Mitochondrial Pathology and Glycolytic Shift During Proximal Tubule Atrophy After Ischemic AKI

BASIC RESEARCH www.jasn.org

Mitochondrial Pathology and Glycolytic Shift during Proximal Tubule Atrophy after Ischemic AKI

† Rongpei Lan,* Hui Geng,* Prajjal K. Singha,* Pothana Saikumar,* Erwin P. Bottinger, ‡ Joel M. Weinberg, and Manjeri A. Venkatachalam*

*Department of Pathology, University of Texas Health Science Center, San Antonio, Texas; †Department of Medicine, Mount Sinai School of Medicine, New York, New York; and ‡Department of Medicine, Veterans Affairs Ann Arbor Healthcare System and University of Michigan Medical Center, Ann Arbor, Michigan

ABSTRACT During recovery by regeneration after AKI, proximal tubule cells can fail to redifferentiate, undergo premature growth arrest, and become atrophic. The atrophic tubules display pathologically persistent signaling increases that trigger production of profibrotic peptides, proliferation of interstitial fibroblasts, and fibrosis. We studied proximal tubules after ischemia-reperfusion injury (IRI) to characterize possible mitochondrial pathologies and alterations of critical enzymes that govern energy metabolism. In rat kidneys, tubules undergoing atrophy late after IRI but not normally recovering tubules showed greatly reduced mitochondrial number, with rounded profiles, and large autophagolysosomes. Studies after IRI of kidneys in mice, done in parallel, showed large scale loss of the oxidant–sensitive mitochondrial protein Mpv17L. Renal expression of hypoxia markers also increased after IRI. During early and late reperfusion after IRI, kidneys exhibited increased lactate and pyruvate content and hexokinase activity, which are indicators of glycolysis. Furthermore, normally regenerating tubules as well as tubules undergoing atrophy exhibited increased glycolytic enzyme expression and inhibitory phosphorylation of pyruvate dehydrogenase. TGF-b antagonism prevented these effects. Our data show that the metabolic switch occurred early during regeneration after injury and was reversed during normal tubule recovery but persisted and became progressively more severe in tubule cells that failed to redifferentiate. In conclusion, irreversibility of the metabolic switch, taking place in the context of hypoxia, high TGF-b signaling and depletion of mitochondria characterizes the development of atrophy in proximal tubule cells and may contribute to the renal pathology after AKI.

J Am Soc Nephrol 27: ccc–ccc, 2016. doi: 10.1681/ASN.2015020177

After death of proximal tubule cells by AKI, surviving N–terminal kinase–MAPK, PI3K, and TGF-b path- epithelial cells dedifferentiate, migrate, and prolifer- ways; decreased PTEN; increased expression of Akt ate. Recovery of normal structure and function and c-Jun, Smad2 and Smad3, TGF-b receptors 1 and occurs by redifferentiation of reconstituted epithe- 2, LPA receptors 1 and 2, lysophospholipase D, and lium.1 However, to varying degrees, proximal tubule cells proliferating after AKI fail to redifferentiate, undergo premature growth arrest, and become atrophic.2–6 Paradoxically, the atrophic epithelium con- Received February 16, 2015. Accepted February 3, 2016. tinues to signal through multiple pathways activated R.L. and H.G. contributed equally to this work. early during proliferation. The pathologically persis- Published online ahead of print. Publication date available at tent signaling increases progressively, triggering pro- www.jasn.org. duction of profibrotic peptides, proliferation Correspondence: Dr. Manjeri A. Venkatachalam, Department of fi fi 2–6 of interstitial broblasts, and brosis. The pheno- Pathology, University of Texas Health Science Center, 7703 Floyd type of failed differentiation of tubules includes Curl Drive, San Antonio, TX 78229. Email: venkatachal@uthscsa. vicarious signaling through EGF receptor (EGFR), edu extracellular signal–regulated kinase–MAPK, c-Jun Copyright © 2016 by the American Society of Nephrology

J Am Soc Nephrol 27: ccc–ccc, 2016 ISSN : 1046-6673/2710-ccc 1 BASIC RESEARCH www.jasn.org b6-integrin; and enhanced production of TGF-b, PDGF-B, and and B). Moreover, atrophic tubules showed large complex au- CTGF.2–7 tophagolysosomes containing degenerate mitochondria, pro- Why regenerating proximal tubules become atrophic after files of partially degraded endoplasmic reticulum, and myelin AKI is unknown. However, a metabolic switch could be in- figures (Figure 1C, Supplemental Figure 1). TGF-b receptor volved. Transport functions in proximal tubules require high antagonist SD208 promotes tubule differentiation during re- turnover of ATP derived almost exclusively through mito- generation after IRI and prevents tubule atrophy,2,4 which is chondrial oxidative phosphorylation.8,9 After ischemic AKI, illustrated in this work by the morphologic phenotype of dif- regenerating proximal tubule cells assume simplified structure ferentiation instead of atrophy in proximal tubules of SD208- as they dedifferentiate. These changes are pronounced in treated rats (SD panels in Figures 1, 2, 4, 5, and 8). Figure 1D tubules that fail to redifferentiate and become atrophic.4 Con- shows that promotion of differentiation by SD208 is accom- ceivably, metabolic alterations are part of the dedifferentiation panied by greater mitochondrial number and normal mito- program that, if not reversed, leads to tubule atrophy. chondrial morphology. Immunohistochemistry (IHC) for Alterations of energy metabolism occur in models of AKI, mitochondrial markers Tom20 (Figure 1) or AIF (not shown) including diminished fatty acid oxidation during folic acid revealed drastic reduction of mitochondrial mass during tu- nephropathy,10 increased glycolysis after mercuric chloride bule atrophy after IRI (Figure 1F) compared with normally AKI,11 increased lactate release into kidney interstitium after recovering tubules (Figure 1E) and tubules of SD208-treated ischemic AKI,12 and elevated pyruvate kinase in kidney ho- rats (Figure 1G). Immunofluorescence confirmed that atro- mogenates after ischemia-reperfusion injury (IRI).13 phic tubules with less mitochondria expressed vimentin (Fig- Although these changes could be components of physiologic ure 1H), the marker that indicates early dedifferentiation as metabolic alterations in regenerating epithelium, they could well as late atrophy of regenerating proximal tubules.4,22–25 become pathologic if not reversed. On the basis of this hy- Severe loss of mitochondrial mass during tubule atrophy pothesis, we studied proximal tubules after ischemic AKI was documented by low-power microscopy covering large with a view to characterize possible mitochondrial pathologies areas of kidney parenchyma (Figure 1, I–K) and semiquanti- and alterations of critical enzymes that govern energy metab- tative grading of mitochondrial granule density (Figure 1L). olism. We found that proximal tubules undergoing atrophy Early dedifferentiation during regeneration of surviving but not tubules recovering normally late after IRI display striking proximal tubules after AKI is marked by massive physiologic mitochondrial alterations, increased glycolysis and loss of mitochondria,26–28 presumably by autophagy. There- glycolytic enzyme expression, and inhibitory pyruvate dehy- fore, redifferentiation during tubule recovery axiomatically drogenase (PDH) phosphorylation. These alterations were requires substantial mitochondrial biogenesis.29 However, accompanied by expression of hypoxia markers and prevented some tubules failed to redifferentiate instead and became atro- by TGF-b antagonism. Hypoxia can trigger TGF-b, and both phic, with persistently low mitochondrial mass and abnormal hypoxia and TGF-b are known to induce glycolysis and mito- autophagy. These findings raised the possibility that a mitochondrial oxidant species in proximal tubule cells and other chondrial pathology had developed that prevented epithelial cell types.14–21 Structural, signaling, and metabolic alterations recovery. Because TGF-b is involved in the pathogenesis of are not unexpected after AKI and may be required for early postischemic tubule atrophy,2,4 we assessed Mpv17L, a mito- tubule regeneration and adaptation. Indeed, they may have chondrial protein sensitive to TGF-b–induced oxidant stress16 protective roles during the early recovery phase. However, that becomes decreased in tubules during TGF-b–induced such modifications should be reversed later by redifferentia- CKD.16,30 Because mouse–directed Mpv17L antibody16 was tion. Our data provide support for a metabolic switch that unreactive with rat antigen, we performed IRI in C57BL/6 takes place physiologically during early regeneration after AKI mice using a protocol similar to that of our rat model.2 De- that progresses in tubules that fail to redifferentiate. The mag- velopment of early fibrosis 14 days after IRI was accompanied nitude, persistence, and progression of mitochondrial and by markedly reduced Mpv17L in kidney lysate (Supplemental metabolic pathologies suggest that they are important com- Figure 2A) and in proximal tubule cells (Supplemental Figure 2B). ponents of proximal tubule atrophy with possible roles in its pathogenesis. IRI Is Followed by Hypoxia Marker Expression That Persists in Tubules as They Become Atropic IRI was followed by enhanced expression of hypoxia–inducible RESULTS factor 1a (HIF1a) as reported before,31,32 remaining persistently increased in kidney lysates until 14 days; kidneys of Mitochondrial Pathology in Atrophic Tubules SD208-treated rats showed modest decrease of HIF1a induc- As we have reported in detail previously, proximal tubules with tion (Figure 2A). There also was increased expression of car- failed differentiation and atrophy after IRI exhibited flat bonic anhydrase 9 (CA9), the hypoxia marker that is directly simplified cytoplasm.4 In such tubules, mitochondria were transcribed by HIF133 (Figure 2A). CA9 showed convincing greatly reduced in number and of smaller size with round increase by Western blotting only at 14 days after IRI in the profiles, unlike tubules that recovered normally (Figure 1, A experiments depicted by Figure 2A. However, CA9 was found

