A reciprocal regulation of spermidine and autophagy in podocytes maintains the filtration barrier
Authors: Wei Liang, Kosuke Yamahara, Camila Hernando-Erhard, Simon Lagies, Nicola Wanner, Huan Liang, Christoph Schell, Bernd Kammerer, Tobias B. Huber, Tillmann Bork
Journal: Kidney International (2020)

Podocyte maintenance and stress resistance are exquisitely based on high basal rates of autophagy making these cells a unique model to unravel mechanisms of autophagy regulation. Polyamines have key cellular functions such as proliferation, nucleic acid biosynthesis and autophagy. Here we test whether endogenous spermidine signaling is a driver of basal and dynamic autophagy in podocytes by using genetic and pharmacologic approaches to interfere with different steps of polyamine metabolism. Translational studies revealed altered spermidine signaling in focal segmental glomerulosclerosis in vivo and in vitro. Exogenous spermidine supplementation emerged as new treatment strategy by successfully activating autophagy in vivo via inhibition of EP300, a protein with an essential role in controlling cell growth, cell division and prompting cells to differentiate to take on specialized functions. Surprisingly, gas chromatography-mass spectroscopy based untargeted metabolomics of wild type and autophagy deficient primary podocytes revealed a positive feedback mechanism whereby autophagy itself maintains polyamine metabolism and spermidine synthesis. The transcription factor MAFB acted as an upstream regulator of polyamine metabolism. Thus, our data highlight a novel positive feedback loop of autophagy and spermidine signaling allowing maintenance of high basal levels of autophagy as a key mechanism to sustain the filtration barrier. Hence, spermidine supplementation may emerge as a new therapeutic to restore autophagy in glomerular disease. Podocyte maintenance and stress resistance are exquisitely based on high basal rates of autophagy making these cells a unique model to unravel mechanisms of autophagy regulation. Polyamines have key cellular functions such as proliferation, nucleic acid biosynthesis and autophagy. Here we test whether endogenous spermidine signaling is a driver of basal and dynamic autophagy in podocytes by using genetic and pharmacologic approaches to interfere with different steps of polyamine metabolism. Translational studies revealed altered spermidine signaling in focal segmental glomerulosclerosis in vivo and in vitro. Exogenous spermidine supplementation emerged as new treatment strategy by successfully activating autophagy in vivo via inhibition of EP300, a protein with an essential role in controlling cell growth, cell division and prompting cells to differentiate to take on specialized functions. Surprisingly, gas chromatography-mass spectroscopy based untargeted metabolomics of wild type and autophagy deficient primary podocytes revealed a positive feedback mechanism whereby autophagy itself maintains polyamine metabolism and spermidine synthesis. The transcription factor MAFB acted as an upstream regulator of polyamine metabolism. Thus, our data highlight a novel positive feedback loop of autophagy and spermidine signaling allowing maintenance of high basal levels of autophagy as a key mechanism to sustain the filtration barrier. Hence, spermidine supplementation may emerge as a new therapeutic to restore autophagy in glomerular disease. Translational StatementAutophagy has emerged as a key maintenance mechanism in podocytes, and impaired autophagy is a common feature in glomerular disease. Thereby, activating autophagy appears a promising therapeutic strategy to prevent podocyte loss. We describe metabolic signaling via spermidine as a key regulatory axis for autophagy activation in podocytes. Our data highlight that autophagy alteration as seen in focal segmental glomerulosclerosis is critically mediated by impaired polyamine metabolism. Exogeneous spermidine supplementation successfully activates autophagy and thereby emerges as a new treatment avenue to prevent or cure glomerular disease. Autophagy has emerged as a key maintenance mechanism in podocytes, and impaired autophagy is a common feature in glomerular disease. Thereby, activating autophagy appears a promising therapeutic strategy to prevent podocyte