Abstract
Prolyl hydroxylase domain enzyme inhibitors (PHDi) stabilize hypoxia-inducible factors (HIF), and are protective in models of acute ischemic and inflammatory kidney disease.Whether PHDi also confer protection in chronic inflammatory kidney disease models remains unknown. Here we investigated long-term effects of PHDi treatment in adenine-induced nephropathy as a model for chronic tubulointerstitial nephritis. After three weeks, renal dysfunction and tubulointerstitial damage, including proximal and distal tubular injury,tubular dilation and renal crystal deposition were significantly attenuated in PHDi-treated (the isoquinoline derivative ICA and Roxadustat) compared to vehicle-treated mice with adenine- induced nephropathy. Crystal-induced renal fibrosis was only partially diminished by treatment with ICA. Renoprotective effects of ICA treatment could not be attributed to changes in adenine metabolism or urinary excretion of the metabolite 2,8-dihydroxyadenine. ICA treatment reduced inflammatory infiltrates ofF4/80+ mononuclear phagocytes in the kidneys and supported a regulatory, anti-inflammatory immune response. Furthermore, interstitial deposition of complement C1q was decreased in ICA-treated mice fed an adenine- enriched diet. Tubular cell-specific HIF-1α and myeloid cell-specific HIF-1α and HIF-2α expression were not required for the renoprotective effects of ICA. In contrast, depletion of mononuclear phagocytes with clodronate largely abolished the nephroprotective effects of PHD inhibition. Thus, our findings indicate novel and potent systemic anti-inflammatory properties of PHDi that confer preservation of kidney function and structure in chronic tubulointerstitial inflammation and might counteract kidney disease progression.
Key words: chronic kidney disease, chronic inflammation, PHD inhibitor, hypoxia-inducible factor, mononuclear phagocyte, adenine
Translational Statement
In this study we investigated prolyl hydroxylase inhibitors (PHDi) for the treatment of experimental chronic tubulointerstitial nephritis. These agents are currently evaluated in phase 3 clinical trials to treat renal anemia. In murine adenine-induced nephropathy PHDi ameliorated crystal-associated renal dysfunction and tissue damage. PHDi treatment supported a regulatory, anti-inflammatory immune response. Its nephroprotective effects relied largely on the presence of mononuclear phagocytes. Our study identified mononuclear phagocytes as important targets of PHDi in chronic tubulointerstitial nephritis. Besides their potential to correct anemia, PHDi may reduce mononuclear phagocyte-driven inflammation and thus delay CKD progression. Further studies will unveil the precise molecular mechanism of PHDi-mediated effects on mononuclear phagocytes.
Introduction
Tubulointerstitial nephritis is an important cause of chronic kidney disease (CKD),1 which is associated with high morbidity and mortality.2 Adenine-induced tubulointerstitial nephritis has been proposed as an experimental model in rodents to study chronic tubulointerstitial kidney diseases and to develop new treatment strategies.3 An adenine-enriched diet leads to deposition and accumulation of 2,8-dihydroxyadenine (DHA) crystals in the kidneys that trigger a mononuclear phagocyte- and inflammasome-dependent, crystal-associated tubulointerstitial nephropathy.Experimental studies revealed that inhibition of prolyl hydroxylase domain enzymes (PHD) is protective in ischemic and inflammatory disease models.2-Oxoglutarate (2-OG) analogues, like the isoquinoline derivative 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA, FG-2216), can effectively block PHD hydroxylation and are well tolerated in isolated cells and animals.11–13 Systemic application of ICA improved organ function and structure in rodent models of renal ischemia-reperfusion injury,13,14 aortic allograft injury,myocardial infarction,16 and hindlimb ischemia.Since PHD inhibition results in accumulation of hypoxia-inducible factors (HIF), which govern the expression of numerous potentially tissue-protective genes, it is assumed that augmentation of HIF-signaling confers tissue protection.18,19 HIF is a heterodimeric basic helix-loop-helix-PAS transcription factor, consisting of an α-subunit (with 2 major protein isoforms, HIF-1α and 2α), and its dimerization partner aryl hydrocarbon receptor nuclear translocator protein (ARNT, also known as HIFβ).18,20 More than 200 HIF target genes have already been identified which regulate complex functions like angiogenesis, erythropoiesis, glucose uptake, metabolism, fibrogenesis and cell fate decisions. When oxygen is available, HIFα is hydroxylated by three prolyl hydroxylase domain enzymes (PHD1-3), which is a prerequisite for its binding to the von Hippel-Lindau (VHL) protein, subsequent ubiquitination and rapid proteasomal degradation.18,20 In the kidney HIFα and PHDs are tightly regulated.