2 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc,2016 www.jasn.org BASIC RESEARCH

to be significantly increased by IHC in regenerating proximal tubules 7 days after IRI, which persisted in atrophic tubules at 14 days, with only modest decreases of the marker in SD208- treated kidneys (Figure 2B–E). Moreover, a separate series of experiments (n=3) showed significant increases at 3, 7, and 14 days after IRI (not shown). Thus, hypoxia, a major driver of glycolysis, was found to affect tubules regenerating after IRI and also, atrophic tubules late after IRI, when fibrosis develops.

Enhanced Kidney Glycolysis Associates with Increased Glycolytic Enzyme Expression in Regenerating Proximal Tubules That Increases Further in Atrophic Tubules Glucose uptake and lactate production by kidneys were reported to be increased over several days after HgCl2-induced AKI in rats coinciding with tubule regeneration accompanied by enhanced glucose phosphorylating activity.11 Kidneys were reported to release more lactate and exhibit increased activities and mRNA content for pyruvate kinase M2 (PKM2; or M2-PK), with com- plementary decreases for pyruvate kinase liver/RBC isoform (PKLR; or L-PK) during early reperfusion after IRI.12,13 We have provided evidence for increased glycolysis in kidneys recovering from IRI. During reperfusion 3, 7, and 14 days after IRI, lactate and pyruvate concentrations in cortex and the outer stripe of outer medulla were increased relative to time controls, and at 7 and 14 days, this was accompanied by increased hexokinase activity (Figure 3). By Western blotting, there was early enhancement and pronounced and progressive late increases in protein expression of rate-limiting enzymes for glycolysis after IRI: hexokinase 2 (HK2), phosphofructokinase platelet isoform (PFKP), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), and PKM2 (Figure 4A). However, PKLR that is expressed by normal proximal tubules34,35 became depleted after an early increase 8 hours after reperfusion (Figure 4A). SD208 treatment largely reversed these changes observed by Western blotting with two exceptions—for PFKP and PKM2, which showed persistent increase, despite TGF-b antagonism (Figure 4A) (see below for results by IHC that do show decrease of both PFKP and PKM2 in SD208–treated IRI kidneys). By

sham left kidney ischemia, (F and H) IRI kidneys, and (G) SD208– protected IRI kidney 14 days after IRI. Tom20 is visualized brown by IHC in E–G and red by immunofluorescence in H. Vimentin, shown in green, indicates dedifferentiation/atrophy of epithelium and is also present in interstitial fibroblasts. Note the abundant red staining of Tom20 in the differentiated vimentin–negative tubule (upper left) but significantly less red staining in flat, undifferentiated vimentin–positive (green) tubules (upper right and lower right) in H. I–K show low-power micrographs of Tom20 staining by IHC in Figure 1. Mitochondrial pathology after IRI. (A–D) Electron mi- kidneys of rats (I) without IRI, (J) with IRI treated with vehicle only, crographs showing (A) normal recovery, (B) atrophy with loss and and (K) with IRI treated with SD208 14 days after surgery. L shows simplification of mitochondria, (C) autophagolysosome associ- results for semiquantitative grading of Tom20 staining by IHC (n=4 ated with atrophy, and (D) SD208-induced recovery in proximal for each group). DAPI, 49,6-diamidino-2-phenylindole; NCtrl, tubule epithelium 14 days after IRI. (E–L) IHC for mitochondrial nephrectomized control; Veh, vehicle; Vim, vimentin. Scale bar, marker Tom20 in (E) kidneys of nephrectomized control with 1 mminA–D; 50 mminE–H; 100 mminI–K. *P,0.01.

J Am Soc Nephrol 27: ccc–ccc, 2016 Metabolic Switch during Proximal Tubule Atrophy 3 BASIC RESEARCH www.jasn.org