loss. We describe metabolic signaling via spermidine as a key regulatory axis for autophagy activation in podocytes. Our data highlight that autophagy alteration as seen in focal segmental glomerulosclerosis is critically mediated by impaired polyamine metabolism. Exogeneous spermidine supplementation successfully activates autophagy and thereby emerges as a new treatment avenue to prevent or cure glomerular disease. Polyamines are ubiquitously found intermediates of L-ornithine metabolism and promote key cellular functions such as proliferation, nucleic acid biosynthesis, and autophagy.1Pegg A.E. Functions of polyamines in mammals.J Biol Chem. 2016; 291: 14904-14912Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 2Madeo F. Eisenberg T. 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Henique C. et al.Endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis.Autophagy. 2015; 11: 1130-1145Crossref PubMed Scopus (181) Google Scholar However, until now it remained unclear how these high levels of autophagy are maintained and what upstream signaling events regulate the adaptive autophagy response in podocytes. In our study, we now provide evidence for metabolic signaling mediated by endogenous spermidine as a key regulator of podocyte autophagy. Of note, we identified a podocyte-specific positive feedback loop of autophagy and transcriptional regulation of key enzymes of polyamine metabolism mediated by the transcription factor MAFB, allowing for a unique self-perpetuating mechanism driving autophagy. Podocytes display high levels of autophagy in vivo as indicated by LC3 fluorescence in mice transgenic for GFP-LC3, a key marker for autophagosomes (Figure 1a). GFP-positive autophagosomes are highly abundant in glomeruli, whereas in the tubular compartment autophagosomes are rarely found. To monitor newly formed autophagosomes and their subsequent lysosomal fusion, mice transgenic for GFP-RFP-LC3 were analyzed for autophagic activity (Figure 1b). Autophagosomes and autophagolysosomes were found to be highly abundant in podocytes, indicating high autophagic flux—much higher than in neighboring mesangial or tubular cells. To test whether spermidine is able to further stimulate autophagy in podocytes, glomeruli obtained from GFP-RFP-LC3 mice were isolated and cultured for 3 days (Figure 1c). Treatment with spermidine resulted in higher levels of GFP- and GFP-RFP–positive puncta compared with vehicle-treated controls (Figure 1d and e). Costaining for NESTIN was used as a podocyte marker. To investigate the effect of spermidine supplementation on induced autophagy, LC3-II levels were assessed after spermidine pretreatment followed by glucose starvation (Supplementary Figure S1A and B). Increased LC3-II levels after spermidine pretreatment indicate the effectivity of spermidine supplementation in increasing both basal and induced autophagy. To confirm the proautophagic effect of spermidine in human podocytes, immortalized podocytes46Saleem M.A. O'Hare M.J. Reiser J. et al.A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression.J Am Soc Nephrol. 2002; 13: 630-638Crossref PubMed Google Scholar stably expressing GFP-LC3 were used. Also, in these human-derived podocytes, treatment with spermidine results in an increased number of GFP-positive autophagosomes (Figure 1f and g) and increased levels of LC3-II in a dose-dependent manner (Figure 1h and i and Supplementary Figure S1C). Lysosomal inhibition with chloroquine revealed increased autophagic flux caused by spermidine. Corresponding findings in mice and human podocytes suggest a conserved role of spermidine in promoting autophagic flux. To further delineate the role of spermidine on autophagy regulation, a primary podocyte culture was generated by isolating glomeruli from mice transgenic for the reporter allele Tomato/eGFP and with podocyte-specific expression of Cre recombinase (hNPHS2-Cre), leading to green-labeled podocytes, which then could be purified using fluorescence-associated cell sorting to obtain pure primary podocyte culture (Figure 1j). Primary podocytes treated with spermidine showed higher levels of LC3-II and decreased levels of SQSTM1/p62 (Figure 1k–m). The same approach in the presence of the lysosomal inhibitor chloroquine revealed an increase in autophagic flux after