Positron emission tomography imaging of renal mitochondria is a powerful tool in the study of acute and progressive kidney disease models
2020; Elsevier BV; Volume: 98; Issue: 1 Linguagem: Inglês
10.1016/j.kint.2020.02.024
ISSN1523-1755
AutoresSatoshi Saeki, Hiroyuki Ohba, Yuko Ube, Kayoko Tanaka, Waka Haruyama, Masako Uchii, Tetsuya Kitayama, Hideo Tsukada, Takashi Shimada,
Tópico(s)Organ Transplantation Techniques and Outcomes
ResumoMitochondrial dysfunction plays a critical role in the pathogenesis of kidney diseases via ATP depletion and reactive oxygen species overproduction. Nonetheless, few studies have reported the renal mitochondrial status clinical settings, partly due to a paucity of methodologies. Recently, a positron emission tomography probe, 18F-BCPP-BF, was developed to non-invasively visualize and quantitate the renal mitochondrial status in vivo. Here, 18F-BCPP-BF positron emission tomography was applied to three mechanistic kidney disease models in rats: kidney ischemia-reperfusion, 5/6 nephrectomy and anti-glomerular basement membrane glomerulonephritis. In rats with ischemia-reperfusion, a slight decrease in the kidney uptake of 18F-BCPP-BF was accompanied by morphological abnormality of the mitochondria in the proximal tubular cells after three hours of reperfusion, when the kidney function was slightly declined. In 5/6 nephrectomy and rats with anti-glomerular basement membrane glomerulonephritis, the kidney uptake of 18F-BCPP-BF cumulatively decreased with impairment of the kidney function, which was accompanied by a reduction of mitochondrial protein and a pathological tubulointerstitial exacerbation rather than glomerular injury. The 18F-BCPP-BF uptake in the injured kidney was suggested to represent the volume of healthy tubular epithelial cells with normally functioning mitochondria. Thus, this positron emission tomography probe can be a powerful tool for studying the pathophysiological meanings of the mitochondrial status in kidney disease. Mitochondrial dysfunction plays a critical role in the pathogenesis of kidney diseases via ATP depletion and reactive oxygen species overproduction. Nonetheless, few studies have reported the renal mitochondrial status clinical settings, partly due to a paucity of methodologies. Recently, a positron emission tomography probe, 18F-BCPP-BF, was developed to non-invasively visualize and quantitate the renal mitochondrial status in vivo. Here, 18F-BCPP-BF positron emission tomography was applied to three mechanistic kidney disease models in rats: kidney ischemia-reperfusion, 5/6 nephrectomy and anti-glomerular basement membrane glomerulonephritis. In rats with ischemia-reperfusion, a slight decrease in the kidney uptake of 18F-BCPP-BF was accompanied by morphological abnormality of the mitochondria in the proximal tubular cells after three hours of reperfusion, when the kidney function was slightly declined. In 5/6 nephrectomy and rats with anti-glomerular basement membrane glomerulonephritis, the kidney uptake of 18F-BCPP-BF cumulatively decreased with impairment of the kidney function, which was accompanied by a reduction of mitochondrial protein and a pathological tubulointerstitial exacerbation rather than glomerular injury. The 18F-BCPP-BF uptake in the injured kidney was suggested to represent the volume of healthy tubular epithelial cells with normally functioning mitochondria. Thus, this positron emission tomography probe can be a powerful tool for studying the pathophysiological meanings of the mitochondrial status in kidney disease. see commentary on page 51 see commentary on page 51 Translational StatementThe present study demonstrates effective mitochondrial imaging with 2-tert-butyl-4-chloro-5-[6-(4-18F-fluorobutoxy)-pyridin-3-ylmethoxy]-2H-pyridazin-3-one (18F-BCPP-BF) in rat kidney disease models. The positron emission tomography (PET) signals are well-correlated to kidney function and are