Dichotomous Responses To Chronic Fetal Hypoxia Lead To A Predetermined Aging Phenotype Ⅱ
Nov 29, 2023
Hypoxic
Fetal Kidneys
Exhibit a Deregulated Protein Expression Profile To elucidate the molecular pathways that underlie hypoxia-driven IUGR, freshly isolated kidneys from hypoxic or normoxic E18.5 fetuses were subjected to bottom-up proteome profiling using a nano-LC system (Dionex UltiMate 3000 RSLC) coupled to a high-resolution orbitrap mass spectrometer (Thermo QExactive). Principal component analysis showed striking differences between the hypoxic and normoxic kidneys (Fig. 2A). In total, 6307 proteins were identified (FDR < 0.01, supplemental Table S2) of which 436 were significantly deregulated (FDR < 0.05); 284 with increased abundance and 152 with decreased abundance. Functional annotation clustering using gene ontology (18–20) revealed enrichment of specific mitochondrial, lysosomal, RNA-, and DNA-binding proteins, as well as proteins involved in specific metabolic processes or innate immune responses (Fig. 2, B– D). We categorized these proteins with regard to (1) nephron formation, (2) metabolic adaptation, and (3) accelerated aging, as presented and discussed in the following paragraphs.
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Repressed DNA Replication and Protein Synthesis Contribute to the Constrained Formation of New Nephrons in Chronic Hypoxia
Adverse events during development such as chronic fetal hypoxia have frequently been shown associated with reduced nephron numbers (27, 29–32). Yet, an underlying mechanism convincingly explaining this finding could rarely be demonstrated. Clustering all 64 deregulated proteins belonging to the GO-terms “DNA-binding” and “RNA-binding” (a heatmap is shown in supplemental Fig. S2) using the STRING database (22) revealed multiple subnetworks including DNA repair, DNA replication, mRNA splicing, and ribosomal proteins (Fig. 3A). A more comprehensive list of enriched processes or pathways is shown in Figure 3B (FDR < 0.05). Of these “Ribosome” and “DNA replication” were among the top terms of repressed proteins, whereas mRNA splicing and “RNA degradation” were among the top terms of induced proteins. DNA repair processes showed a bipartite expression pattern. Here, proteins involved in nucleotide excision or mismatch repair were repressed, but those mediating “Base excision repair” were induced. Furthermore, the abundance level of the cell cycle inhibitor p27Kip1 was twofold increased (Fig. 3C) and that of the proliferation marker Ki67 was 3.4-fold reduced (Fig. 3D), which together indicates a slowdown of the cell division process. In particular, the mRNA expression level of Mki67, the gene encoding for Ki67, was significantly repressed in mouse primary proximal tubular cells cultured under hypoxic conditions (Fig. 3E). This repression was mediated through hypermethylation of the Mki67 promoter region in hypoxic fetal kidneys (Fig. 3F). Thus, in fetal hypoxic kidneys, DNA synthesis, mRNA translation, and most DNA repair processes seem to be turned down, reducing the ability of the cells to grow and proliferate. In aggregate here, for the first time, proteome profiling data provide molecular evidence potentially explaining the diminished formation of nephrons.