spermidine treatment in these primary podocytes (Figure 1n). To test whether endogenous spermidine plays a role in podocyte autophagy regulation, primary podocytes were treated with the ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) (Figure 2a). ODC inhibition reduced basal autophagy, as demonstrated by decreased levels of LC3-II and increase of SQSTM1 (Figure 2b). Spermidine exposure could restore autophagy after DFMO pretreatment (Supplementary Figure S1D). Lysosomal inhibition by chloroquine revealed reduced autophagic flux resulting from DFMO (Figure 2c). The effect of ODC inhibition on endogenous spermidine production and thereby on autophagy is not limited to primary murine podocytes. Human podocytes stably transfected with GFP-LC3 showed reduced autophagosome formation after treatment with DFMO and a significant reduction in autophagic flux (Figure 2d and e). Confirmatory, ODC inhibition by the ornithine decarboxylase inhibitor POB (BOC-protected pyridoxyl-ornithine conjugate47Wu F. Grossenbacher D. Gehring H. New transition state-based inhibitor for human ornithine decarboxylase inhibits growth of tumor cells.Mol Cancer Ther. 2007; 6: 1831-1839Crossref PubMed Scopus (21) Google Scholar) with and without lysosomal inhibition by chloroquine revealed decreased autophagosome formation and reduced autophagic flux (Supplementary Figure S1E and F). Accordingly, knockdown of spermidine synthase by siRNA reduced autophagosome formation and autophagic flux (Figure 2f and g). Western blot for autophagy markers revealed decreased LC3-II abundance in response to SRM reduction in the presence of lysosomal inhibition (Figure 2h and i). To confirm the presence of key enzymes of polyamine metabolism in podocytes in vivo, kidney sections of 16-week-old wild-type (WT) mice were stained for SRM and ODC by using immunofluorescence (Figure 2j and k). Costaining for the podocyte-specific protein NEPRHIN revealed ODC and SRM expression in podocytes, suggesting endogenous spermidine production in podocytes in vivo as observed in vitro (Figure 2f, Supplementary Figure S2D and E). To prove the in vivo relevance of polyamine metabolism for podocyte autophagy, 6-week-old GFP-LC3 mice were treated with either spermidine or DFMO (Figure 3a). Glomerular lysates clearly revealed proautophagic effects of spermidine, and DFMO inhibited autophagy (Figure 3b–d). Mechanistically, spermidine did not affect AMPK activity or mTORC1 activation but clearly decreased p300 expression and acetylation (Figure 3e and f). Transcriptomic analysis from human podocytes treated with spermidine did not show a broad transcriptional impact of spermidine on key ATGs (Figure 3g and h), thereby supporting posttranslational acetylation via p300 as the major downstream mechanism for spermidine action. Further metabolic profiling revealed a distinct impact of spermidine on podocyte metabolism (Figure 3i). Glucose metabolism to fructose via the polyol pathway was clearly reduced by exogenous spermidine supplementation, suggesting a critical involvement of endogenous polyamine metabolism in diabetic nephropathy (Figure 3j and k). To test for the potential impact of hyperglycemic environment on the expression of key enzymes of polyamine metabolism in podocytes, human podocytes were exposed to different glucose concentrations (Figure 4a). Neither enzyme expression levels nor direct assessment of endogenous spermidine by gas chromatography followed by mass spectrometry suggested any effect of glucose on polyamine metabolism in podocytes (Figure 4b and c). Human transcriptomic data as well did not show consistent changes in the expression patterns of ODC or SRM in DN (Supplementary Figure S1G); also, ODC levels in murine DN showed no obvious difference (Supplementary Figure S1H). To test for the involvement of polyamine metabolism in FSGS, human podocytes were exposed to Adriamycin for 24 hours or for 3 days to mimic early-onset FSGS and chronic disease. Targeted gas chromatography followed by mass spectrometry profiling revealed significantly increased levels of spermidine, putrescine, and spermine in ADR short-term treatment, whereas activation of polyamine metabolism was blunted after long-term exposure (Figure 4e). Correspondingly, ODC levels significantly decreased after long-term