HIF-1α is primarily found in renal tubular epithelial cells, particularly in distal tubular cells, whereas HIF-2α is predominantly found in interstitial and endothelial cells.21 The PHD enzymes are mainly found in distal tubular cells of the renal medulla,22 where low physiological oxygen tensions prevail.Recently, pharmacological PHD inhibitors (PHDi) have been applied in phase 2 and 3 clinical trials for the treatment of renal anemia.18,24,25 However, more experience with long-term effects of PHDi in CKD is needed, as these agents may have consequences beyond the correction of renal anemia due to the variety of HIF-regulated adaptive responses.In this study we examined two PHDi for the treatment of experimental chronic tubulointerstitial nephritis. PHDi had protective effects on kidney function and structure, which required the presence of mononuclear phagocytes. PHDi shifted renal mononuclear phagocytes towards a regulatory, anti-inflammatory phenotype and reduced mononuclear phagocyte-driven renal inflammation. Our findings highlight PHDi as potential therapeutics for targeting inflammation in CKD.
Results
ICA treatment improved kidney function and structure in adenine-induced nephropathy We previously reported that the PHDi ICA was protective in rodent ischemic disease models, when up to two doses were applied before the ischemic insults.13,14,16,17 Here, long-term daily administration of ICA to C57BL/6 wild-type (WT) mice for 6 weeks was well tolerated. It neither affected kidney function nor induced fibrosis, inflammation or microvascular alterations in the kidney (Figure S1). Feeding WT mice with an adenine-enriched diet for 3 weeks resulted in crystal-induced renal failure with glomerular and tubular dysfunction, as evidenced by elevated plasma levels of creatinine and urea (Figure 1A) as well as increased
urinary excretion of proteins and sodium (Figure 1B), in parallel with polyuria, polydipsia and body weight loss (Figure 1C). Daily treatment with ICA remarkably improved all these parameters. Furthermore, ICA caused an increase in hematocrit levels (Figure 1A), which was associated with upregulation of renal Epo mRNA expression (Figure S2A). ICA did not affect urinary excretion of 2,8-dihydroxyadenine (DHA) (Figure 1B), which is the end product of adenine metabolism and excreted into the urine.