suggested to reflect the mass of remnant functional mitochondria in tubular epithelial cells. Once the safety of the probe and its compatibility with the human kidney have been confirmed, 18F-BCPP-BF can be a powerful tool for analyzing the mitochondrial status quantitatively, directly, and less invasively in clinical settings, thereby improving our understanding of the pathophysiological importance of renal mitochondria and possibly leading to novel diagnoses and therapies. The present study demonstrates effective mitochondrial imaging with 2-tert-butyl-4-chloro-5-[6-(4-18F-fluorobutoxy)-pyridin-3-ylmethoxy]-2H-pyridazin-3-one (18F-BCPP-BF) in rat kidney disease models. The positron emission tomography (PET) signals are well-correlated to kidney function and are suggested to reflect the mass of remnant functional mitochondria in tubular epithelial cells. Once the safety of the probe and its compatibility with the human kidney have been confirmed, 18F-BCPP-BF can be a powerful tool for analyzing the mitochondrial status quantitatively, directly, and less invasively in clinical settings, thereby improving our understanding of the pathophysiological importance of renal mitochondria and possibly leading to novel diagnoses and therapies. The kidney is a highly energy-demanding and mitochondria-rich organ.1Che R. Yuan Y. Huang S. et al.Mitochondrial dysfunction in the pathophysiology of renal diseases.Am J Physiol Renal Physiol. 2014; 306: F367-F378Crossref PubMed Scopus (251) Google Scholar Specifically, proximal tubules consume large amounts of energy supplied from mitochondria to handle large amounts of fluid and solutes to maintain homeostasis. Adenosine triphosphate (ATP) production in the proximal tubular epithelial cells mostly depends on mitochondrial oxidative phosphorylation rather than the glycolytic pathway.2Bagnasco S. Good D. Balaban R. et al.Lactate production in isolated segments of the rat nephron.Am J Physiol. 1985; 248: F522-F526PubMed Google Scholar In addition to the physiological role as a powerhouse, mitochondrial dysfunction is often associated with the increased production of reactive oxygen species, which causes tissue damage.3Small D.M. Coombes J.S. Bennett N. et al.Oxidative stress, anti-oxidant therapies and chronic kidney disease.Nephrology (Carlton). 2012; 17: 311-321Crossref PubMed Scopus (332) Google Scholar Indeed, mitochondrial dysfunction is reported to play a critical role in the pathogenesis of kidney diseases such as acute kidney injury (AKI) and chronic kidney disease (CKD).1Che R. Yuan Y. Huang S. et al.Mitochondrial dysfunction in the pathophysiology of renal diseases.Am J Physiol Renal Physiol. 2014; 306: F367-F378Crossref PubMed Scopus (251) Google Scholar,4Hall A.M. Unwin R.J. The not so “mighty chondrion”: emergence of renal diseases due to mitochondrial dysfunction.Nephron Physiol. 2007; 105: 1-10Crossref PubMed Scopus (97) Google Scholar,5Hallan S. Sharma K. The role of mitochondria in diabetic kidney disease.Curr Diab Rep. 2016; 16: 61Crossref PubMed Scopus (54) Google Scholar Thus, much attention has been paid to mitochondria in efforts to understand the pathogenesis of kidney diseases and thereby identify therapeutic targets.6Granata S. Dalla Gassa A. Tomei P. et al.Mitochondria: a new therapeutic target in chronic kidney disease.Nutr Metab (Lond). 2015; 12: 49Crossref PubMed Scopus (81) Google Scholar, 7Martin J.L. Gruszczyk A.V. Beach T.E. et al.Mitochondrial mechanisms and therapeutics in ischaemia reperfusion injury.Pediatr Nephrol. 2019; 34: 1167-1174Crossref PubMed Scopus (33) Google Scholar, 8Szeto H.H. Pharmacologic approaches to improve mitochondrial function in AKI and CKD.J Am Soc Nephrol. 