CXCL12 and MYC control energy metabolism to support adaptive responses after kidney injury

Abstract

Kidney injury is a common complication of severe disease. Here, we report that injuries of the zebrafish embryonal kidney are rapidly repaired by a migratory response in 2-, but not in 1-day-old embryos. Gene expression profiles between these two developmental stages identify cxcl12a and myca as candidates involved in the repair process. Zebrafish embryos with cxcl12a, cxcr4b, or myca deficiency display repair abnormalities, confirming their role in response to injury. In mice with a kidney-specific knockout, Cxcl12 and Myc gene deletions suppress mitochondrial metabolism and glycolysis, and delay the recovery after ischemia/reperfusion injury. Probing these observations in zebrafish reveal that inhibition of glycolysis slows fast migrating cells and delays the repair after injury, but does not affect the slow cell movements during kidney development. Our findings demonstrate that Cxcl12 and Myc facilitate glycolysis to promote fast migratory responses during development and repair, and potentially also during tumor invasion and metastasis.

Fig. 1

Pronephric ducts injured 1 day after fertilization fail to repair. a In situ hybridization for the pronephros-specific probe cadherin17 (cadh17) reveals that an injury applied 24 h after fertilization (24 hpf) is not repaired. The in situ hybridization was performed 24 h after injury. b Frames taken from a time-lapse movie of cldn2b:lyn-GFP transgenic embryo injured 30 hpf. Cells (blue and green asterisks) next to the gap exhibit little net movement. The gap persisted for 9 h after the injury. The red asterisk marks a somite border serving as a landmark. Tracking lines are color coded, ranging from blue to white (0–9 h) (scale bars, 10 µm). c Repair occurs in the absence of fluid flow. Frames were taken from a time-lapse movie injured first 1 day post fertilization (dpf) (green bracket), with two subsequent injuries (blue brackets) applied 2 dpf. Despite the lack of fluid flow both secondary injuries (blue brackets) recovered after 4 h. d Staining of cldn2b:lyn-GFP transgenic zebrafish embryos wounded at 1 or 2 dpf, fixed 30 min post wounding, and stained for phospho-myosin light chain-2 (MyoP). Activated myosin was detected at the edges of the pronephros injury in 1-day old embryos (red arrows), but not in 2-day-old embryos (white arrows). The bottom panel of each image shows the phospho-myosin levels using a color-coded range indicator (scale bars, 100 µm). e Staining of cldn2b:lyn-GFP transgenic zebrafish embryos wounded 1 or 2 dpf, fixed 4 h after wounding, and stained with phalloidin for actin. Actin staining was detected on the apical surface of cells close to the injury in both 1-day and 2-day old embryos. Actin staining was more prominent in 1-day embryos, and appeared perpendicular to the duct lumen (red arrows) (scale bars, 100 µm). f Quantification of the pixel intensity within the marked region of a single representative confocal plane from the MyoP staining (middle panel) of an embryo injured 1 dpf. The histogram depicts two peaks with increased levels of phosphorylated myosin adjacent to the injury. The bottom panel represents the merged image of GFP/MyoP staining

Fig. 2

Pronephric duct injuries are repaired by migration. a Frames from Supplementary Movie 1, showing recovery after ablation of a pronephric duct fragment (ablated cells are marked in red). The free ends of the duct move towards each other, whereby the leading cells extend protrusions and exhibit active cell migration. The bottom panel depicts tracking of individual cells over time. Tracking lines are color coded, ranging from blue to white (0–9 h); the green arrows summarize migration direction and cell displacement (scale bars, 10 µm). b Frames from Supplementary Movie 2. The panel depicts a two-sided cell ablation (red-labeled cells), isolating a patch of pronephric cells. The free ends of the isolated duct migrated in opposite direction, stretching the isolated segment. Whereas the cells in the terminal regions exhibited prominent net displacement, the cells in the middle segment remained almost stationary. Tracking lines are color coded, ranging from blue to white (0–9 h) (scale bars, 10 µm). c To compare repair response and collective cell migration, 2-day-old Tg(-8.0cldnb:LY-EGFP), TgBAC(cxcr4b:h2b-RFP) transgenic zebrafish embryos were mounted on the side to image pronephric ducts by confocal microscopy. The pronephros was injured with a two-photon laser at 36 h, and track speed (left panel) (p = 0.0023, t-test) and track displacement length (right panel) (p = 0.0034, t-test) was measured over 2 h. After injury, tubular epithelial cells adjacent to the injury almost tripled their speed. The cell number (n) was derived from three control and six injured pronephric tubules