H&E stainings of kidney sections from mice with DHA nephropathy revealed focal tubulointerstitial damage comprising tubular atrophy and dilatation, interstitial fibrosis and inflammation (Figure 2A). No remarkable microscopical alterations in glomeruli were observed. ICA treatment attenuated the overall tubulointerstitial damage (Figure 2A, C). Extensive tubulointerstitial damage (score ≥4) was almost exclusively found in vehicle- treated mice (Figure 2D). Dilation of tubular lumina, due to obstruction by DHA crystals, was decreased in ICA-treated mice (Figure 2E). Reduction of tubulointerstitial damage in mice treated with ICA was also reflected by diminished renal mRNA transcripts of neutrophil gelatinase-associated lipocalin (Ngal) (Figure S2B) and fewer Kim-1 positive proximal tubular epithelial cells (Figure 3), which are both biomarkers of acute and chronic kidney injury.26,27 Tubulointerstitial damage, tubular dilation and proximal tubular injury were closely correlated with the number of DHA crystals in the kidneys (Figure S3A, B, C). The number and average size of birefringent DHA crystal deposits were reduced in ICA-treated mice (Figure 2B, F, G, H). Since the histological site of renal crystal deposits might give insight into the pathomechanism of crystal-induced nephropathies,28 we analyzed the effect of PHDi treatment on the intrarenal localization of DHA crystals. Crystals were detectable mainly in renal tubules and the interstitium, and in a few isolated foreign body granulomas. ICA did not change the intrarenal localization of these crystal deposits (Figure 2I).Next, we examined whether the PHDi roxadustat (FG-4592), which is currently investigated in phase 3 clinical trials,25 also protects against adenine-induced nephropathy. In mice fed an adenine-enriched diet, daily treatment with roxadustat increased hematocrit levels (Figure 4A) and ameliorated renal dysfunction (Figure 4B, C) and tissue injury (Figure 4D), confirming the results obtained with ICA. Thus, both PHDi attenuated renal dysfunction and structural kidney damage in DHA nephropathy.
ICA did not inhibit xanthine oxidoreductase
Oxidation of adenine into the extremely insoluble DHA by xanthine oxidoreductase (XOR) is prerequisite for precipitation of DHA crystals in the kidney (Figure 5A).29 To test if ICA reduced DHA crystal formation by inhibiting XOR, we determined the activity of this enzyme in the presence of ICA or the XOR inhibitor allopurinol in vitro. In contrast to allopurinol, ICA did not block XOR activity (Figure 5B, C), indicating that reduced crystal formation in ICA-treated mice was not due to inhibition of adenine metabolism.
Treatment with ICA did not reduce renal fibrosis in adenine-induced nephropathy
Next, we analyzed if collagen deposition and pro-fibrotic gene expression in DHA nephropathy were modified by ICA treatment. Staining of kidney sections with Sirius red, collagen IV and fibronectin did not show differences in the extent of interstitial fibrosis between vehicle- and ICA-treated mice (Figure 6A, B, C). ICA treatment reduced mRNA expression of α2-chain of type-1 collagen (Col1a2) and TGF- β1 (Tgfb1) (Figure 6D) as well as the collagen content (Figure 6E), but these changes were not statistically significant. Our data suggest that ICA can potentially prevent renal fibrosis in DHA nephropathy, albeit the results were not conclusive after 3 weeks.
Treatment with ICA attenuated inflammation in adenine-induced nephropathy
H&E-stained kidney sections (Figure 2A) suggested that ICA treatment reduced renal leucocyte infiltrates in mice fed an adenine-enriched diet. CD3+ T cells, Nimp-R14+ cells (mainly representing neutrophils and monocytes) and F4/80+ mononuclear phagocytes (MNPs) were identified by immunohistochemical stainings (Figure 7A, B, C). Treating mice with ICA did not affect the number of T cells in the kidneys (Figure 7A). Renal infiltration of Nimp-R14+ cells tended to be reduced (Figure 7B), whereas renal F4/80+ MNPs were profoundly decreased in ICA-treated mice (Figure 7C). The numbers of MNPs and DHA crystals were highly correlated (Figure S3D). In line with these data, renal mRNA expression of MNP markers F4/80, Ccl2 and Ccr5 were lower in ICA-treated animals (Figure 8A). As HIFα isoforms influence MNP polarization,30 we analyzed markers for inflammatory (M1-like) and regulatory, anti-inflammatory (M2-like) MNP activation by quantitative RT-PCR. In kidneys of ICA-treated mice the markers of inflammatory MNPs interleukins 1b (Il1b) and 6 (Il6) were downregulated, whereas Nos2 (iNOS) was induced (Figure 8B), most likely because it is a direct HIF-1α target gene.30,31 The markers of regulatory, anti-inflammatory MNPs Mrc1, Arg1 and Il10 were all increased in kidneys of ICA-treated mice (Figure 8C). Overall, these data suggested that ICA treatment favored activation of a regulatory, anti-inflammatory MNP phenotype.Next, we characterized renal MNPs of vehicle- and ICA-treated animals by flow cytometry. We used CD206 (MRC1) to identify regulatory, anti-inflammatory MNPs and determined the ratio of CD206- and CD206+ cells. ICA treatment resulted in a lower CD206-:CD206+ ratio indicating that ICA shifted the renal MNP population towards an anti-inflammatory phenotype (Figure S4).