2017; 28: 2856-2865Crossref PubMed Scopus (110) Google Scholar In animal studies, several methods are used to evaluate the intrinsic mitochondrial status of the kidney; however, these have some limitations in quantitative capability and precision. One direct method is to measure the oxygen consumption rate of isolated mitochondria.9Sun L. Yuan Q. Xu T. et al.Pioglitazone improves mitochondrial function in the remnant kidney and protects against renal fibrosis in 5/6 nephrectomized rats.Front Pharmacol. 2017; 8: 545Crossref PubMed Scopus (32) Google Scholar, 10Aparicio-Trejo O.E. Tapia E. Molina-Jijón E. et al.Curcumin prevents mitochondrial dynamics disturbances in early 5/6 nephrectomy: relation to oxidative stress and mitochondrial bioenergetics.Biofactors. 2017; 43: 293-310Crossref PubMed Scopus (50) Google Scholar, 11Thomas J.L. Pham H. Li Y. et al.Hypoxia-inducible factor-1α activation improves renal oxygenation and mitochondrial function in early chronic kidney disease.Am J Physiol Renal Physiol. 2017; 313: F282-F290Crossref PubMed Scopus (24) Google Scholar However, the complicated process of mitochondrial isolation may cause data variation. The mitochondrial morphology (e.g., fragmentation or hyperfusion) is a widely accepted indicator of their functional status.12Galvan D.L. Green N.H. Danesh F.R. The hallmarks of mitochondrial dysfunction in chronic kidney disease.Kidney Int. 2017; 92: 1051-1057Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 13Zhan M. Brooks C. Liu F. et al.Mitochondrial dynamics: regulatory mechanisms and emerging role in renal pathophysiology.Kidney Int. 2013; 83: 568-581Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 14Westermann B. Bioenergetic role of mitochondrial fusion and fission.Biochim Biophys Acta. 2012; 1817: 1833-1838Crossref PubMed Scopus (400) Google Scholar However, electron microscopic analysis of the morphology hardly demonstrates quantitative results. Thus, it is more common to use the amount of mitochondrial proteins or mitochondrial DNAs as quantitative surrogate indicators of the mitochondrial status. In the clinical setting, few studies have reported the renal mitochondrial status. In patients with diabetic nephropathy, the analysis of urinary exosomes demonstrated lower levels of mitochondrial proteins and mitochondrial DNAs that were probably derived from kidneys, and the urinary metabolite profile differed from that of healthy controls, suggesting a dysregulated mitochondrial function in the kidney.15Sharma K. Karl B. Mathew A.V. et al.Metabolomics reveals signature of mitochondrial dysfunction in diabetic kidney disease.J Am Soc Nephrol. 2013; 24: 1901-1912Crossref PubMed Scopus (352) Google Scholar Furthermore, dysfunction of extrarenal mitochondria (e.g., in peripheral blood or muscle) was observed in CKD patients.16Tin A. Grams M.E. Ashar F.N. et al.Association between mitochondrial DNA copy number in peripheral blood and incident CKD in the Atherosclerosis Risk in Communities Study.J Am Soc Nephrol. 2016; 27: 2467-2473Crossref PubMed Scopus (73) Google Scholar, 17Lee J.E. Park H. Ju Y.S. et al.Higher mitochondrial DNA copy number is associated with lower prevalence of microalbuminuria.Exp Mol Med. 2009; 41: 253-258Crossref PubMed Scopus (27) Google Scholar, 18Rao M. Li L. Demello C. et al.Mitochondrial DNA injury and mortality in hemodialysis patients.J Am Soc Nephrol. 2009; 20: 189-196Crossref PubMed Scopus (52) Google Scholar, 19Granata S. Zaza G. Simone S. et al.Mitochondrial dysregulation and oxidative stress in patients with chronic kidney disease.BMC Genomics. 2009; 10: 388Crossref PubMed Scopus (168) Google Scholar, 20Gamboa J.L. Billings 4th, F.T. Bojanowski M.T. et al.Mitochondrial dysfunction and oxidative stress in patients with chronic kidney disease.Physiol Rep. 2016; 4e12780Crossref PubMed Scopus (124) Google Scholar Thus, studies on mitochondrial involvement in kidney diseases are limited, mainly due to a paucity of versatile methods to directly and noninvasively quantify the renal mitochondrial status. We previously developed a novel positron emission tomography (PET) probe, 18F-BCPP-BF, which binds specifically to mitochondrial complex I (MC-I)21Harada N. Nishiyama S. Kanazawa M. et al.Development of novel PET probes, [18F]BCPP-EF, [18F]BCPP-BF, and [11C]BCPP-EM for mitochondrial complex 1 imaging in the living brain.J Labelled Comp Radiopharm. 