The Innate Immune System and Oxidative Stress Are Activated in Chronic Hypoxic Conditions
One of the most enriched terms appearing in the functional annotation analysis of all 436 proteins and the topmost for induced proteins was “Neutrophil degranulation” (Fig. 2, B and C), indicating an ongoing activation of the innate immune system in hypoxic fetal kidneys. Of the 34 associated proteins, 29 were induced and five repressed (pink in Fig. 4A). Among the induced proteins were many glycolytic and lysosome-related enzymes, several S100A protein family members, the inflammatory cytokine macrophage migration inhibitory factor (MIF), as well as the primary and secondary granule proteins neutrophilic granule protein (NGP), myeloperoxidase (MPO), Cathelicidin (CAMP), peptidoglycan recognition protein 1 (PGLYRP1), and lactoferrin (LTF). To verify the anticipated invasion of neutrophils in hypoxic fetal kidneys and to uncover their location within the tissue, renal sections were stained for MPO. MPO-positive cells formed clusters in the nephrogenic zone of the renal cortex, in the proximity of blood vessels, adjacent to the proximal tubule, and occasionally could be found in medullary regions of hypoxic samples (Fig. 4D and supplemental Fig. S3, A–C). In contrast, normoxic fetal kidneys did not show infiltration or accumulation of neutrophils (Fig. 4C). Caveolin-1 (CAV1) was shown to enhance the transcellular migration of immune cells (33). Accordingly, CAV1 was induced in hypoxic fetal kidneys (supplemental Fig. S3D), showing enhanced staining of renal blood vessels including those traversing the cortical region of the fetal kidney (Fig. 4, E and F). Degranulation of neutrophils produces a local burst of reactive oxygen species leading to increased oxidative stress and tissue damage including DNA oxidation. An important marker of DNA oxidation is 8-hydroxy-2′ -deoxyguanosine (8-OHdG), which was enhanced in proximal tubules and in the nephrogenic zone of hypoxic fetal kidneys (Fig. 4, G and H), in the vicinity of neutrophil clustering. This rise in 8-OHdG damaged DNA occurred despite a simultaneous induction of MGMT and OGG1 (Fig. 4, I and J), two enzymes responsible for the removal of 8-OHdG. These results demonstrate an ongoing activation of the innate immune system in the developing kidney, which causes even more tissue damage in addition to the hypoxic stress. Along with the ineffective repair of these tissue lesions, a vicious circle might be initiated that further impairs nephrogenesis.
FIG. 2. Proteomic profiling reveals multiple changes associated with hypoxia. A, principal component analysis of the proteomic data showed a clear separation between normoxic and hypoxic fetal kidneys. B–D, functional annotation clustering of all significantly deregulated proteins (B), induced proteins (C), and reduced proteins (D) The significantly changed fraction of a pathway (x-axis) is plotted against its significance of enrichment (y-axis). The size of each point encodes the total number of members in that pathway. Induced proteins showed enrichment of metabolic processes (glycolysis), mitochondrial or lysosomal proteins, and proteins involved in immune system response (neutrophil degranulation), whereas reduced proteins are enriched for DNA- and RNA-binding pathways (a structural constituent of the ribosome).
(PGLYRP1), and lactoferrin (LTF). To verify the anticipated invasion of neutrophils in hypoxic fetal kidneys and to uncover their location within the tissue, renal sections were stained for MPO. MPO-positive cells formed clusters in the nephrogenic zone of the renal cortex, in the proximity of blood vessels, adjacent to the proximal tubule, and occasionally could be found in medullary regions of hypoxic samples (Fig. 4D and supplemental Fig. S3, A–C). In contrast, normoxic fetal kidneys did not show infiltration or accumulation of neutrophils (Fig. 4C). Caveolin-1 (CAV1) was shown to enhance the transcellular migration of immune cells (33). Accordingly, CAV1 was induced in hypoxic fetal kidneys (supplemental Fig. S3D), showing enhanced staining of renal blood vessels including those traversing the cortical region of the fetal kidney (Fig. 4, E and F). Degranulation of neutrophils produces a local burst of reactive oxygen species leading to increased oxidative stress and tissue damage including DNA oxidation. An important marker of DNA oxidation is 8-hydroxy-2′ -deoxyguanosine (8-OHdG), which was enhanced in proximal tubules and in the nephrogenic zone of hypoxic fetal kidneys (Fig. 4, G and H), in the vicinity of neutrophil clustering. This rise in 8-OHdG damaged DNA occurred despite a simultaneous induction of MGMT and OGG1 (Fig. 4, I and J), two enzymes responsible for the removal of 8-OHdG. These results demonstrate an ongoing activation of the innate immune system in the developing kidney, which causes even more tissue damage in addition to the hypoxic stress. Along with the ineffective repair of these tissue lesions, a vicious circle might be initiated that further impairs nephrogenesis.