As PHDi was reported to suppress complement factor C1q secretion from macrophages,we tested the impact of ICA on intrarenal complement deposition in DHA nephropathy. In the renal cortex from mice with adenine-induced nephropathy, mild interstitial deposition of complement factors C1q and C3c was detected (Figure 9), indicating activation of the classical complement pathway. Expression of complement factor C1q, which requires prolyl- 4-hydroxylation for its activation,32 was reduced in ICA-treated mice (Figure 9A), whereas interstitial C3c deposition was not affected by ICA treatment (Figure 9B).Overall, these findings demonstrated that ICA treatment reduced DHA-induced inflammation.
ICA treatment did not change HIFα expression patterns in adenine-induced nephropathy Both PHDi treatment and renal inflammation are known to stabilize HIFα and regulate hypoxic gene expression.33–35 Therefore, we investigated whether DHA nephropathy itself leads to HIFα accumulation and whether this response might differ from ICA-induced renal HIFα stabilization.In vehicle-treated control mice HIF-1α was occasionally found in cortical collecting ducts (Figure S5A), whereas HIF-2α signals were absent (Figure S5B). ICA administration to control mice stabilized HIF-1α in almost all tubular segments and HIF-2α in interstitial fibroblasts and endothelial cells (Figure S5), as previously described.13 In DHA nephropathy (treated with either vehicle or ICA) HIF-1α was stabilized in a limited number of preserved tubular cells (Figure 10A), HIF-2α in endothelial cells (Figure 10B) and both isoforms in inflammatory cells of foreign body granulomas (insets in Figure 10A, B). Atrophic tubules did not upregulate HIF-1α , presumably due to metabolic shutdown suspending cellular oxygen consumption and energy generation.36 Stabilized HIF-1α was functionally active, as shown by upregulation of its target genes Glut1 and Phd3 (Figure 10C),37–39 which did not reach statistical significance in total kidney lysates, probably because renal HIF-1α stabilization occurred only focally in DHA nephropathy. However, immunohistochemistry showed co-localization of HIF-1α and GLUT1 signals in cortical renal tubules (Figure 10D). ICA treatment enhanced HIFα protein stabilization but did not modulate the pattern of HIFα accumulation in different cell types.
Renoprotective effects of ICA were independent of HIFα in tubular or myeloid cells
Based on HIFα detection in tubular and inflammatory cells with and without ICA treatment, we determined if ICA-induced renoprotection in DHA nephropathy was dependent on HIF-1α in tubular cells. For this purpose, we used mice with targeted deletion of Hif1a in tubular cells (KspCre Hif1afl/fl), as previously described.40 Deficiency of Hif1a in renal tubular cells impaired ICA-induced upregulation of HIF-1α target genes Glut1 and Pgk1 (Figure S6A, B),37,41 but did not have an impact on kidney function, tubulointerstitial damage, renal crystal deposition or MNP infiltration in ICA-treated mice with DHA nephropathy (Figure 11A).Then, we generated mice with lysozyme M (LysM)-Cre-mediated myeloid-specific deletion of either Hif1a or Hif2a (LysMCre Hif1afl/fl or LysMCre Hif2afl/fl). In isolated bone marrow-derived macrophages from LysMCre Hif1afl/fl and LysMCre Hif2afl/fl mice, we confirmed deletion of Hif1a (93%) or Hif2a (88%) (Figure S6C, E), which was associated with reduced expression of typical HIF-1α (Glut1, Pgk1) or HIF-2α (Adm, Adm2) target genes upon stimulation with ICA (Figure S6D, F).37,41,42 Inactivation of Hif1a or Hif2a in myeloid cells did also not affect kidney function, tubulointerstitial damage, renal DHA crystal deposition or MNP abundance in ICA-treated mice with DHA nephropathy (Figure 11B, C).These data indicated that in tubular and myeloid cells HIF-1α and/or HIF-2α were not required for ICA-mediated renoprotection in adenine-induced nephropathy.