for PKM2 in Supplemental Figure 3). Im- munofluorescence confirmed the specificity of the strikingly increased glycolytic enzyme in atrophic proximal tubule epithelium. In mosaic tubules lined partially by well differentiated and atrophic epithelium,4 vimentin–positive atrophic cells but not well differentiated cells with intact PHAE-lectin staining brush borders expressed PKM2 (Figure 6). In a notable exception, proximal tubules that had recovered normal structure after SD208 treatment also expressed PKM2 focally, albeit to a lesser degree (Figure 5A), presumably accounting for persistently increased PKM2 seen by Western blotting in SD208-treated kidneys (Figure 4A). IHC for PKLR confirmed the decrease of this enzyme during reperfusion seen by Western blotting; atrophic tubules, but not well differentiated tubules, showed the decrease of PKLR protein expression (Figure 5B). Notably, fibroblasts that had proliferated in the interstitium around atrophic tubules also showed increased glycolytic enzyme expression, most pronounced for PKM2 (Figures 5A and 6). Treatment with SD208 largely prevented these alterations (Figures 4 and 5, Supplemental Figure 3). Strikingly, PKM2 colocalized with hypoxia marker CA9 in atrophic tubules (Supplemental Figure 4). In varying degrees, distal nephron segments but not proximal tubules in normal kidneys expressed glycolytic enzymes as might be expected from measurements of enzyme ac- Figure 2. Expression of hypoxia markers after IRI. (A) Western blotting of kidney lysates tivities in microdissected nephrons and func- for HIF1a and CA9 in normal control, 14-day nephrectomy sham left kidney ischemia 8,9,36 control, kidneys at serial time intervals up to 14 days of IRI, and kidney 14 days after IRI tional assays. Also in varying degrees, with SD208 treatment. In this blot, CA9 appears increased only after 14 days of IRI. A proximal tubules that recovered normally af- separate series of experiments (n=3) showed significant increases at 3, 7, and 14 days after ter IRI and proximal tubules from kidneys of IRI (not shown). (B–E) IHC for CA9 in nephrectomy control, kidneys 7 and 14 days after IRI, sham ischemia controls that received contra- and SD208-treated kidney 14 days after IRI. (B) Compared with the time control 14 days lateral nephrectomy showed modest in- after nephrectomy (NCtrl 14d), there is markedincreaseofstainingforCA9in(C)re- creases of glycolytic enzyme expression, generating tubules (with flat epithelium) and fibroblasts proliferating in markedly widened never approaching the dramatic increases interstitium of kidneys of rats treated with vehicle only 7 days after IRI (IRI 7d + Veh) and (D) seen in atrophic tubules (Figures 4 and 5). flat atrophic epithelium of tubules in a fibrotic area of rat kidney treated with vehicle only 14 days after IRI (IRI 14d + Veh). The corresponding staining of kidney tubules from kid- Hypoxic Induction of Glycolytic neys of rats treated with SD208 14 days after IRI (IRI 14d + SD) in (E) is reduced only fi Enzymes in Cultured Proximal Tubule modestly. These IHC ndings are representative of a series of four separate experiments. a ctrl, Control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NCtrl, nephrectom- Cells Involves HIF1 ized control; neph, nephrectomy; Veh, vehicle. Scale bar, 100 mm. Proximal tubules regenerating after IRI are subjected to chronic hypoxia.37–39 Hypoxic tubule microenvironments were shown by IHC, there was increased expression of hexokinase 2, PFKP, pimonidazole adduct IHC early after reperfusion as well as PFKFB3, and PKM2 in regenerating tubules 7 days after IRI, late, when tubules become atrophic and fibrosis de- with striking further increases in atrophic tubules at 14 days velops.37,39,40 Increased hypoxia marker CA9 in atrophic tu- (Figures 4–6). Increases were seen also at 3 and 5 days (shown bules (Figure 2B, Supplemental Figure 4) suggested that

4 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc,2016 www.jasn.org BASIC RESEARCH

data suggest or HIF1a-dependent processes as proposed,44 these results highlight a fundamentally important effect of hypoxia in proximal tubule cells—the potential for suppressing the gatekeeper function of PDH in permitting pyruvate entry into the Krebs cycle for mitochondrial oxidative metabolism. Fatty acid oxidation also supplies acetyl- CoA substrate for the Krebs cycle. We, therefore, asked if carnitine palmitoyl transferase 1, the rate-limiting enzyme for mitochondrial fatty acid import, is affected by hypoxia. Expression of CPT1A, the isoform significant for fatty acid metabolism in kidney, was strongly suppressed by hypoxia in an HIF1a-independent manner (data not shown), but we obtained conflicting findings in vivo after IRI (see below).

PDHE1a S293 Phosphorylation Is Figure 3. Renal pyruvate and lactate content and hexokinase activity are increased Increased during Late Regeneration after IRI. Concentrations of pyruvate and L-lactate and activity of hexokinase in kidney cortex and outer stripe of outer medulla 3, 7, and 14 days after IRI and corresponding and during the Development of time controls (n=3 for each group). Ctrl, control. *P,0.05; **P,0.01. Atrophy in Tubules Regenerating after IRI By Western blotting, phospho-PDHE1a increased glycolytic enzymes in atrophic tubules (Figures 4–6) (S293) showed early decreases of overall signal in kidney lysates were induced by hypoxia, a possibility consistent with a role for for several days after IRI followed by a slight increase after 14 HIF1a in controlling metabolism.41,42 As such, we examined the days; SD208 treatment did not reduce the signal compared with effects of shRNA HIF1a knockdowninculturedproximaltubule that in IRI rats that received vehicle alone, whereas the corre- cells. Exposure to 0.5% O2 for 48 hours increased the expression of sponding 14-day nephrectomy control with sham ischemia also PFKP,PFKFB3,PKM2,andPKLR;HIF1a knockdown abrogated showed a modest increase. PDHE1a remained unaltered the hypoxic effect for PFKP, PFKFB3, and PKM2 but increased throughout (Figure 8). By IHC, the signal for phospho- further the expression of PKLR (Figure 7A). These data showed PDHE1a was correspondingly reduced in regenerating tubules that PKLR, the pyruvate kinase isoform normally expressed by 3 days after IRI (not shown). Subsequently, the signal increased proximal tubules, is regulated differently relative to PKM2. but still remained low (not shown). However, at day 7, strong staining was seen in regenerating tubules (Figure 8). After 14 Hypoxia Induces Phosphorylation at the Inhibitory days of IRI, when tubules were atrophic and surrounded by S293 Site of PDH E1a-Subunit in Cultured Proximal fibrosis, the atrophic tubule epithelium showed markedly in- Tubule Cells creased signal for phospho-PDHE1a far exceeding the control; Entry of pyruvate into the Krebs cycle is controlled by PDH. signals in tubules in SD208-treated rats were significantly lower Phosphorylation of PDH by pyruvate dehydrogenase kinases than those in rats with vehicle alone (Figure 8). The specificity of (PDKs) at inhibitory sites S232, S293, and S300 of pyruvate increased phospho-PDHE1a signal in atrophic tubule epithe- dehydrogenase E1a–subunit (PDHE1a) inhibits PDH activity lium was clearly shown by sharp transitions of high to low signal and thereby, suppresses mitochondrial metabolism by de- from atrophic to differentiated epithelium (arrows in Figure 8, creasing the production of acetyl-CoA.43–45 Hypoxia induces upper panel), paralleling similarly sharp transitions for PKM2 PDKs.44,45 It was reported that hypoxic activation of HIF1 (Figure 6). induces PDK1 in mouse embryo fibroblasts; phosphorylation Because hypoxia suppressed CPT1A in cultured proximal of PDHE1a then inhibits PDH to slow the Krebs cycle and tubule cells, we assessed CPT1A protein after IRI. Western decrease mitochondrial oxidant stress.44 We found that expo- blotting of lysates showed no alterations of expression sure of cultured proximal tubule cells to 0.5% O2 for 48 hours throughout 14 days after IRI; by IHC, staining was strong in induced the S293 phosphorylation of PDHE1a,whichwas, distal nephron segments but weak in proximal tubules of however, not decreased by HIF1a knockdown (Figure 7B). normal controls before IRI. Counterintuitively, with progres- Regardless of whether hypoxic PDHE1a phosphorylation sion of time during reperfusion, staining in regenerating was mediated by HIF1a-independent mechanisms as our proximal tubules showed increase rather than decrease (not

J Am Soc Nephrol 27: ccc–ccc, 2016 Metabolic Switch during Proximal Tubule Atrophy 5 BASIC RESEARCH www.jasn.org

Figure 4. Glycolytic enzyme protein expression in kidney tissue and tubules after IRI. (A) Western blotting of kidney lysates for hexokinase 2 (HK2), PFKP, PFKFB3, PKM2, and PKLR. Protocol and conditions are as outlined in Figure 2A. (B–D): IHC for (B) HK2, (C) PFKP, and (D) PFKFB3 in nephrectomy control, kidneys 7 and 14 days after IRI, and kidneys with SD208 treatment 14 days after IRI. Results for HK2 and PFKP are representative of four separate experiments. The accentuation of staining for PFKFB3 at the apical membranes of tubules seen after IRI in addition to increased cytoplasmic staining is not an artifact as shown by control IHC studies and the absence of such staining in the apical membranes of adjacent normally redifferentiating tubules as well as control tubules. ctrl, Control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NCtrl, nephrectomized control; neph, nephrectomy; Veh, vehicle. Scale bar, 100 mm. shown). Similarly, we assessed PGC1a, a regulator of mito- They also provide additional perspective on the phenotype of chondrial biogenesis, and PPARa; both of these transcription failed differentiation that often develops in such tubules and factors regulate fatty acid metabolism.29 Neither protein that, importantly, contributes to chronic progression. Cells of showed alterations post-IRI (not shown). this unique phenotype exhibited greatly diminished numbers of mitochondria, altered mitochondrial shape and structure, and abnormally large autophagic bodies. Decrease of a mito- DISCUSSION chondrial protein that is sensitive to TGF-b–induced oxidant stress, Mpv17L, suggested that mitochondria suffer free radi- Our ﬁndings provide pathologic and biochemical basis for a cal damage during tubule atrophy. Furthermore, increased metabolic switch in proximal tubules regenerating after AKI. phosphorylation of PDH, the rate-limiting enzyme for

