein, we early and of both and with CsA 8 weeks of CsA, we obtained and and of the injury and and in of kidney PubMed Scopus Google Scholar Moreover, 4 weeks of CsA, in renal suggesting a et loss a of and renal J Physiol 2012; PubMed Scopus Google Scholar was in and in suggesting a of the to were at 8 weeks of CsA, suggesting that to CsA changes over CsA treatment S.J. et of the injury Transplant. PubMed Scopus Google Scholar a of kidney the collected in may CsA toxicity, some of which is amenable to Although the of kidney injury under CsA treatment are of CsA-induced anemia. The latter by V. Junior A.P. Coste J. et al.Impaired erythropoietin production in liver transplant recipients: the role of calcineurin inhibitors.Liver Transpl. 2006; 12: 1649-1654Crossref PubMed Scopus (16) Google Scholar,10Lei D.M. Piao S.G. Jin Y.S. et al.Expression of erythropoietin and its receptor in kidneys from normal and cyclosporine-treated rats.Transplant Proc. 2014; 46: 521-528Crossref PubMed Scopus (6) Google Scholar most treated with CsA anemia, and in experiments blood is CsA-induced anemia may be that long-term to CsA blood kidney function is as by plasma studies to CsA to a of or to Interestingly, at in the daprodustat the effect of CsA on protein phosphorylation. as a as protein among the top and the mechanisms Although daprodustat reduced it is that was limiting for calcineurin inhibition through the is among the most and of CsA to is et of in normal 1991; PubMed Scopus Google Scholar Remarkably, the a for and hypoxia was to upregulate in J. et a gene of the of 2014; Scholar In the 8 weeks of daprodustat like and suggesting was suggesting a from the in was designed to a of an early of long-term CsA-induced kidney Therefore, the 1360 for analysis pathways in injury and and CsA to to kidney capillary Interestingly, CsA both expression and phosphorylation of the key angiogenesis factor in renal is a PubMed Scopus Google Scholar including receptor and phosphorylation of Although daprodustat restored kidney capillaries, to microcirculation and were by daprodustat, a selective of daprodustat in the of CsA Daprodustat had effect on CsA-induced and it that daprodustat restored angiogenesis through However, daprodustat the effect of CsA on key of of CsA-induced kidney toxicity is as changes are and by C. N. M. et to the of renal 2018; PubMed Scopus Google Scholar the of kidney transplant biopsies, for of or and analysis in renal Nephrol. 2006; 2: PubMed Scopus (23) Google Scholar However, the of is well J. M. et in renal a of and J Transplant. 2009; PubMed Scopus Google Scholar the fingerprint of calcineurin inhibitor toxicity is based on their of effects of calcineurin inhibitors to be by by we phospho-PRKAR2A as a potential in CsA toxicity. Moreover, analysis that may daprodustat’s on the CsA-induced and as a for A of 1360 were the or daprodustat effects CsA Although kidney injury for by et factor in J Physiol 2005; PubMed Scopus Google Scholar or M. Mathia S. A. et Am Soc Nephrol. 2013; PubMed Scopus Google Scholar the is Moreover, of in and CsA treatment incompletely the the but in CsA in mice at 4 weeks of daprodustat/CsA, is in studies be to the mechanisms the protective effects of daprodustat in CsA kidney toxicity, as well as the of combined therapy in In anemia and kidney capillary rarefaction are of early CsA toxicity. are amenable to treatment with the activator daprodustat, which virtually offsets the CsA-induced changes of the phospho-proteome. 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