Effects of ICA are driven by mononuclear phagocytes in adenine-induced nephropathy
Next, we investigated the role of MNPs in DHA nephropathy and PHDi-mediated renoprotection. We depleted MNPs by repetitive administration of liposomal clodronate. In line with previous results (Figures 1, 2, 7), treatment with ICA alone strongly decreased plasma creatinine and urea levels (Figure 12A, B), renal MNP numbers (Figure 12C),tubulointerstitial damage (Figure 12D) and deposition of DHA crystals (Figure 12E).Depletion of MNPs with clodronate also moderately ameliorated renal dysfunction and tissue damage, although renal crystal deposition increased. However, in the absence of MNPs ICA treatment was largely ineffective in ameliorating renal dysfunction and tubulointerstitial damage.These findings suggested that MNPs have a crucial role in both kidney damage and PHDi-mediated renoprotection in adenine-induced nephropathy.
Discussion
In this study we investigated the long-term effects of PHDi treatment on adenine-induced nephropathy as a model for chronic tubulointerstitial nephritis.
PHDi in steady state
Long-term use of PHDi might have adverse effects due to the continuous activation or suppression of HIF-regulated genes. In particular, HIF-1α and/or HIF-2α may promote pro- fibrogenic and pro-tumorigenic signaling pathways.43 The effect of genetic HIF stabilization in tubular cells is still controversial.33,44–47 However, in this study administration of the PHDi ICA to control mice was well tolerated and did not alter renal morphology. In contrast to genetic approaches globally inactivating Phd2 or Phd2 and Phd3, we also did not observe lethal
hyperviscosity syndrome or dilated cardiomyopathy.48,49 These observations may depend on the duration of exposure or may indicate distinct effects of pharmacological inhibition and
genetic knockout of PHD proteins.
PHDi in experimental kidney disease
Pharmacological PHD inhibition has been shown to ameliorate renal failure in various acute and chronic disease models.In this study, we employed the model of adenine-induced nephropathy, which closely mimics human CKD and is characterized by tubular epithelial cell damage, interstitial fibrosis and inflammation resulting from renal deposition and accumulation of DHA crystals.3,4 PHDi treatment mitigated renal dysfunction and tubulointerstitial damage including proximal and distal tubular injury,tubular dilation and renal DHA crystal deposition.Besides direct cytotoxicity, crystals can obstruct the tubular lumen which adds to the tubulointerstitial injury.52 In the unilateral ureter obstruction (UUO) model, tubular dilation correlates with tubular cell death and interstitial fibrosis.53,54 Renal fibrosis may also been driven by tubulointerstitial inflammation.55,56 Although ICA treatment unequivocally reduced both tubular dilation and tubulointerstitial inflammation, renal fibrosis was only partially prevented in ICA-treated mice after 3 weeks. However, effects on renal fibrosis might become significant at later time points.
The mechanism underlying the reduced deposition of DHA crystals in kidneys of ICA-treated mice is still unclear. We did not detect changes in adenine metabolism or urinary excretion of the metabolite 2,8-DHA. In hyperoxaluric mice, migrating F4/80+ MNPs contributed to crystal removal by phagocytosis and digestion,57 and particularly M2 macrophages were identified to suppress renal crystal formation.58,59 M2-like regulatory, anti-inflammatory MNPs, which we found to be induced by ICA treatment, might also contribute to increased removal and/or reduced formation of DHA crystals, but further studies are needed to clarify this hypothesis.