6 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc,2016 www.jasn.org BASIC RESEARCH pyruvate entry into the Krebs cycle, indicated that oxidative cycle) decreased during ischemia and remained decreased un- metabolism of pyruvate carbons derived from lactate, amino til 18 hours of reperfusion. Indeed, pyruvate infusions protec- acids, and glucose must become inhibited in the fewer mito- ted against ischemic AKI. Differences between these findings chondria that remained. In the other arm of the switch, kidney and the concurrent increases of pyruvate and lactate that we tissue glycolysis assessed as pyruvate and lactate content and found during IRI are likely attributable to timing—our mea- hexokinase activity was enhanced, and the expression of key surements were done at and after 3 days of IRI when tubules enzymes that are rate limiting for glycolysis was markedly are dedifferentiated, regenerating and expressing more glyco- increased in the same tubules that showed advanced mito- lytic enzymes, in contrast to in the study by Zager et al.,18 in chondrial pathology. The switch was remarkably cell specific. which differentiated proximal tubules with predominantly ox- Mitochondrial pathology and increased glycolytic enzyme ex- idative metabolism were in the earliest stages of active injury pression affected vimentin–positive whole tubules, clusters of and cell death (up to only 18 hours of reperfusion after ische- cells, and even single cells sharply demarcated from normally mia). In other studies, basal levels of lactate released normally recovering neighbors (shown for PKM2 in Figure 6). Together into the interstitium of renal cortex were found to be much with older data showing increased renal glucose utilization for lower than those in the medulla, but these levels became lactate production during tubule regeneration after toxic in- increased during ischemia and reperfusion.12 Such measure- jury,11 our results suggest that energy metabolism in proximal ments cannot be ascribed to the production of lactate by tubules becomes profoundly altered as they dedifferentiate, proximal tubules alone, because vigorously glycolytic distal neph- migrate, and proliferate after AKI. This phase of early regen- ron segments,8,36,46 present in the cortex, will also contribute. eration is marked by substantial loss of mitochondria that The most persuasive evidence for increased glycolysis after AKI occurs as highly differentiated proximal tubule cells become was provided by increased glucose utilization, lactate production, simplified in structure.26–28 Thereafter, instead of the reversal and glucose phosphorylation over many days as tubules regen- expected for normal recovery, tubules that failed to rediffer- erated after toxic AKI.11 However, interpretation of results from entiate continued to exhibit mitochondrial pathology and these studies is fraught with inability to discriminate between persistently showed marked and progressively increased ex- AKI events taking place in uninjured proximal tubules, distal pression of glycolytic enzymes. tubules, and proximal tubules that recover normally after injury Because fatty acids are mitochondrial substrates for oxi- and AKI events taking place in those that become atrophic. IHC dative metabolism in proximal tubules, we probed for key in conjunction with protein analysis of lysates now show that proteins that globally control fatty acid oxidation. CPT1A increased glycolysis during tubule regeneration and atrophy after regulates fatty acid entry into mitochondria; PGC1a and AKI can be explained by enhanced glycolytic enzyme expression PPARa are master regulators of mitochondrial biogenesis in tubules of the same undifferentiated phenotype, which show and transcription of proteins required for fatty acid metabo- reduced mitochondrial mass and subsequently, display inhibi- lism.29 All three were downregulated during folic acid ne- tory PDH phosphorylation. phropathy,10 but we failed to detect altered expression early Normal proximal tubules use pyruvate derived from lactate as well as late after IRI, when tubule atrophy developed. These (also from amino acids, oxaloacetate, and other metabolites) results do not exclude a role for diminished fatty acid oxida- to make glucose in a predominantly gluconeogenic pathway. tion during tubule atrophy after IRI, because mitochondrial Inferring from overall increase of glycolysis and the markedly pathology after IRI and folic acid nephropathy could be increased glycolytic enzyme expression in regenerating tu- caused differently. Importantly, extreme reduction of mito- bules, and the enhanced glycolysis reported for kidneys chondria in each atrophic cell implies overall decrease of mi- recovering from toxic AKI,11 we surmise that lactate to pyru- tochondrial metabolism per cell for all substrates, including vate conversion for gluconeogenesis in regenerating proximal fatty acids. It seems possible that actions of the most proximal tubules would be suppressed. Instead, lactate dehydrogenase upstream defect that causes failed differentiation—a defect would work in reverse to generate lactate from pyruvate, that remains unknown—somehow induce uncontrolled accommodating the needs of glycolysis. Later, when some tu- autophagy, a process known to be involved in atrophy. bules recover normal structure and biochemical function, The other component of the metabolic switch—increased others do not; the metabolic anomaly in tubules with failed glycolysis and glycolytic enzyme expression—is more easily differentiation is likely made worse by progressive mitochon- explained. Unlike the distal nephron, proximal tubules express drial pathology and autophagy as well as inhibitory PDH phos- very low levels of glycolytic enzymes,8,36 and although they do phorylation. Pyruvate made by glycolysis would be denied entry exhibit some glycolysis during hypoxia17 and hypoxia reoxy- into the Krebs cycle and converted largely to lactate. Further- genation,18 their capacity for glycolysis is limited, unlike in the more, in view of reduced mitochondrial mass, oxaloacetate sub- distal nephron.36,46 Zager et al.18 showed that lactate increased strate for gluconeogenesis would also fall, because oxaloacetate is in kidneys during clamp ischemia but fell thereafter during made in mitochondria, and the Krebs cycle would be slowed by reperfusion; conversely, pyruvate (an intermediary metabolite PDH inhibition. More evidence for the metabolic switch was that can be a product of glycolysis, gluconeogenesis, or PDH provided by observing that PKLR, the pyruvate kinase isoform inhibition, causing decreased pyruvate entry into the Krebs expressed by normal proximal tubules, decreased during tubule

J Am Soc Nephrol 27: ccc–ccc, 2016 Metabolic Switch during Proximal Tubule Atrophy 7 BASIC RESEARCH www.jasn.org

