The Nephrotoxin Puromycin Aminonucleoside Induces Injury in Kidney Organoids Differentiated from Induced Pluripotent Stem Cells.

Kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD), which can progress to end stage renal disease (ESRD), are a worldwide health burden. Organ transplantation or kidney dialysis are the only effective available therapeutic tools. Therefore, in vitro models of kidney diseases and the development of prospective therapeutic options are urgently needed. Within the kidney, the glomeruli are involved in blood filtration and waste excretion and are easily affected by changing cellular conditions. Puromycin aminonucleoside (PAN) is a nephrotoxin, which can be employed to induce acute glomerular damage and to model glomerular disease. For this reason, we generated kidney organoids from three iPSC lines and treated these with PAN in order to induce kidney injury. Morphological observations revealed the disruption of glomerular and tubular structures within the kidney organoids upon PAN treatment, which were confirmed by transcriptome analyses. Subsequent analyses revealed an upregulation of immune response as well as inflammatory and cell-death-related processes. We conclude that the treatment of iPSC-derived kidney organoids with PAN induces kidney injury mediated by an intertwined network of inflammation, cytoskeletal re-arrangement, DNA damage, apoptosis and cell death. Furthermore, urine-stem-cell-derived kidney organoids can be used to model kidney-associated diseases and drug discovery.

Plasminogenuria is associated with podocyte injury, edema, and kidney dysfunction in incident glomerular disease.

Urinary plasminogen/plasmin, or plasmin (ogen) uria, has been demonstrated in proteinuric patients and exposure of cultured podocytes to plasminogen results in injury via oxidative stress pathways. A causative role for plasmin (ogen) as a "second hit" in kidney disease progression has yet to have been demonstrated in vivo. Additionally, association between plasmin (ogen) uria and kidney function in glomerular diseases remains unclear. We performed comparative studies in a puromycin aminonucleoside (PAN) nephropathy rat model treated with amiloride, an inhibitor of plasminogen activation, and measured changes in plasmin (ogen) uria. In a glomerular disease biorepository cohort (n = 128), we measured time-of-biopsy albuminuria, proteinuria, and plasmin (ogen) uria for correlations with kidney outcomes. In cultured human podocytes, plasminogen treatment was associated with decreased focal adhesion marker expression with rescue by amiloride. Increased glomerular plasmin (ogen) was found in PAN rats and focal segmental glomerulosclerosis (FSGS) patients. PAN nephropathy was associated with increases in plasmin (ogen) uria and proteinuria. Amiloride was protective against PAN-induced glomerular injury, reducing CD36 scavenger receptor expression and oxidative stress. In patients, we found associations between plasmin (ogen) uria and edema status as well as eGFR. Our study demonstrates a role for plasmin (ogen)-induced podocyte injury in the PAN nephropathy model, with amiloride having podocyte-protective properties. In one of the largest glomerular disease cohorts to study plasminogen, we validated previous findings while suggesting a potentially novel relationship between plasmin (ogen) uria and estimated glomerular filtration rate (eGFR). Together, these findings suggest a role for plasmin (ogen) in mediating glomerular injury and as a viable targetable biomarker for podocyte-sparing treatments.

Inhibition of p53 deSUMOylation exacerbates puromycin aminonucleoside-induced apoptosis in podocytes.

Apoptosis is a major cause of reduced podocyte numbers, which leads to proteinuria and/or glomerulosclerosis. Emerging evidence has indicated that deSUMOylation, a dynamic post-translational modification that reverses SUMOylation, is involved in the apoptosis of Burkitt's lymphoma cells and cardiomyocytes; however, the impact of deSUMOylation on podocyte apoptosis remains unexplored. The p53 protein plays a major role in the pathogenesis of podocyte apoptosis, and p53 can be SUMOylated. Therefore, in the present study, we evaluated the effect of p53 deSUMOylation, which is regulated by sentrin/SUMO-specific protease 1 (SENP1), on podocyte apoptosis. Our results showed that SENP1 deficiency significantly increases puromycin aminonucleoside (PAN)-induced podocyte apoptosis. Moreover, SENP1 knockdown results in the accumulation of SUMOylated p53 protein and the increased expression of the p53 target pro-apoptotic genes, BAX, Noxa and PUMA, in podocytes during PAN stimulation. Thus, SENP1 may be essential for preventing podocyte apoptosis, at least partly through regulating the functions of p53 protein via deSUMOylation. The regulation of deSUMOylation may provide a novel strategy for the treatment of glomerular disorders that involve podocyte apoptosis.