PHDi may exert their protective effects through HIF target genes. However, cell type-specific functions of HIF-1α and HIF-2α and their individual target genes in PHDi-mediated renoprotection remain elusive. Tubular Vhl deficiency was sufficient to ameliorate acute or chronic kidney injury and phenocopy PHDi effects.46,60,61 In the UUO model Hif1a deficiency in proximal tubular cells reduced renal fibrosis,33 whereas myeloid cell-specific Hif1a/2a and Vhl deletion increased and decreased macrophage infiltration, respectively.62 In this study, tubular HIF-1α as well as myeloid HIF-1α and HIF-2α were not required for renoprotective responses induced by PHDi in DHA nephropathy. However, it cannot be excluded that HIFα in other cell types contributed to PHDi-mediated renoprotection in adenine-induced nephropathy.PHDi can also inhibit other 2OG-dependent dioxgenases than HIF-PHDs.18,64 PHDi have recently been shown to suppress secretion of the complement protein C1q from macrophages, and this process involved inhibition of collagen prolyl-4-hydroxylases.32 In line with this report, we found that ICA reduced complement C1q deposition in mice fed an adenine-enriched diet. Reduced complement activation may contribute to the decrease of renal inflammation and dysfunction in ICA-treated mice. However, the role of complement activation in DHA nephropathy is, to the best of our knowledge, still indefinite. Monosodium urate (MSU) crystals haven been shown to activate the complement cascade which in turn sustains MSU-triggered inflammasome activation.65,66 Similar this website mechanisms might also be involved in DHA nephropathy.
PHDi have also been shown to protect mice from colitis in various experimental models.67–70 Recent cell culture experiments demonstrated reduced NF-κB signaling and NLRP3 expression through hypoxia-induced autophagy, which could be mimicked by PHDi treatment of mice with colitis.70 Both NF-κB and inflammasome signaling also have a pathogenic role in adenine-induced nephropathy, and inhibition of each pathway ameliorated kidney disease.5,6 Thus, ICA might interfere with NF-κB and/or inflammasome signaling ultimately contributing to its renoprotective effects in DHA nephropathy.
Mononuclear phagocytes and PHDi-mediated kidney protection
MNPs (including macrophages and dendritic cells) form a contiguous network throughout the kidney in steady state and play an important role in maintenance of kidney function.71,72 Upon injury MNPs infiltrate into the damaged tissue area. Stimulated MNPs adopt context- dependent phenotypes that either promote or inhibit inflammatory responses.73 In DHA nephropathy, F4/80+ MNPs accumulated in the kidneys, as previously reported.4 ICA treatment reduced renal abundance of F4/80+ MNPs and skewed the MNP population towards an (M2-like) regulatory, anti-inflammatory phenotype. Our results are consistent with recent reports demonstrating that HIFα isoforms affect macrophage phenotypes.In line with the inflammatory and tissue damaging function of MNPs, depletion of these cells with clodronate-liposomes attenuated kidney damage in DHA nephropathy, confirming previous reports under different experimental conditions.7 On the other hand, clodronate- depletion rendered ICA treatment largely ineffective indicating that mononuclear phagocytes are also required for ICA-mediated renoprotective effects. Our findings demonstrate that MNPs contributed to both kidney damage and PHDi-mediated renoprotection in DHA nephropathy. This is consistent with emerging evidence that resident and infiltrating MNPs are able to promote tissue inflammation and damage,74,75 but also curtail inflammatory responses after various insults.76,77 We hypothesize that MNPs with a certain activated phenotype promote DHA nephropathy, while others orchestrate ICA-mediated renoprotection. Alterations in the expression of marker proteins support the notion that ICA shifts the balance from an inflammatory to a regulatory, anti-inflammatory phenotype. Further studies are needed to clarify the effects of ICA on MNPs. PHDi might be suitable to control MNP functions and have an impact on inflammatory kidney diseases.