Figure 6. Glycolytic enzyme PKM2 protein expression is increased in vimentin positive atrophic epithelium but not in well differentiated proximal tubule epithelium in whole tubules as well as in clusters of cells or single cells lining the same tubule. Fluorescence images for PHAE lectin brush border staining and immunofluorescence images for vimentin and PKM2 localization in an IRI kidney 14 days after IRI shown singly or merged. Small arrows point to sharp transitions between isolated cells or clusters of cells with vimentin–positive undifferentiated proximal tubule epithelium and adjacent well differentiated proximal tubule epithelium with PHAE staining brush border. Note that interstitial fibroblasts (arrowheads) as well as undifferentiated proximal tubule epithelium (large arrows) stain for both vimentin and PKM2. Distal tubules stain strongly for PKM2 but lack vimentin. DT, distal tubule; Figure 5. Glycolytic enzyme protein expression in atrophic kid- PT, proximal tubule; Vim, vimentin. Scale bar, 50 mm. ney tubules after IRI showing increase for PKM2 but decrease for PKLR. (A and B) IHC for (A) PKM2 and (B) PKLR in nephrectomy gluconeogenic flux in the opposite direction.50 Not surprisingly, control, kidneys 7 and 14 days after IRI, and kidney with SD208 PFKFB3/iPFK2 is highly expressed in contexts associated with treatment 14 days after IRI. Results for PKM2 are representative 49 of a series of four separate experiments. NCtrl, nephrectomized markedly increased glycolysis, including cancer. control; Veh, vehicle. Scale bar, 100 mm. Stimulated glycolytic enzyme expression in vivo after IRI becomes easier to understand in the context of chronic hypoxia and enhanced TGF-b signaling, both of which come into regeneration and atrophy in opposite direction from PKM2, the play during tubule regeneration after IRI.2,3,37–39,51 Both isoform responsible for glycolysis in the distal nephron. Notably, HIF1–mediated hypoxic actions and TGF-b signaling have modulation of PKLR activity is required to regulate gluconeo- pronounced effects to enhance glycolytic gene expression genic flux in the normal state—to convert excess phosphoenol and glycolysis in proximal tubule cells.14,15,17,18 The para- pyruvate back to pyruvate and avoid futile cycles.35,47 Decreased mount role of HIF1 in giving rise to increased enzyme expres- PKLRis,therefore,notunexpectedafterIRIwhenPKM2,ama- sion required to sustain enhanced glycolysis is well established jor engine for glycolysis, was overexpressed. Also in support of for diverse cell types.41,42 Particularly interesting, given the the metabolic switch after IRI is increased expression in atrophic dramatic increase of PKM2 expression, enhanced expression tubules of PFKFB3, the hypoxia–induced PFK2 isoform48 also of this isoform was shown to form part of a positive hypoxic known as inducible phosphofructokinase 2 (iPFK2).49 PFKFB3 feedback loop to potentiate HIF1 signaling.42 In this study, we has the highest 6-phosphofructo-2-kinase/fructose-2-to-6- show specific effects of HIF1 to regulate glycolytic gene ex- bisphosphatase-3 activity ratio (700:1) among PFK2 isozymes, pression using shRNA knockdown of HIF1a in cultured prox- greatly favoring glycolytic PFK1 activity and decreasing imal tubule cells. HIF1 also regulates glycolytic genes of kidney

8 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc,2016 www.jasn.org BASIC RESEARCH

Figure 8. Phosphorylation of pyruvate dehydrogenase in kidney tissue and tubules after IRI. (Upper panel) IHC for phosphopyruvate dehydrogenase E1a (pPDHE1a) in nephrectomy control, kidneys 7 and 14 days after IRI, and kidney with SD208 treatment 14 days after Figure 7. HIF1 dependent and independent effects on glycolytic IRI. Arrows point to sharp transitions between pPDHE1a staining in enzyme protein expression and phosphorylation of pyruvate atrophic epithelium and adjacent thicker differentiated epithelium dehydrogenase. (A) Western blotting for HIF1a, PFKP, PFKFB3, with weak or no staining. Scale bar, 100 mm. (Lower panel) Western PKM2, and PKLR in two groups of BUMPT cultured proximal tu- blotting of kidney lysates for pPDHE1a and PDHE1a in the time bule cells without and with HIF1a shRNA knockdown exposed to course and protocol outlined for Figure 2A. ctrl, Control; GAPDH,

0.5% O2 for 48 hours. (B) Western blotting for HIF1a, phospho- glyceraldehyde-3-phosphate dehydrogenase; NCtrl, nephrectomized pyruvate dehydrogenase E1a (pPDHE1a; S293), and PDHE1a in control; neph, nephrectomy; Veh, vehicle. two groups of BUMPT cultured proximal tubule cells without and with HIF1a shRNA knockdown exposed to 0.5% O2 for 48 hours. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. redifferentiation of kidney tubules early after injury but pathologically causing tubule atrophy and fibrosis late after injury are likely to tubules in vivo.Specific and stable induction of HIF1a in kidney be complex as well. For example, EGF gives rise to downstream TGF-b tubules (by Pax8-rtTA–based VHL deletion) led to increased ex- signaling, but the reverse (i.e., transactivation of EGFR by TGF-b) pression of glycolytic enzymes and protected proximal tubules at has also been reported.55 IRI does not heal completely and transitions risk for injury and cell death during AKI.52 Increased EGF sig- to CKD (the AKI-CKD transition), which is accompanied by sus- naling could also contribute importantly to glycolytic shift in tained EGFR activation.7 Relationships between the several factors proximal tubules during AKI. Recovery from AKI is facilitated that promote glycolysis (EGF, TGF-b,HIF1,andothers),themet- by increased signaling through EGFR during early AKI.53–55 abolic switch that we now show, and kidney fate (normal recovery of EGF signaling promotes proximal tubule cell dedifferentiation, tubule or tubule atrophy) are, therefore, likely to be complex, variable migration, and proliferation after injury.55–58 EGF stimulates during the course of disease, and difficult to unravel. Additional glycolysis14,59 and furthermore, gives rise to downstream TGF-b detailed investigation of these relationships is clearly required. signaling,54,55 which also stimulates glycolysis in kidney tu- The information presented here provides a basis for additional bule cells.14 The situation is complicated by the fact that EGFR research on why tubules often fail to enter a differentiation signaling that increases glycolysis and protects kidneys early program during recovery from AKI. Because a metabolic switch during AKI can also promote tubule atrophy and fibro- occurs as an early component of the physiologic program of sis.54,55,60 Moreover, interactions between the several signaling dedifferentiation in regenerating epithelium, it needs to be deter- pathways physiologically required for dedifferentiation and mined why such a program does not reverse in tubules that