Fig. 3

cxcr4b or cxcl12a deletion does not affect pronephros cell migration. a Collective cell migration in the zebrafish pronephros was quantified, using a cxcr4b:H2B-RFP; cldnb:GFP double transgenic zebrafish line (upper panel, see Supplementary Movie 4). The transgene cxcr4b:H2B-RFP labels the nuclei of cells that express RFP under the control of the endogenous cxcr4b promoter; cldnb:GFP labels the membranes of pronephric duct cells. The pronephros and the corpuscle of Stannius are outlined with dashed lines. The arrow points to the corpuscle of Stannius (scale bars, 10 µm). A model of collective cell migration was generated (lower panel), using the image tool Imaris. The migration of ca. 40 pronephric nuclei was tracked over 8 h, starting 48 hpf. The tracks for each cell were calculated and represented as lines. The observation times were coded by blue-to-green colors; blue are early time points and green are later ones (see Supplementary Movie 5). b The mean track speed and displacement length, i.e., the length that a cell traveled over the observation period were measured at 36 and 41 hpf for 3 h, and at 48 hpf for 8 h. Deficiency of cxcr4b (left panel) or cxcl12a (right panel) did not affect track speed (left panel). c Deficiency of cxcr4b (left panel) or cxcl12a (right panel) did not affect displacement length. Note that the displacement length is larger in the 48–56-hour-interval, because cells were tracked for a longer time period (8 h) in comparison with the earlier time points (3 h each). In contrast, the speed did not change significantly over time (n.s., not significant; t-test)

Fig. 4

Defective repair in cxcl12a (−/−) and cxcr4b (−/−) zebrafish embryos. a While control zebrafish embryos repair a lased-induced injury, more than 60% of cxcr4b-deficient zebrafish embryos (means ± SEM; ***p < 0.001; t-test) and b more than 40% of cxcl12a-deficient zebrafish embryos did not repair the wound, but instead revealed various abnormalities (means ± SEM; **p < 0.01; t-test). c The pronephros of 2-day old wild-type cldnb:GFP control zebrafish embryos rapidly regenerates after a laser-induced injury. The arrow points to the regenerated area at 24 h after the injury. The arrowhead points to the cloaca. The pronephros is outlined with dashed lines (scale bar, 100 µm). d Magnification of the regenerating region depicted in c (scale bar, 20 µm). e cxcl12a (−/−) zebrafish embryos fail to regenerate and the pronephric tubule remains discontinued. The arrow points to the gap in the tubule. The pronephros is outlined with discontinued lines. The arrowhead points to the cloaca. Note the dilation of the anterior duct due to ongoing filtration (scale bar, 100 µm). f Magnification of the region that failed to regenerate depicted in d (scale bar, 20 µm). g In some cases, cxcl12a mutant embryos showed aberrant regeneration and formed dorsal pouches. The arrow points to the abnormally regenerated tubule. The pronephros is outlined with discontinued lines. The arrowhead points to the cloaca (scale bar, 100 µm). h Magnification of the abnormally regenerated region depicted in e (scale bar, 20 µm). i cxcr4b mutant zebrafish embryos failed to re-establish the tubular patency similar to cxcl12a mutant embryos. The arrow points to the gap in the tubule. The pronephros is outlined with discontinued lines. The arrowhead points to the cloaca (scale bar, 100 µm). j Magnification of the region that failed to regenerate depicted in f (scale bar, 20 µm)

Fig. 5

Cxcr4b expression in response to injury. a The GFP-RFP-tagged Cxcr4b (cxcr4b:cxcr4b-tFT) was up-regulated in cells adjacent to the laser-induced injury. The arrowhead points to the corpuscle of Stannius. Arrows point to RFP-positive cells, accumulating distally after the injury. Sequential frames were taken from a time-lapse video at t1 = 96 min, t2 = 120 min, and t3 = 160 min after injury (scale bar, 20 µm). b In situ hybridization revealed up-regulated cxcr4b expression in the leading ends of the regenerating tubule 2 h after laser-induced ablation. The cxcr4b up-regulation is outlined in a dashed box. Note that cxcr4b is strongly expressed in the corpuscle of Stannius (arrow) and in the lateral line. c Magnification of the injury region depicted in b. The white arrows point towards the up-regulated cxcr4b, the red arrowhead towards the corpuscle of Stannius

Fig. 6

Cxcl12 or Myc deletion impairs the recovery after injury. a Male mice with the genotype Cxcl12^fl/fl*Pax8rtTA*TetOCre (n = 5) and control mice (n = 5) with the genotype Cxcl12^fl/fl*Pax8rtTA were subjected to I/R injury. Histology and biochemical analysis were performed 12 h later. b Serum urea concentrations were significantly higher in mice lacking Cxcl12 (*p < 0.05). c Urinary NGAL excretion, normalized for urinary creatinine, was significantly higher in mice lacking Cxcl12 than in the control animals (means ± SEM; *p < 0.05; t-test). d PAS-stained kidney sections from Cxcl12^fl/fl*Pax8rtTA (controls) and Cxcl12^fl/fl*PAX8rtTA*TetOCre mice were obtained 12 h after I/R injury. Squares labeled 1 and 3 show enlarged images of the cortex, squares labeled 2 and 4 show enlarged images of the cortico-medullary junction. e Male mice with the genotype Myc^fl/fl*Pax8rtTA*TetOCre (n = 5) and control mice (n = 5) were subjected to I/R injury. Histology and biochemical analysis were performed 12 h later. f Serum urea concentrations were significantly higher in mice lacking Myc (*p < 0.05). g Urinary NGAL excretion, normalized for urinary creatinine, was significantly higher in mice lacking Myc than in the control animals (means ± SEM; *p < 0.05; t-test). h PAS-stained kidney sections from Myc^fl/fl*Pax8rtTA (controls) and Myc^fl/fl*PAX8rtTA*TetOCre mice were obtained 12 h after I/R injury. Squares labeled 1 and 3 show enlarged images of the cortex, squares labeled 2 and 4 show enlarged images of the cortico-medullary junction (scale bars, 1 mm and 100 µm)