Methods
Animal experiments
All animal experiments were approved by the animal care and use committee of local government authorities (Regierung von Mittelfranken, Ansbach, Germany; Az 54-2532.1- 11/13, 54-2532.1-24/13) and conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animals were housed on a 12:12 hour light-dark cycle at constant temperature (221 °C) and humidity (55±5 %) in standard cages and surrogate medical decision maker had free access to tap water and standard rodent chow immune sensor (V1534-0, ssniff Spezialdiäten GmbH) unless otherwise stated. The body weight of all mice was recorded daily. To monitor water and food intake and to collect urine samples, mice were individually housed in metabolic cages for 24 h at the beginning and end of the experiment. Animals used in this study were male mice aged between 12 and 20 weeks weighing 20 to 25 g at the beginning of the experiments. C57BL/6NCrl mice were obtained from Charles River. Generation and genotyping of mice carrying loxP-flanked conditional alleles of Hif1a and Hif2a has been described elsewhere.78,79 To delete Hif1a or Hif2a in renal tubular or myeloid cells, Hif1afl/fl and Hif2afl/fl mice were crossed to transgenic mice expressing Cre-recombinase under control of the kidney-specific (Ksp) cadherin (Cdh16) or lysozyme M (LysM)mpromoter.80,81 Thus, the following genotypes were generated in a C57BL/6 background:KspCre Hif1afl/fl, LysM Hif1afl/fl, and LysM Hif2afl/fl. Respective Cre-negative littermates were used as controls.
Tubulointerstitial nephritis was induced by feeding mice an adenine-enriched diet 0.2% (w/w) for 3 weeks.4,82 Mice were treated intraperitoneally (i. p.) with the PHD inhibitors 2-(1-chloro- 4-hydroxyisoquinoline-3-carboxamido) acetate (ICA; 40 mg/kg), roxadustat (FG-4592, Cayman Chemical; 10 mg/kg) or their vehicle (0.5 M Tris buffer with 3% DMSO for ICA, PBS with 4% DMSO for roxadustat) once daily as previously described.13,83,84 The roxadustat dose used in mice (10 mg/kg daily) was approx. 6-fold higher than in human clinical trials (up to 3.5 mg/kg thrice weekly).85 The last i.p. injection of vehicle or PHDi was given the day before removal of the kidneys.
For depletion of mononuclear phagocytes liposome-encapsulated clodronate (200 µl; Liposoma) was administered i.p. every 3 days starting 1 day before the onset of the adenine-enriched diet.86 PBS-liposomes were used as vehicle control.Animal experiments with different mouse lines and treatments were performed in 1-3 independent series with a least 4 mice in each group.After 3 weeks mice were sacrificed by exsanguination under deep isoflurane anesthesia. Blood was drawn from the inferior vena cava into EDTA-containing syringes, kidneys were harvested and either snap-frozen in liquid nitrogen or fixed by immersion in 4% paraformaldehyde.
Laboratory testing
Hematocrit was measured using a Compur M1100 micro-centrifuge (Compur Electronic GmbH). Creatinine, urea, and sodium concentrations were measured in plasma and/or urine on a Cobas Integra 800 autoanalyzer (Roche). Urinary protein concentration was assessed using Bradford’s method (DC Protein Assay, Bio-Rad Laboratories GmbH).