J Am Soc Nephrol 27: ccc–ccc, 2016 Metabolic Switch during Proximal Tubule Atrophy 9 BASIC RESEARCH www.jasn.org become atrophic. Given that increased TGF-b signaling may for 4 days. SD208 is a specific inhibitor of Alk5 kinase used for in vivo contribute to altered metabolism through mitochondrial dam- studies to inhibit TGF signaling given according to this dose regimen.61,62 age and stimulated glycolysis, the question must be asked: what Controls were rats with right nephrectomy and sham surgery to the left are the upstream events that drive TGF-b signaling in regener- kidney. IRI was monitored by serum creatinine and renal histology. In a ating cells that, if not reversed, lead to atrophy? Does chronic separate series of studies, rats were studied after 60 minutes of ischemia hypoxia have HIF1-independent effects that damage tubules to using a similar IRI protocol. Studies were also done on 11- to 12-week-old the extent that it produces irreversible dedifferentiation? Addi- male C57BL/6 mice after a surgery protocol for IRI exactly the same as tional studies are needed to answer these questions using both in that used for rats. Mice were euthanized after 3, 7, or 14 days. vivo models of tubule injury and cultured proximal tubule cells. A suitable proximal tubule culture model, in which dedifferen- Cell Culture and shRNA Knockdown tiation and redifferentiation can be modulated at will under Boston University mouse proximal tubule cells (BUMPT-Clone 306; normoxic and hypoxic conditions, will be particularly informa- from W. Lieberthal and J. Schwartz) were grown at 37°C in DMEM tive to unravel the interplay of TGF-b with HIF1-dependent and (Gibco, Carlsbad, CA) with 10% FBS. BUMPT cells were derived -independent processes in the pathogenesis of tubule atrophy. from primary cultures of kidney proximal tubules of F1 hybrid mice with single copies of the H-2Kb-tsA58 transgene.63 Expression of large T antigen by the transgene at 39.5°C without g-IFN is CONCISE METHODS inhibited by .95% relative to cells at 33°C with the cytokine.63 Con- fluent BUMPT cells display proximal tubule characteristics and de- Antibodies and Reagents velop transepithelial resistance of approximately 300 V/cm2 when Antibody sources were HIF1a (Cayman Chemicals, Ann Arbor, MI); grown at 37°C.64 For HIF1a knockdown, BUMPT cells were infected CA9 and phospho-PDHE1a (S293; Novus Biologicals, Littleton, CO); with lentiviral particles with scrambled or HIF1a-specific shRNA and hexokinase 2, C64G5, and PKM2 (D78A4; Cell Signaling Technology, selected with puromycin. Two cultures, one with the lowest expres- Danvers, MA); PFKP (H44), iPFK2 br/pl (C-11), PKLR (E-2), and sion, and another with very low but perceptibly higher expression of Tom20 (FL-145; Santa Cruz Biotechnology, Santa Cruz, CA); PFKFB3 HIF1a, and one culture with scrambled control were used for studies. (iPFK-2 and uPFK-2; Sigma-Aldrich, St. Louis, MO); pyruvate kinase L, AD12, BH3, and PKM2 DF4 (ScheBo Biotech, Giessen, Germany); Morphology, IHC, and Immunofluorescence PDHE1a (GeneTex, Irvine, CA); CPT1A and PGC1a (Abcam, Inc., Cam- Rat kidneys were perfusion fixed with periodic acid–lysine- bridge,MA);PPARa (Rockland Immunochemicals Inc., Gilbertsville, PA); paraformaldehyde using methods identical to those described in vimentin clone V9 (Thermo Fisher Scientific, Vernon Hills, IL); glyceral- our previous studies.65 After overnight fixation at 4°C, kidney slices dehyde-3-phosphate dehydrogenase (Research Diagnostics, Flanders, NJ); were washed twice in PBS and once in PBS and 100 mM glycine, and ImmPRESS HRP polymer–conjugated secondary antibodies (Vector Lab- they were transferred to 70% ethanol for dehydration in increasing oratories, Burlingame, CA); and HRP–labeled secondary antibodies concentrations of ethanol and embedding in paraffin. After antigen (Jackson ImmunoResearch Laboratories, West Grove, PA). Sources of retrieval in 99°C 1 mM Tris-EDTA for 20–30 minutes, deparaffinized other reagents were MISSION shRNA Lentiviral Transduction Particles sections were blocked with 2.5% horse serum before IHC with pri- (Sigma-Aldrich), FITC–conjugated Phaseolus vulgaris Lectin PHA-E (EY mary antibodies followed by ImmPRESS HRP polymer–conjugated Laboratories, Inc., San Mateo, CA), SD208 (2-[5-chloro-2-fluorophenyl] secondary antibodies. For immunofluorescence, deparaffinized sections pteridin-4-yl)pyridin-4-yl amine; SCIOS, Inc., Johnson and Johnson, with antigen retrieval were used. Sections were examined by confocal New Brunswick, NJ), and ImmPACT DAB reagent (Vector Laboratories). fluorescence microscopy. Periodic acid–lysine-paraformaldehyde–fixed tissue was postfixed in 2% glutaraldehyde in 0.1 M Na Cacodylate buffer Animals (pH 7.2) and 1% OsO4 and processed for electron microscopy.2 Animals were cared for in accordance with National Institutes of Health and institutional guidelines, and studies were approved by the Institutional Semiquantitative Grading for Mitochondrial Mass Animal Care and Use Committee at University of texas Health Science Mitochondrial mass was assessed semiquantitatively in four separate Center,SanAntonio,TX.Studieswereperformedontissueobtainedfrom experiments. After IHC for mitochondrial marker Tom20, five dif- rats subjected to IRI without or with SD208 treatment during reperfusion ferent fields in kidney cross-sections from each kidney of each group using methods identical to those we reported before.2 SD208 (SCIOS, were graded (by M.A.V.) on a zero to five scale using a system identical Inc.) is a highly specificAlk5TGF-b receptor antagonist that suppresses to that we described before in a previous study.25 The five fields TGF-b signaling61,62 and TGF-b–dependent organ fibrosis,61,62 which we evaluated covered the entire inner cortex and outer stripe of the outer found to promote tubule differentiation and protect against tubulointer- medulla (where most of the injury took place after IRI). They were stitial fibrosis after IRI.2 Briefly, male Sprague–Dawley rats under isoflur- examined at low power (3100) and then, high power (3200 and as ane anesthesia received 45 minutes of left kidney ischemia and right needed, 3400) to grade the density of brown–staining mitochondrial nephrectomy followed by reperfusion for periods up to 14 days. The granules in entire tubule profiles. The grades represented an index of procedures used here were identical to those we have reported in previous approximate mitochondrial number per tubule profile (or per tubule studies.2 After 4 hours of reperfusion, SD208 in 1% methylcellulose at cell). Averaged grades from each of four kidneys from each group 60 mg/kg body wt or vehicle alone was provided by gavage two times daily were used for quantitation and statistical analysis.

10 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc,2016 www.jasn.org BASIC RESEARCH