Fig. 7

Retinoic acid accelerates the recovery after injury. a GO term analysis of Cxcl12^fl/fl- and Myc^fl/fl*Pax8rtTA*TetOCre compared to control mice revealed suppressed retinol acid metabolic and retinol metabolic processes, respectively; the individual genes of the GO term are depicted. b Myc^fl/fl*PAX8rTA/TetOCre (KO) (n = 13) and Myc^fl/fl*PAX8rTA (control animals) (n = 7), lacking the Cre recombinase were treated with doxycycline for 2 weeks, followed by a washout period of 1 week. Tretinoin (RA, 40 mg/kg) was applied 12 h before and during the ischemia/reperfusion injury to seven control and six KO mice. Eight KO mice were injected with an equal volume of cottonseed oil, only (solvent). One of the untreated animals was removed from further analysis due to bleeding and incomplete clamping. Urea levels were measured 24 h after surgery. Differences between mice treated with RA and untreated animals were statistically significant (means ± SEM; *p < 0.05; t-test), while there were no differences between RA-treated control and Myc KO mice. c Histology of the cortical and medullary region revealed significant tubular injury with intraluminal debris (*) and casts (#) in all three groups after ischemia/reperfusion injury (scale bars, 1 mm and 100 µm)

Fig. 8

Key metabolite changes after Cxcl12 or Myc deletion. a While lactate and glucose were elevated after ischemia/reperfusion (I/R) injury in the urine of wild-type animals (red box), leucine/isoleucine, α-hydroxy-isovalerate and citrate were elevated in the urine of knockout (KO) animals. b In humans, approximately 20 mmol of citrate are filtered daily; 70–90% of the filtered citrate are reabsorbed by di- and tricarboxylate transporters (e.g., NaDC1/SLC13A2), and metabolized by mitochondria. Slowing entry of citrate into mitochondria due to mitochondrial dysfunction reduces tubular citrate uptake, contributing to increased urinary citrate concentrations^, . c Lactate production is rapidly increased during renal ischemia due to enhanced glycolysis. The efflux of lactate drives glycolysis and maintains intracellular pH, increasing urinary lactate concentrations. Defective glycolysis results in reduced lactate production and lactate efflux in KO animals. Gluconeogenesis is enhanced during early reperfusion, resulting in increased glucose production and urinary glucose concentrations in wild-type mice, while defective pyruvate and lactate production during ischemia prevents gluconeogenesis in KO animals. d Knockout of Cxcl12 or Myc compromises mitochondrial catabolism of branched-chain amino acids (e.g., valine), resulting in increased accumulation of α-hydroxy-isovalerate. The transamination by branched-chain amino acid transaminase (BCAT) as well as the conversion to α-hydroxy-isovalerate by lactate dehydrogenase occur in the cytoplasm, while the rate-limiting step of aerobic catabolism of branched-chain amino acids (valine, leucine, isoleucine) by branched-chain alpha-keto acid dehydrogenase (BCKDH) occurs in mitochondria. Thus, defective mitochondrial functions cause increased leucine, isoleucine, and α-hydroxy-isovalerate concentrations in the urine

Fig. 9

Inhibition of glycolysis slows migration. a Depicted are frames from Supplementary Movie 10, demonstrating the analysis of migration speed in the posterior Lateral Line Primordium (pLLP). A similar imaging approach was used to determine the speed of migrating cells in the zebrafish pronephros. b The small molecule 3-(3-pyri-dinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) at concentrations that had no recognizable developmental effects (20 µM) delayed pLLP migration, but did not affect pronephros cell migration. The box plots represent the median, the first and third quartile (boxed area), and 1.5× interquartile range (whiskers) (n.s., not significant; t-test). c 3PO delayed the repair of a pronephros injury determined at 6 h, but no effect was observed 24 h after the injury (Fisher’s exact test). d The glucose analog 2-deoxy-D-glucose (2-DG) at 40 µM slowed pLLP migration similar to 3PO, but had no effect on pronephros collective cell migration. The box plots represent the median, the first and third quartile (boxed area), and 1.5× interquartile range (whiskers) (n.s., not significant; t-test). e While almost all injuries were repaired 6 h after wounding of control embryos, 40 µM 2-DG reduced the number of repaired injuries to 70% and 80 µM 2-DG to 58%. At 24 h after wounding, all injuries were repaired. Full bars depict not repaired injuries, open bars repaired injuries. The graphs represent the summary of at least three independent experiments (Fisher’s exact test)