ADR, and autophagy was inhibited, as suggested by SQSTM1 accumulation and reduced LC3-II (Figure 4e). In murine FSGS, ODC1 and SRM levels decreased in podocytes in case of massively increased proteinuria, as seen in later/chronic stages of FSGS (Figure 4e–g, Supplementary Figure S1I). Transcriptomic data obtained from human glomeruli showed significant reduction for ODC1 and a clear trend toward lower SRM levels in FSGS (Figure 4h). To test for protective effects of spermidine on podocyte survival in FSGS, murine podocytes were exposed to ADR and spermidine (Supplementary Figure S1J). Spermidine reduced podocyte apoptosis, as suggested by reduced levels of cleaved caspase 3, and cleaved poly ADP ribose polymerase. In Atg5-deficient podocytes, spermidine showed no protective effect, suggesting autophagy activation as the key downstream effector mechanism of spermidine action (Supplementary Figure S1K). To determine the effect of endogenous spermidine on induced autophagy, ODC was inhibited by DFMO followed by glucose starvation (Supplementary Figure S2A). Autophagy response as indicated by LC3-II and SQSTM1 levels was decreased by DFMO, suggesting a role of endogenous spermidine in dynamic autophagy regulation in podocytes (Supplementary Figure S2B). Because spermidine is derived from ornithine metabolism, and autophagy itself promotes protein catabolism with potentially concomitant ornithine production, we further aimed to ravel the role of this crosstalk of autophagy and polyamine metabolism by gas chromatography followed by mass spectrometry. Therefore, we generated primary podocyte culture from WT mice and—as controls—from mice with podocyte-specific knockout of Atg5 both additionally harboring a Tomato/EGFP reporter allele for podocyte-specific green fluorescence (Figure 5a). The knockout of Atg5 was confirmed by western blot (Figure 5b and c). As expected, LC3-II levels decreased and SQSTM1 abundance increased in Atg5-deficient podocytes (Figure 5d and e). Lysosomal inhibition revealed decreased autophagic flux if Atg5 is deleted (Figure 5f and g). Different starvation conditions were tested to determine the most effective response in autophagy activation in WT podocytes (Figure 5h). The highest levels of autophagy as indicated by LC3-II abundance were detected after glucose starvation (Figure 5h and i). According to this approach, untargeted metabolomic analysis was performed with and without glucose starvation in WT and Atg5-deficient podocytes (Figure 5j). As expected, starvation and Atg5 deficiency had a significant impact on intracellular metabolite content (Figure 5k). Glucose starvation resulted in a clear decrease of glucose and intermediates of glycolysis in both Atg5-deficient and WT podocytes (Figure 5ln). The intracellular levels of most detected amino acids, including ornithine, increased in both WT and Atg5-deficient podocytes after starvation, suggesting a proteolytic response on glucose withdrawal independent of autophagy. After starvation, the levels of ornithine and the ornithine-derived metabolite putrescine were similar in WT and Atg5-deficient podocytes (Figure 5o, p). Spermidine levels, however, were found to be significantly lower in Atg5-deficient podocytes both in resting conditions and after starvation (Figure 5Q). In conclusion, these data suggest impaired spermidine synthesis in Atg5-deficient podocytes, likely being caused by reduced metabolic flux from ornithine to spermidine. To further study the underlying mechanism of impaired spermidine synthesis in Atg5-deficient podocytes, the abundance of ODC and SRM, and the abundance of the regulatory proteins ODC antizyme and ODC antizyme inhibitor, were assessed in primary podocytes lacking Atg5 and respective controls (Figure 6a, Supplementary Figure S2C–E). Levels of SRM were found to be strongly reduced in Atg5-deficient podocytes, and ODC levels were slightly lower in podocytes lacking Atg5 (Figure 6b). Also, the regulatory enzymes ODC antizyme and antizyme inhibitor-1 were less expressed if Atg5 was missing (Supplementary Figure S2C). Abundance of SRM, ODC, and ODC antizyme of antizyme inhibitor-1 did not further change under glucose starvation (Supplementary Figure S2C). We next tested for the podocyte-specific character of this polyamine–autophagy crosst