Western Blotting 7. Geng H, Lan R, Singha PK, Saikumar P, Weinberg JM, Venkatachalam Inner cortex and outer stripe of outer medulla were dissected, frozen, MA: Lysophosphatidic acid (LPA) transactivates epidermal growth fac- ground in liquid nitrogen, and extracted with 43 SDS Laemmli buffer. tor receptors (EGFR) via LPAR1/Gai/o signaling to potentiate LPAR2/ Gaq/avb6 integrin dependent TGFb signaling and increase the pro- Protein loading was assessed by densitometry of Coomassie Blue–stained 3 duction of PDGF-B and CTGF by proximal tubule (PT) cells [abstract]. J gels and loading controls. Cultured cells were extracted with 1 SDS Am Soc Nephrol 24: 115A, 2013 buffer. SDS-PAGE and Western blotting were done as described.2 8. Guder WG, Ross BD: Enzyme distribution along the nephron. Kidney Int 26: 101–111, 1984 Lactate and Pyruvate Content and Hexokinase Activity 9. Weinberg JM, Molitoris BA: Illuminating mitochondrial function and dysfunction using multiphoton technology. JAmSocNephrol20: Measurements were made using kits purchased from BioVision, Inc. 1164–1166, 2009 (Milipitas, CA). Kidney cortex and outer stripe of outer medulla were 10. Kang HM, Ahn SH, Choi P, Ko YA, Han SH, Chinga F, Park AS, Tao J, dissected on a cold plate, cut into small pieces, frozen in liquid nitrogen, Sharma K, Pullman J, Bottinger EP, Goldberg IJ, Susztak K: Defective and ground under liquid nitrogen with pestle and mortar. Aliquots of fatty acid oxidation in renal tubular epithelial cells has a key role in fi – ground tissue were stored at 280°C. For analysis, ground tissue was kidney brosis development. Nat Med 21: 37 46, 2015 11. Ash SR, Cuppage FE: Shift toward anaerobic glycolysis in the re- rapidly homogenized in specific assay buffers provided by the manu- generating rat kidney. Am J Pathol 60: 385–402, 1970 3 facturer. Supernatants after centrifugation at 15,000 g were then pro- 12. Eklund T, Wahlberg J, Ungerstedt U, Hillered L: Interstitial lactate, in- cessed according to the manufacturer’s instructions for assay with minor osine and hypoxanthine in rat kidney during normothermic ischaemia modifications. Lactate and pyruvate were assayed by fluorimetry, and and recirculation. Acta Physiol Scand 143: 279–286, 1991 hexokinase activity was assayed by colorimetry. 13. FukuharaY,YamamotoS,YanoF,OritaY,FujiwaraY,UedaN,Kamada T, Noguchi T, Tanaka T: Changes in activities and mRNA levels of glycolytic enzymes of ischemia-reperfused rat kidney. Contrib Nephrol 95: 222–228, 1991 14. Nowak G, Schnellmann RG: Autocrine production and TGF-beta 1- ACKNOWLEDGMENTS mediated effects on metabolism and viability in renal cells. Am J Physiol 271: F689–F697, 1996 E.P.B. received National Institutes of Health (NIH) grant 5R01DK060043, 15. de Laplanche E, Gouget K, Cléris G, Dragounoff F, Demont J, Morales A, and M.A.V. is funded by NIH grant 1 R01 DK104128. J.M.W was Bezin L, Godinot C, Perrière G, Mouchiroud D, Simonnet H: Physiological was supported by Merit Review I01 BX002367 from the US De- oxygenation status is required for fully differentiated phenotype in kidney cortex proximal tubules. Am J Physiol Renal Physiol 291: F750–F760, 2006 partmentofVeteransAffairs.Thecontentsdonotrepresenttheviews 16. Krick S, Shi S, Ju W, Faul C, Tsai SY, Mundel P, Böttinger EP: Mpv17l of the US Department of Veterans Affairs or the US Government. protects against mitochondrial oxidative stress and apoptosis by activation of Omi/HtrA2 protease. Proc Natl Acad Sci U S A 105: 14106–14111, 2008 17. Dickman KG, Mandel LJ: Differential effects of respiratory inhibitors on – DISCLOSURES glycolysis in proximal tubules. Am J Physiol 258: F1608 F1615, 1990 18. Zager RA, Johnson AC, Becker K: Renal cortical pyruvate depletion None. during AKI. JAmSocNephrol25: 998–1012, 2014 19. Casalena G, Daehn I, Bottinger E: Transforming growth factor-b,bio- energetics, and mitochondria in renal disease. Semin Nephrol 32: 295– REFERENCES 303, 2012 20. Jain M, Rivera S, Monclus EA, Synenki L, Zirk A, Eisenbart J, Feghali- Bostwick C, Mutlu GM, Budinger GR, Chandel NS: Mitochondrial re- 1. Bonventre JV, Weinberg JM: Recent advances in the pathophysiology active oxygen species regulate transforming growth factor-b signaling. – of ischemic acute renal failure. JAmSocNephrol14: 2199 2210, 2003 J Biol Chem 288: 770–777, 2013 2. Geng H, Lan R, Wang G, Siddiqi AR, Naski MC, Brooks AI, Barnes JL, 21. Finkel T: Signal transduction by mitochondrial oxidants. JBiolChem Saikumar P, Weinberg JM, Venkatachalam MA: Inhibition of autor- 287: 4434–4440, 2012 egulated TGFbeta signaling simultaneously enhances proliferation and 22. Suzuki T, Kimura M, Asano M, Fujigaki Y, Hishida A: Role of atrophic differentiation of kidney epithelium and promotes repair following re- tubules in development of interstitial fibrosis in microembolism-in- – nal ischemia. Am J Pathol 174: 1291 1308, 2009 duced renal failure in rat. Am J Pathol 158: 75–85, 2001 3. Geng H, Lan R, Singha PK, Gilchrist A, Weinreb PH, Violette SM, 23. Gröne HJ, Weber K, Gröne E, Helmchen U, Osborn M: Coexpression of Weinberg JM, Saikumar P, Venkatachalam MA: Lysophosphatidic acid keratin and vimentin in damaged and regenerating tubular epithelia of increases proximal tubule cell secretion of profibrotic cytokines PDGF- the kidney. Am J Pathol 129: 1–8, 1987 BandCTGFthroughLPA2-andGaq-mediated Rho and avb6 integrin- 24. Ward JM, Stevens JL, Konishi N, Kurata Y, Uno H, Diwan BA, Ohmori T: dependent activation of TGF-b. Am J Pathol 181: 1236–1249, 2012 Vimentin metaplasia in renal cortical tubules of preneoplastic, neo- 4. Lan R, Geng H, Polichnowski AJ, Singha PK, Saikumar P, McEwen DG, plastic, aging, and regenerative lesions of rats and humans. Am J Pathol Griffin KA, Koesters R, Weinberg JM, Bidani AK, Kriz W, Venkatachalam 141: 955–964, 1992 MA: PTEN loss defines a TGF-b-induced tubule phenotype of failed 25. Polichnowski AJ, Lan R, Geng H, Griffin KA, Venkatachalam MA, Bidani differentiation and JNK signaling during renal fibrosis. Am J Physiol AK: Severe renal mass reduction impairs recovery and promotes fi- Renal Physiol 302: F1210–F1223, 2012 brosis after AKI. J Am Soc Nephrol 25: 1496 –1507, 2014 5. Yang L, Besschetnova TY, Brooks CR, Shah JV, Bonventre JV: Epithelial 26. Ormos J, Elemér G, Csapó Z: Ultrastructure of the proximal convo- cell cycle arrest in G2/M mediates kidney fibrosis after injury. Nat Med luted tubules during repair following hormonally induced necrosis in 16: 535–543, 2010 rat kidney. Virchows Arch B Cell Pathol Incl Mol Pathol 13: 1–13, 6. Yang L, Humphreys BD, Bonventre JV: Pathophysiology of acute 1973 kidney injury to chronic kidney disease: Maladaptive repair. Contrib Nephrol 27. Kempczinski RF, Caulfield JB: A light and electron microscopic study of 174: 149–155, 2011 renal tubular regeneration. Nephron 5: 249–264, 1968

J Am Soc Nephrol 27: ccc–ccc, 2016 Metabolic Switch during Proximal Tubule Atrophy 11 BASIC RESEARCH www.jasn.org