Metabolic Adaptations to Hypoxia Result in Pronounced Glycolysis in the Fetal Kidney
Chronic hypoxia is a severe condition to which cells have to adapt via metabolic changes in order to survive. Foremost to this is the maintenance of cellular ATP production that in the absence of sufficient oxygenation requires a shift from oxidative phosphorylation to glycolysis. Of all terms resulting from the functional annotation, “glycolytic process” was the most significant (Fig. 2B). All ten enzymes (red ①–⑩ in Fig. 5A) required for the conversion of glucose to pyruvate were induced in fetal hypoxic kidneys compared with normoxic controls (a heatmap is shown in Fig. 5C). Furthermore, the expressions of glucose transporter 1 (SLC2A1, orange in Fig. 4A) and lactate dehydrogenase A (LDHA, dark red in Fig. 5A) were also enhanced, which should facilitate increased uptake of glucose into the cell and the augmented reduction of pyruvate to lactate, respectively. The alternative utilization of pyruvate in the citric acid cycle seemed to be impeded (1) by enhanced expression of pyruvate dehydrogenase kinase 1 (PDK1, dark red in Fig. 5A), which inactivates the pyruvate dehydrogenase complex in mitochondria and thus the oxidation of pyruvate to acetyl-CoA; and (2) by reduced levels of pyruvate carboxylase (PC), which catalyzes the conversion of pyruvate to oxaloacetate. On the other hand, fructose-1,6-bisphosphatase 1 (FBP1, blue in Fig. 5A) was reduced, further augmenting the potential flux of glucose toward pyruvate. All these enzymatic changes favor the production of lactate, and indeed, lactate concentration was increased in hypoxic fetal kidneys (Fig. 5B). Enhanced lactate production may lead to unfavorable acidification. Yet, we found among the enriched proteins the monocarboxylate transporters SLC16A3 and SLC5A8 (orange in Fig. 5A), which are known to excrete lactate into the extracellular space to avoid toxic effects of cytoplasmic acidification. Thus, our model demonstrates the remarkable capability of fetal kidneys to adapt to chronic hypoxia by increasing glycolytic activity,
FIG. 3. The formation of new nephrons in hypoxia is associated with repressed DNA replication and protein synthesis. A, protein interaction network of significantly changed DNA- and RNA-binding proteins derived from the STRING database shows several protein clusters: ribosomal proteins (blue), proteins involved in DNA replication (green), DNA repair (purple), and mRNA splicing (red). The proliferation marker Ki67 (Mki67) is marked in black, highlighting its close relationship to DNA repair and DNA replication. B, a selection of pathways of significantly changed DNA- and RNA-binding proteins from KEGG and Reactome databases that exhibited the most prominent alterations in hypoxic kidneys, depicted in decreasing order of significance. RNA splicing and RNA degradation processes were enriched (red), while RNA translation, DNA replication, and repair pathways were repressed (blue). Only processes with an FDR <0.05 are shown. C and D, the cell cycle inhibitor p27Kip1 was enhanced (unpaired two-tailed t test, Welch’s correction, p = 0.0095), whereas the expression of the proliferation marker Ki67 was reduced (unpaired two-tailed t test, Welch’s correction, p = 0.0078) in hypoxic fetal kidneys. E, hypoxia reduced the mRNA expression level of Mki67 in mouse primary proximal tubular cells (unpaired two-tailed t test, p < 0.0001). F, this reduction was mediated by hypermethylation of the Mki67 promoter in hypoxic fetal kidneys. Open circle unmethylated, black circle methylated (Fisher's exact test, p = 0.0016; Mann–Whitney U-test, p = 0.0431)
FIG. 4. Proteins involved in inflammatory response and oxidative stress are enriched in newly forming nephrons under hypoxia. A, depiction of all 34 significantly changed proteins for the annotation termed neutrophil degranulation (pink circles) among all significant (larger gray circles) and not significant (smaller gray circles) proteins of our data set. 29 had a higher abundance level, whereas five were reduced. B, a heatmap showing all pink proteins in (A) and their induction or repression in hypoxia, depicted in decreasing order of protein abundance. C–H, representative immunohistochemical images of E18.5 normoxic or hypoxic kidneys showing the infiltration of neutrophils and oxidative stress. C and D, immunohistochemistry for the neutrophil marker myeloperoxidase (MPO) revealed clustering of these cells in the proximity of newly forming nephrons (asterisk) and adjacent to renal blood vessels (arrowhead) of hypoxic fetal kidneys (D). In normoxic controls (C), MPO-positive cells were rarely present. (Scale bars 100 μm). E and F, immunohistochemistry depicting enhanced expression of caveolin-1 (CAV1) in renal blood vessels of hypoxic fetal kidneys (F) compared with controls (E) (Scale bars 200 μm). G and H, 8-hydroxy-2′ -deoxyguanosine (8-OHdG), a marker for oxidative DNA damage, was prominently enhanced in newly forming nephrons (asterisk) in the renal cortex and in cells of the proximal tubules (arrow) of hypoxic kidneys (H), whereas only few cells were stained in normoxic tissue (G) (Scale bars 200 μm). I and J, this increase in DNA damage occurred despite a simultaneous rise in O-6-methylguanine-DNA methyltransferase (MGMT; unpaired two-tailed t test, p = 0.0002) (I) and 8-oxo guanine glycosylase (OGG1; unpaired two-tailed t-test, p = 0.0063) (J), two enzymes involved in the repair of oxidized DNA.
which ensures sufficient ATP production and survival under this adverse condition.