2013; 56: 553-561Crossref PubMed Scopus (38) Google Scholar even in in vivo studies (Supplementary Figure S1). Unlike currently available PET probes, 18F-BCPP-BF enables kidney imaging with a low level of background due to its favorable pharmacokinetic properties (i.e., a rapid uptake and long retention in kidney tissue).22Tsukada H. Nishiyama S. Fukumoto D. et al.Novel PET probes 18F-BCPP-EF and 18F-BCPP-BF for mitochondrial complex I: a PET study in comparison with 18F-BMS-747158-02 in rat brain.J Nucl Med. 2014; 55: 473-480Crossref PubMed Scopus (43) Google Scholar It is therefore preferable to the noninvasive evaluation and quantification of the renal mitochondrial status. A previous study showed that the renal uptake of 18F-BCPP-BF was reduced in an acetaminophen-induced liver and kidney injury model.23Ohba H. Kanazawa M. Kakiuchi T. et al.Effects of acetaminophen on mitochondrial complex I activity in the rat liver and kidney: a PET study with 18F-BCPP-BF.EJNMMI Res. 2016; 6: 82Crossref PubMed Scopus (5) Google Scholar Because that finding was obtained under drug-induced toxic conditions, in the present study, we further evaluated the clinical usefulness of 18F-BCPP-BF in typical models of kidney disease caused by completely different clinical pathogeneses but sharing a common pathway of disease progression in the later phase (renal ischemia reperfusion [I/R], 5/6 nephrectomy [5/6 Nx], and anti-glomerular basement membrane [GBM] glomerulonephritis). Our I/R rat model exhibits typical serial changes seen in oxidative stress-induced AKI. The serum levels of blood urea nitrogen (BUN) and serum levels of creatinine (sCre) peaked within 24 hours after reperfusion (Figure 1a and b).Figure 1Organ uptake of 2-tert-butyl-4-chloro-5-[6-(4-18F-fluorobutoxy)-pyridin-3-ylmethoxy]-2H-pyridazin-3-one (18F-BCPP-BF) in renal ischemia-reperfusion (I/R) rats, and functional and morphologic analysis of the kidneys. (a,b) Serial changes in blood renal parameters, blood urea nitrogen (BUN), and serum creatinine (sCre). Open symbols represent individual values. Closed symbols represent average values (n = 3). (c) Representative 18F-BCPP-BF positron emission tomography–computed tomography fusion images of I/R rats at 3 hours. (d) Averaged standard uptake values (SUVs) of 18F-BCPP-BF by livers or the outer portion of kidneys after 3 hours of reperfusion. Circles represent individual values (n = 4). ∗P < 0.05 versus sham, pairwise comparison by Student’s t test. (e) Representative micrographic images of kidneys. After 3 hours of reperfusion followed by 1-hour positron emission tomography imaging, kidney sections were obtained. Representative sections from the renal cortex and outer stripe of the outer medulla (OSOM) were stained with hematoxylin and eosin. Bars = 50 μm. (f) Western blotting of mitochondrial proteins. Protein samples were extracted from kidneys after 3 hours of reperfusion. The blood parameters of each animal are shown in Supplementary Table S1. (g) Representative electron micrographs of S3 proximal tubular cells in kidneys. Kidneys were obtained from untreated, sham-operated, and I/R injury animals after 3 hours of reperfusion. Bars = 2 μm. ATP5A-CV, ATP synthase mitochondrial F1 complex alpha subunit 1, complex V; MTCO1-CIV, mitochondrially encoded cytochrome c oxidase I, complex IV; NDUFB8-CI, NADH:ubiquinone oxidoreductase subunit B8, complex I; SDHD-CII, succinate dehydrogenase complex iron sulfur subunit B, complex II; STD, standard; UQCRC2-CIII, ubiquinol cytochrome c reductase core protein 2, complex III. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PET imaging was conducted at 3 hours after reperfusion. In the sham-operated group, PET measurement detected a strong uptake of 18F-BCPP-BF by the liver and outer portion of the kidney (Figure 1c), which presumably corresponds to the renal cortex and outer stripe of the outer medulla, which are abundant in mitochondria. With I/R surgery, the uptake of 18F-BCPP-BF was significantly reduced in the outer portion of the kidney (75% of that in sham, P < 0.05), while it was not affected in liver (98% of that in sham) (Figure 1d). After 3 hours of reperfusion, the I/R group already showed moderate elevation of BUN (30 mg/dl vs. sham 17 mg/dl) and sCre (0.88 mg/dl vs. sham 0.34 mg/dl). The histopathologic examination of I/R rats demonstrated minimal shedding of the tubular epithelium in the outer stripe of the outer medulla, the tissue most susceptible to I/R damage, and necrosis or debris was minimal (Figure 1e), suggesting that the decreased PET signal was not simply due to loss of the mitochondria-rich tubular epithelium. Immunoblotting revealed no obvious change in the mitochondrial protein levels in the I/R kidney (Figure 1f; Supplementary Table S1). However, electron microscopic examination of the S3 segment of proximal tubular cells located at the outer stripe of the outer medulla revealed that mitochondria were densely packed in the sham-operated group, whereas those were dramatically swollen in the I/R group (Figure 1g). We next conducted a PET study with 5/6 Nx rats, a model of CKD. To compare the mitochondrial status between the early and progressive phases of CKD, the 18F-BCPP-BF uptake was evaluated at 4 and 15 weeks after surgery, respectively. In addition, we treated one 5/6 Nx group with angiotensin II receptor blocker (ARB) to analyze the influence of the renoprotective effects on the mitochondrial condition. Four weeks after nephrectomy, the 18F-BCPP-BF uptake was significantly reduced in the outer portion of the kidney (78% of that in sham, P < 0.001), but not in the liver (124% of that in sham) (Figure 2a and b). At this point, 5/6 Nx rats demonstrated mild kidney disease (BUN, 62 mg/dl for 5/6 Nx vs. 21 mg/dl for sham; sCre, 0.59 mg/dl for 5/6 Nx vs. 0.24 mg/dl for sham) (Figure 2c and d, respectively). A histopathologic examination demonstrated enlarged glomeruli, minimal tubular dilatation, and the proliferation of tubular epithelium in the renal cortex of 5/6Nx rats, while tubulo-interstitial damage had not yet developed (Figure 2e). Fifteen weeks after nephrectomy, 5/6 Nx rats developed progressive kidney disease, which was indicated by the distinct elevation of BUN (95 mg/dl vs. 19 mg/dl in sham) and sCre (2.2 mg/dl vs. 0.27 mg/dl in sham) (Figure 3a and b, respectively), and PET measurements demonstrated the significantly reduced uptake of 18F-BCPP-BF in the outer portion of the remnant kidney (44% of that in sham, P < 0.01) (Figure 3c and d), which was more robust than that in the early phase of 5/6 Nx. The uptake was not reduced in the liver (130% of that in sham) in the 5/6 Nx group. Immunoblotting demonstrated decreases in each protein component of all mitochondrial complexes (Figure 3e; Supplementary Table S2), suggesting the reduction of mitochondrial mass per tissue volume. Notably, ARB treatment remarkably protected the kidney function (BUN, 48 mg/dl; sCre, 0.69 mg/dl), which was accompanied by the relatively preserved uptake of 18F-BCPP-BF in the kidney (82% of that in sham), suggesting that the PET signal reflected the remnant kidney function. Actually, the standard uptake value (SUV) of individual kidneys, whether in the early or late phase, showed a very good negative correlation with the renal parameters, including those in ARB-treated animals (Figure 3f and g; Supplementary Figure S2).Figure 3Organ uptake of 2-tert-butyl-4-chloro-5-[6-(4-18F-fluorobutoxy)-pyridin-3-ylmethoxy]-2H-pyridazin-3-one (18F-BCPP-BF) in 5/6 nephrectomy (5/6 Nx) rats at the late phase, and functional and morphologic analysis