28. Venkatachalam MA, Bernard DB, Donohoe JF, Levinsky NG: Ischemic 49. Chesney J, Mitchell R, Benigni F, Bacher M, Spiegel L, Al-Abed Y, Han damage and repair in the rat proximal tubule: Differences among the JH, Metz C, Bucala R: An inducible gene product for 6-phosphofructo- S1, S2, and S3 segments. Kidney Int 14: 31–49, 1978 2-kinase with an AU-rich instability element: Role in tumor cell glycolysis 29. Weinberg JM: Mitochondrial biogenesis in kidney disease. JAmSoc and the Warburg effect. Proc Natl Acad Sci U S A 96: 3047–3052, 1999 Nephrol 22: 431–436, 2011 50. Sakakibara R, Kato M, Okamura N, Nakagawa T, Komada Y, Tominaga 30. Ju W, Eichinger F, Bitzer M, Oh J, McWeeney S, Berthier CC, Shedden N, Shimojo M, Fukasawa M: Characterization of a human placental K, Cohen CD, Henger A, Krick S, Kopp JB, Stoeckert CJ Jr., Dikman S, fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase. J Bio- Schröppel B, Thomas DB, Schlondorff D, Kretzler M, Böttinger EP: chem 122: 122–128, 1997 Renal gene and protein expression signatures for prediction of kidney 51. Spurgeon KR, Donohoe DL, Basile DP: Transforming growth factor- disease progression. Am J Pathol 174: 2073–2085, 2009 beta in acute renal failure: Receptor expression, effects on proliferation, 31. Conde E, Alegre L, Blanco-Sánchez I, Sáenz-Morales D, Aguado-Fraile cellularity, and vascularization after recovery from injury. Am J Physiol E, Ponte B, Ramos E, Sáiz A, Jiménez C, Ordoñez A, López-Cabrera M, Renal Physiol 288: F568–F577, 2005 del Peso L, de Landázuri MO, Liaño F, Selgas R, Sanchez-Tomero JA, 52. Fähling M, Mathia S, Paliege A, Koesters R, Mrowka R, Peters H, García-Bermejo ML: Hypoxia inducible factor 1-alpha (HIF-1 alpha) is Persson PB, Neumayer HH, Bachmann S, Rosenberger C: Tubular von induced during reperfusion after renal ischemia and is critical for Hippel-Lindau knockout protects against rhabdomyolysis-induced proximal tubule cell survival. PLoS One 7: e33258, 2012 AKI. J Am Soc Nephrol 24: 1806–1819, 2013 32. Heyman SN, Rosenberger C, Rosen S: Experimental ischemia-reperfusion: 53. Chen J, Chen JK, Harris RC: Deletion of the epidermal growth factor Biases and myths-the proximal vs. distal hypoxic tubular injury debate receptor in renal proximal tubule epithelial cells delays recovery from revisited. Kidney Int 77: 9–16, 2010 acute kidney injury. Kidney Int 82: 45–52, 2012 33. Potter C, Harris AL: Hypoxia inducible carbonic anhydrase IX, marker of 54. Zeng F, Singh AB, Harris RC: The role of the EGF family of ligands and tumour hypoxia, survival pathway and therapy target. Cell Cycle 3: receptors in renal development, physiology and pathophysiology. Exp 164–167, 2004 Cell Res 315: 602–610, 2009 34. Domingo M, Einig C, Eigenbrodt E, Reinacher M: Immunohistological 55. Tang J, Liu N, Zhuang S: Role of epidermal growth factor receptor in demonstration of pyruvate kinase isoenzyme type L in rat with mono- acute and chronic kidney injury. Kidney Int 83: 804–810, 2013 clonal antibodies. J Histochem Cytochem 40: 665–673, 1992 56. Zhuang S, Dang Y, Schnellmann RG: Requirement of the epidermal 35. Schering B, Reinacher M, Schoner W: Localization and role of pyruvate ki- growth factor receptor in renal epithelial cell proliferation and migra- nase isoenzymes in the regulation of carbohydrate metabolism and pyru- tion. Am J Physiol Renal Physiol 287: F365–F372, 2004 vate recycling in rat kidney cortex. Biochim Biophys Acta 881: 62–71, 1986 57. Zhuang S, Yan Y, Han J, Schnellmann RG: p38 kinase-mediated trans- 36. Wirthensohn G, Guder WG: Renal substrate metabolism. Physiol Rev activation of the epidermal growth factor receptor is required for de- 66: 469–497, 1986 differentiation of renal epithelial cells after oxidant injury. JBiolChem 37. Basile DP, Donohoe DL, Roethe K, Mattson DL: Chronic renal hypoxia 280: 21036–21042, 2005 after acute ischemic injury: Effects of L-arginine on hypoxia and sec- 58. Hallman MA, Zhuang S, Schnellmann RG: Regulation of dedifferentiation ondary damage. Am J Physiol Renal Physiol 284: F338–F348, 2003 and redifferentiation in renal proximal tubular cells by the epidermal 38. Basile DP: Rarefaction of peritubular capillaries following ischemic growth factor receptor. J Pharmacol Exp Ther 325: 520–528, 2008 acute renal failure: A potential factor predisposing to progressive ne- 59. Nowak G, Schnellmann RG: Integrative effects of EGF on metabolism phropathy. Curr Opin Nephrol Hypertens 13: 1–7, 2004 and proliferation in renal proximal tubular cells. Am J Physiol 269: 39. Goldfarb M, Rosenberger C, Abassi Z, Shina A, Zilbersat F, Eckardt KU, C1317–C1325, 1995 Rosen S, Heyman SN: Acute-on-chronic renal failure in the rat: Functional 60. Chen J, Chen JK, Nagai K, Plieth D, Tan M, Lee TC, Threadgill DW, compensation and hypoxia tolerance. Am J Nephrol 26: 22–33, 2006 Neilson EG, Harris RC: EGFR signaling promotes TGFb-dependent 40. Abdelkader A, Ho J, Ow CP, Eppel GA, Rajapakse NW, Schlaich MP, renal fibrosis. J Am Soc Nephrol 23: 215–224, 2012 Evans RG: Renal oxygenation in acute renal ischemia-reperfusion in- 61. Bonniaud P, Margetts PJ, Kolb M, Schroeder JA, Kapoun AM, Damm D, jury. Am J Physiol Renal Physiol 306: F1026–F1038, 2014 Murphy A, Chakravarty S, Dugar S, Higgins L, Protter AA, Gauldie J: 41. Semenza GL, Roth PH, Fang HM, Wang GL: Transcriptional regulation Progressive transforming growth factor beta1-induced lung fibrosis is of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J blocked by an orally active ALK5 kinase inhibitor. Am J Respir Crit Care Biol Chem 269: 23757–23763, 1994 Med 171: 889–898, 2005 42. Semenza GL: Regulation of metabolism by hypoxia-inducible factor 1. 62. KapounAM,GasparNJ,WangY,DammD,LiuYW,O’young G, Quon D, Cold Spring Harb Symp Quant Biol 76: 347–353, 2011 LamA,MunsonK,TranTT,MaJY,MurphyA,DugarS,ChakravartyS, 43. Rardin MJ, Wiley SE, Naviaux RK, Murphy AN, Dixon JE: Monitoring Protter AA, Wen FQ, Liu X, Rennard SI, Higgins LS: Transforming growth phosphorylation of the pyruvate dehydrogenase complex. Anal Bio- factor-beta receptor type 1 (TGFbetaRI) kinase activity but not p38 acti- chem 389: 157– 164, 2009 vation is required for TGFbetaRI-induced myofibroblast differentiation 44. Kim JW, Tchernyshyov I, Semenza GL, Dang CV: HIF-1-mediated and profibrotic gene expression. Mol Pharmacol 70: 518–531, 2006 expression of pyruvate dehydrogenase kinase: A metabolic switch 63. JatPS,NobleMD,AtaliotisP,TanakaY,YannoutsosN,LarsenL,KioussisD: required for cellular adaptation to hypoxia. Cell Metab 3: 177–185, Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 2006 transgenic mouse. Proc Natl Acad Sci U S A 88: 5096–5100, 1991 45. Lu CW, Lin SC, Chen KF, Lai YY, Tsai SJ: Induction of pyruvate de- 64. Sinha D, Wang Z, Price VR, Schwartz JH, Lieberthal W: Chemical anoxia hydrogenase kinase-3 by hypoxia-inducible factor-1 promotes meta- of tubular cells induces activation of c-Src and its translocation to the bolic switch and drug resistance. JBiolChem283: 28106–28114, 2008 zonula adherens. Am J Physiol Renal Physiol 284: F488–F497, 2003 46. Bagnasco S, Good D, Balaban R, Burg M: Lactate production in isolated 65. McLean IW, Nakane PK: Periodate-lysine-paraformaldehyde fixative. A segments of the rat nephron. Am J Physiol 248: F522–F526, 1985 new fixation for immunoelectron microscopy. J Histochem Cytochem 47. Kraus-Friedmann N: Hormonal regulation of hepatic gluconeogenesis. 22: 1077–1083, 1974 Physiol Rev 64: 170–259, 1984 48. MinchenkoA,LeshchinskyI,OpentanovaI,SangN,SrinivasV,ArmsteadV, Caro J: Hypoxia-inducible factor-1-mediated expression of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) gene. Its pos- This article contains supplemental material online at http://jasn.asnjournals. sibleroleintheWarburgeffect.J Biol Chem 277: 6183–6187, 2002 org/lookup/suppl/doi:10.1681/ASN.2015020177/-/DCSupplemental.

12 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc,2016