Fetal Hypoxia Promotes Compensatory MitochondrialProtein Import and Respiratory Chain Assembly
Besides enhanced glycolysis, we found multiple alterations in mitochondrial protein abundance levels (a heatmap is shown in supplemental Fig. S4). Generation of protein networks using STRING revealed several clusters comprising proteins involved in the translocation of proteins into mitochon.dria, oxidative phosphorylation, and mitochondrial ribosomal proteins (Fig. 5, D and E). Of note, multiple proteins linked to the inner mitochondrial membrane were induced, the site where oxidative phosphorylation (OXPHOS) takes place.OXPHOS encompasses five multiprotein complexes arranged along the inner mitochondrial membrane. Among the induced proteins were components of OXPHOScomplexes(NDUSF6), III(UQCR10), IV (COX6B1, COXC, and COX7A2),and V (ATP5J, MTATP8), but also UQCC3 and SCO2, which are required for the correct assembly and function of complex and IV, respectively (Fig. 5D and supplemental Fig. S4). SDHC and SDHD, subunits of complex ll, were also upregu.lated (1.6- and 2.5-fold, respectively), but did not reach statistical significance. Moreover, not only components of the respiratory chain were enriched in fetal hypoxic kidneys, but also a multitude of proteins mediating their import into mitochondria. This included members of the outer membrane translocase (TOM - TOMM22),and the inner membrane translocase complex TIM22 (TIMM22， TIMM9, and TIMM10and the associated TIMM8-TIMM13-complex (Fig. 5D and supplemental Fig. S4). Among the 21 mitochondrial proteins with reduced abundance were four mitochondrial ribosomal proteins, as well as proteins involved in amino acid catabolism, vitamin biosynthesis, or fatty acid beta-oxidation (Fig. 5, D and E, and supplemental Fig. S4). These findings point toward potential mitochondrial dysfunction and apparent efforts to regenerate damaged proteins (34), despite enhanced glycolysis and overall reduced protein synthesis.
Lysosomal Biogenesis and Autophagy Are Enhanced in Hypoxic Fetal Kidneys The lysosome was the second organelle that seems to be enriched under hypoxic conditions. However, in comparison to mitochondria, where 36% of the proteins were reduced, almost all lysosome-related proteins were upregulated (Fig. 6, A and B). Among them were nine lysosomal acid hydrolases, representing nearly 20% of the lysosomal acid hydrolases annotated in the KEGG pathway for lysosomes: four proteases (CTSA, CTSF, CTSZ, TPP1), three glycosidases (GAA, NAGA, NEU1), the lysosomal acid phosphatase 2 (ACP2), and the lysosomal acid lipase A (LIPA). Furthermore, we found evidence for increased biogenesis of lysosomes and related organelles. Three of the eight BLOC1 (biogenesis of lysosome-related organelles complex 1) components were significantly induced (Fig. 6, C–E), as well as the H/Cl exchange transporter 5 (CLCN5), which is a crucial player in the acidification of endosomes (Fig. 6F). Of note, Snapin (BLOC1 subunit 7) also plays a role in lysosomal acidification and in autophagosome maturation and function. Other induced proteins known to play a role in autophagy were Atg7 and Bnip3 (Fig. 6, G and H). Another requirement for autophagic flux is the perinuclear clustering of lysosomes, mediated by the lysosomal Ragulator complex (35, 36) and two opposing motor protein families. Strikingly, four of the five members of the Regulator scaffolding subunits (LAMTOR1, 2, 3, and 5) were significantly induced (Fig. 6, A and B); LAMTOR4 was also 1.81-fold induced, but did not reach statistical significance. Furthermore, kinesins, which mediate the outward movement of organelles, showed a tendency to be repressed, whereas dynein family members that facilitate the inward movement were induced, although not statistically significant (supplemental Fig. S5). Collectively these findings provide strong evidence that the housekeeping function of lysosomes is enhanced in fetal hypoxic kidneys, fully compatible with the anticipated need to renew damaged mitochondria.