of the kidneys. (a,b) Serial changes in blood renal parameters, blood urea nitrogen (BUN), and serum creatinine (sCre). Open symbols represent individual values. Closed symbols represent average values (n = 4). (c) Representative 18F-BCPP-BF positron emission tomography–computed tomography fusion images of 5/6 Nx rats at 15 weeks after surgery. (d) Averaged standard uptake values (SUVs) of 18F-BCPP-BF by livers or the outer portion of kidneys. Circles represent individual values (n = 4). ∗∗P < 0.01 versus sham, pairwise comparison by Student’s t test or Aspin-Welch's t test. (e) Western blotting of mitochondrial proteins. Protein samples were extracted from kidneys at 15 weeks after 5/6 Nx surgery. Blood parameters of each animal are shown in Supplementary Table S2. (f,g) Correlation of the kidney uptake of 18F-BCPP-BF with blood renal parameters of rats at 15 weeks after 5/6 Nx surgery. Each SUV of 18F-BCPP-BF was plotted against BUN or sCre of sham-operated (triangles), vehicle-treated 5/6 Nx (circles), or angiotensin II receptor blocker (ARB)-treated (squares) animals. (h) Representative micrographic images of the kidneys at 15 weeks after 5/6 Nx surgery. Kidney sections were obtained from sham-operated (Sham), vehicle-treated 5/6 Nx (5/6 Nx), and ARB-treated (5/6 Nx + ARB) animals. Representative sections from the renal cortex were stained with hematoxylin and eosin. Bars = 200 μm. ATP5A-CV, ATP synthase mitochondrial F1 complex alpha subunit 1, complex V; MTCO1-CIV, mitochondrially encoded cytochrome c oxidase I, complex IV; NDUFB8-CI, NADH:ubiquinone oxidoreductase subunit B8, complex I; SDHD-CII, succinate dehydrogenase complex iron sulfur subunit B, complex II; STD, standard; UQCRC2-CIII, ubiquinol cytochrome c reductase core protein 2, complex III. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The histopathologic examination of the renal cortex in 5/6 Nx animals demonstrated progressive damage in the glomeruli and tubular epithelium (Figure 3h). More than half of the glomeruli exhibited crescent formation, and 20% to 50% of glomeruli developed glomerular sclerosis. Severe interstitial fibrosis and moderate hyaline casts were observed. All pathologic changes were minimal in ARB-treated animals. Next, we conducted a PET study with the anti-GBM model, a well-known model of progressive glomerulonephritis. PET measurement was performed in both the acute and late phases, at 2 and 6 weeks after anti-GBM antibody injection, respectively. Two weeks after anti-GBM injection, PET demonstrated the significantly reduced renal uptake of 18F-BCPP-BF (74% of that in normal control, P < 0.05) (Figure 4a and b) with no reduction of the SUV of the liver (94% of that in normal control). At this point, the kidney function was still well preserved (BUN, 29 mg/dl vs. 20 mg/dl in normal control; sCre, 0.26 mg/dl vs. 0.22 mg/dl in normal control) (Figure 4d and e, respectively), although the animals had already developed massive albuminuria (albumin creatinine ratio, 23 mg/mg vs. 0.063 mg/mg in normal control) within 1 week after the administration of anti-GBM serum (Figure 4f). The histopathologic examination of the renal cortex demonstrated segmental (20%–50%) or diffuse (<20%) glomerular sclerosis (Figure 4h). More than one-half of glomeruli exhibited crescent formation, and approximately one-half of the glomeruli exhibited endocapillary proliferation. In contrast, interstitial fibrosis was not observed, and hyaline casts were minimal, suggesting minimal tubulointerstitial damage.Figure 42-tert-butyl-4-chloro-5-[6-(4-18F-fluorobutoxy)-pyridin-3-ylmethoxy]-2H-pyridazin-3-one (18F-BCPP-BF) in anti–glomerular basement membrane (anti-GBM) glomerulonephritis rats at the acute and late phases, and functional and morphologic analysis of the kidneys. (a) Representative 18F-BCPP-BF positron emission tomography–computed tomography fusion images of anti-GBM rats at 2 or 6 weeks after