Analysis of 2,8-dihydroxyadenine and adenine in mouse urine
Urinary 2,8-dihydroxyadenine (DHA) and adenine were measured using the ultra- performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) assay developed as previously described.87 Briefly, a standard curve and quality control (QC) samples were prepared using urine from healthy C57BL/6 mice (purchased from BioIV). Mouse urine samples were diluted 1:15 (v/v) in 10 mM NH4OH and 50 µl of the diluted urine sample was pipetted into a 96-well plate, followed by the addition of 100 µl of 10 mM NH4OH and 50 µl of internal standard working solution. The samples were subsequently mixed in the plate for 3 min and centrifuged at 3100 rpm for 10 min at 20 °C before injection into the UPLC–MS/MS system. The injection volume was 5 µl. All mouse urine samples were stored at -80 °C prior to analysis.
Histomorphological analyses
At least 10 non-overlapping digital microphotographs of the renal cortex were obtained at 50- to 200-fold magnification using a Leica DM6000 B microscope and Leica DFC450 C digital camera (Leica Microsystems). The area stained positive for Sirius red, collagen 4, fibronectin, Nimp-R14, and MECA-32 was quantified as percentage of the total cortical area using ImageJ software version 1.51.88For quantitative assessment of F4/80 immunostaining, a digital line grid consisting of 252 evenly distributed intersections was superimposed over each image, and the number of grid intersections overlying positive brown staining was manually counted regardless of staining intensity. CD3 positive nuclei were manually counted per high-power field without regard to staining intensity.
As the intensity and extent of immunohistochemical stainings differed between experimental series due to technical reasons, stainings were separately evaluated for each experimental series. Then, results were normalized as percentage relative to the mean values of vehicle-treated mice in each series. Finally, data were pooled and analyzed.Tubular dilation was measured as cross-sectional tubular luminal area in the renal cortex using computer image analysis as described previously,and results were expressed as percentage of total cortical area.
The number of Kim-1 positive renal tubules as well as the number and size of DHA crystals (in polarized light) were automatically quantified using computer image analysis.
Tubulointerstitial damage was scored semiquantitatively using a 0 to 5 scale depending on the percentage of the total cortical area affected by tubular dilatation, tubular atrophy, interstitial fibrosis and interstitial inflammation at 200-fold magnification as follows: score 0, no changes; score 1, lesions involving less than 25%; score 2, lesions involving 25-50%; score 3, lesions involving 50-75%; score 4, lesions involving more than 75%; and score 5,lesions involving the entire area.Intensity and distribution of complement factor C1q and C3c staining in the tubulointerstitial compartment were graded at 200x magnification using a semiquantitative score ranging from 0 to 4: score 0, no deposits; score 1, weak staining affecting up to 25%; score 2, moderate staining affecting up to 50%; score 3, substantial staining affecting up to 75%; and score 4,highest staining intensity affecting more than 75% of the investigated compartment.All histomorphological analyses were performed in a blinded fashion by independent observers.
Tissue hydroxyproline content
Hydroxyproline content of kidney samples was determined by a colorimetric assay as a quantitative measure of collagen deposition.90 Data are expressed as µg collagen/mg dry
weight.
Xanthine oxidoreductase activity
Xanthine oxidoreductase (XOR) activity was measured spectrophotometrically based on uric acid formation using a kit available from Sigma-Aldrich. Xanthine (0.05 mM) and recombinant XOR (0.05 U/ml) were incubated in the presence of vehicle, the PDH inhibitor ICA (100 µM) or the XOR inhibitor allopurinol (10 µg/ml, Sigma-Aldrich).
Generation of bone marrow-derived macrophages
Bone marrow-derived macrophages (BMDM) were generated from myeloid cell-specific Hif1a- or Hif2a-deficient mice and respective Cre- littermate controls in hydrophobic Teflon®
bags (FT FEP 100 C (DuPont), American Durafilm) as described earlier.
Statistical analysis
Statistical analysis was performed using GraphPad Prism version 5.04 for Windows (GraphPad Software). All data are presented as mean ± standard error. An unpaired Student’s t-test was used for comparison of two independent groups. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. Statistical significance was defined as p value <0.05.