Premature
Aging Exemplifies the Janus-Faced Aspects of Hypoxic Adaptation Opposed to the compensatory repair and rejuvenation mechanisms, we found 15 of the deregulated proteins belonging to the GO term “Aging” (Fig. 7A), representing the third category of hypoxic adaptations: accelerated aging. While most of these proteins play a role in one of the processes described above neutrophil degranulation and lysosomes (MIF, MPO, PSEN1), mitochondria (NDUFS6, FADS1, MTCO1, CYP27B1), glycolysis (ALDOC), and DNA repair (OGG1); some have been described to directly effect on the life span of mice. In particular, the protein abundance of both klotho and sirtuin 6 was decreased in E18.5 hypoxic fetal kidneys (Fig. 7, B and C). Sirtuin 6 is a ubiquitously expressed enzyme with protein deacetylase and mono-ADP ribosyltransferase activity involved in the regulation of several cellular functions, including inflammation, glycolysis, and DNA repair. Its knockout leads to severe progeria in mice with a reduced life span of 1 to 3 months (37, 38). The kidney is the major site of klotho synthesis (i.e., distal convoluted tubule—DCT contributes the majority of the protein with additional synthesis in the proximal convoluted tubules), where it acts locally as a membrane-bound beta-glucuronidase. The reduced abundance of klotho seems to be a specific process since other proteins of the DCT including CALB1 were not altered. Klotho is also secreted into the circulation either by cleavage of the extracellular part of the membrane-bound form or by translation of an alternative splice variant. The level of circulating klotho (sKL) decreases with age (39, 40), which prompted us to assess its concentration and that of sirtuin 6 in trunk blood of hypoxic or normoxic E18.5 fetuses. Indeed, the concentrations of sKL and sirtuin 6 were significantly reduced in hypoxic E18.5 fetuses (Fig. 7, D and E). Furthermore and importantly, serum levels of klotho and sirtuin 6 were also still significantly reduced in aged mice (Fig. 7, F and G), indicating a permanent reduction of these two proteins throughout life. For Klotho, this seems to be due to significantly reduced mRNA expression levels in the kidneys of 15-month-old hypoxic offspring (Fig. 7H). However, renal mRNA expression levels of sirtuin 6 were unchanged between aged normoxic and hypoxic mice (Fig. 7I). Furthermore, changes in DNA methylation have been shown to be one of the most important mechanisms not only for nephron progenitor cell renewal and differentiation (41), but also for klotho expression (42, 43). However, in contrast to the hypermethylation of Mki67 (Fig. 3F), the klotho promoter was hypomethylated during chronic hypoxia (supplemental Fig. S6). The methylation pattern of Sirt6 could not be determined. To the best of our knowledge, our IUGR model is the first to delineate a mechanism leading to a premature aging phenotype, through synergy of inflammatory damage, ineffective repair, altered metabolism, and reduced abundance of antiaging proteins taking place at birth already.
The Reduction of Antiaging Proteins in Response to Chronic Hypoxia Is Conserved Between Mice and Men In a last set of experiments, we asked whether the interplay between chronic hypoxia and reduced serum levels of antiaging proteins is an evolutionarily conserved phenomenon. To this end, serum samples from a controlled study (10) of nine healthy volunteers (eight males, one female) were collected 2 weeks before (sea level, SL), at three-time points during an uninterrupted 28-day sojourn at 3454 m (high altitude, HA3, HA9, HA28), and 1, 7, and 14 days after their return to SL (RSL1, RSL7, RSL14) were analyzed for sKL and SIRT6 (Fig. 8). The samples obtained at SL served as controls for each participant. sKL and SIRT6 serum levels declined at high altitudes and both reached statistical relevance at H28. Upon return to sea level, sKL increased to levels higher than before the stay at high altitude at RSL7 and returned to normal at RSL14 (Fig. 8A). On the other hand, SIRT6 continued to decline upon return to sea level and only started to incline again at RSL14 (Fig. 8B), suggesting differential regulation of these two anti-aging proteins. In summary, these findings suggest that reduced serum klotho and sirtuin 6 levels may generally require exposure to chronic hypoxic conditions and may thus represent an evolutionary highly conserved process.
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