anti-GBM serum injection. (b,c) Averaged standard uptake values (SUVs) of 18F-BCPP-BF by livers or the outer portion of kidneys. Circles represent individual values (n = 4 or 8). ∗P < 0.05 versus normal, ∗∗P <0.01 versus normal, pairwise comparison by Student’s t test. (d,e) Changes in blood renal parameters, blood urea nitrogen (BUN), and serum creatinine (sCre), after anti-GBM serum administration. Symbols represent individual values (n = 4 or 8). (f) Serial changes in the albumin-creatinine ratio (ACR). Open symbols represent individual values. Closed symbols represent average values (n = 4 or 8). (g) Correlation of kidney uptake of 18F-BCPP-BF at 6 weeks after the anti-GBM serum injection with urinary N-acetyl-beta-D-glucosaminidase (NAG) at 4 weeks after the serum injection. Each SUV of 18F-BCPP-BF was plotted against urinary NAG-to–creatinine ratio of normal control (triangles) or anti-GBM (circles) animal. (h) Representative micrographic images of kidneys from anti-GBM glomerulonephritis model rats. Representative renal cortex sections were stained with hematoxylin and eosin. Bars = 100 μm. (i) Western blotting of mitochondrial proteins. Protein samples were extracted from kidneys excised immediately after positron emission tomography measurement at 2 or 6 weeks after the anti-GBM serum administration. Blood parameters of each animal are shown in Supplementary Table S3. ATP5A-CV, ATP synthase mitochondrial F1 complex alpha subunit 1, complex V; MTCO1-CIV, mitochondrially encoded cytochrome c oxidase I, complex IV; NDUFB8-CI, NADH:ubiquinone oxidoreductase subunit B8, complex I; SDHD-CII, succinate dehydrogenase complex iron sulfur subunit B, complex II; STD, standard; UQCRC2-CIII, ubiquinol cytochrome c reductase core protein 2, complex III. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) At the second PET measurement, in the late phase, the reduced kidney uptake of 18F-BCPP-BF was more remarkable (33% of that in normal control, P < 0.001) (Figure 4c). The kidney dysfunction progressed further (BUN, 56 mg/ml vs. 19 mg/ml in normal control; sCre, 0.83 mg/dl vs. 0.25 mg/dl in sham) (Figure 4d and e), and a histopathologic examination of the renal cortex demonstrated the progressive damages in Bowman’s capsule and the tubular epithelium (Figure 4h). Most glomeruli (>80%) exhibited crescent formation, and more than one-half of glomeruli developed global glomerular sclerosis. Severe basophilic tubules and moderate hyaline casts were observed, demonstrating remarkable tubulointerstitial damage. It is of note that SUVs were inversely correlated with both the renal functions (Supplementary Figure S3), and urinary N-acetyl-beta-D-glucosaminidase, a marker of renal tubular dysfunction (Figure 4g; Supplementary Figure S4). Immunoblotting demonstrated that the mitochondrial complex component protein levels were slightly decreased in the early phase. This decrease became more remarkable in the late phase (Figure 4i). SUVs were correlated with the protein levels of mitochondrial complex component in the kidney of each animal (Supplementary Table S3 and Supplementary Figure S5). In this study, 18F-BCPP-BF, a new PET probe for MC-I imaging, was utilized to investigate the kidney mitochondrial status in the I/R, 5/6Nx, and anti-GBM models. In these models, PET imaging demonstrated a common reduction in the renal uptake of 18F-BCPP-BF, suggesting some defect in the mitochondrial status was involved in the process of disease development despite their distinct pathologic triggers. In addition, the present study demonstrates that the mitochondrial defects probably reflect the decrease in the mitochondrial mass or qualitative changes in the tubular epithelium rather than the glomerular cells or both. The renal tubular epithelial damage caused by I/R injury is a major pathophysiological event in AKI,24Gueler F. Gwinner W. 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