Podocyte expression of membrane transporters involved in puromycin aminonucleoside-mediated injury.

Several complex mechanisms contribute to the maintenance of the intricate ramified morphology of glomerular podocytes and to interactions with neighboring cells and the underlying basement membrane. Recently, components of small molecule transporter families have been found in the podocyte membrane, but expression and function of membrane transporters in podocytes is largely unexplored. To investigate this complex field of investigation, we used two molecules which are known substrates of membrane transporters, namely Penicillin G and Puromycin Aminonucleoside (PA). We observed that Penicillin G pre-administration prevented both in vitro and in vivo podocyte damage caused by PA, suggesting the engagement of the same membrane transporters by the two molecules. Indeed, we found that podocytes express a series of transporters which are known to be used by Penicillin G, such as members of the Organic Anion Transporter Polypeptides (OATP/Oatp) family of influx transporters, and P-glycoprotein, a member of the MultiDrug Resistance (MDR) efflux transporter family. Expression of OATP/Oatp transporters was modified by PA treatment. Similarly, in vitro PA treatment increased mRNA and protein expression of P-glycoprotein, as well as its activity, confirming the engagement of the molecule upon PA administration. In summary, we have characterized some of the small molecule transporters present at the podocyte membrane, focusing on those used by PA to enter and exit the cell. Further investigation will be needed to understand precisely the role of these transporter families in maintaining podocyte homeostasis and in the pathogenesis of podocyte injury.

Podocyte-specific expression of organic cation transporter PMAT: implication in puromycin aminonucleoside nephrotoxicity.

Plasma membrane monoamine transporter (PMAT) is a novel polyspecific organic cation transporter that transports organic cations and the purine nucleoside, adenosine. PMAT is expressed in the kidney, but the specific localization and function of this transporter in renal cells are unclear. In this study, we developed a polyclonal antibody toward a 14-amino acid sequence in the last intracellular loop of PMAT and determined the precise cellular localization of PMAT in human and rat kidneys. Surprisingly, we found that the PMAT protein was predominantly expressed in the glomerulus with minimal expression in tubular cells. Within the glomerulus, dual-color immunofluorescence labeling showed that the PMAT protein was specifically localized to the visceral glomerular epithelial cells, i.e., podocytes. There was no significant PMAT immunoreactivity in mesangial or glomerular endothelial cells. We further showed that puromycin aminonucleoside (PAN), a classic podocyte toxin that induces massive proteinuria and severe glomerulopathy, is transported by PMAT. Expression of PMAT in Madin-Darby canine kidney cells significantly increased cell sensitivity to PAN. Decynium 22, a potent PMAT inhibitor, abolished PAN toxicity in PMAT-expressing cells. Together, our data suggest that PMAT is specifically expressed in podocytes and may play an important role in PAN-induced kidney injury.

Interaction of the spectrin-like repeats of alpha-actinin-4 with humanin peptide.

BACKGROUND: Podocyte alpha-actinin-4 (actinin-4) is an essential component of the glomerular filtration barrier. We recently reported that the central rod spectrin-like repeats (R1-R4) of actinin-4 have a high affinity to puromycin aminonucleoside (PAN), which can induce nephro-sis in animals. The aim of this study was to identify endogenous molecules that interact with the actinin-4 R1-R4 domain. METHODS: To identify such molecules, we performed a bacterial two-hybrid screening of a human kidney cDNA library using as a bait human actinin-4 R1-R4. We further verified the identified interactions by in vitro affinity assays and immunofluorescent studies of cultured human embryonic kidney HEK293 cells. To investigate the expression of the identified molecules in podocytes, in situ hybridization, and immunohistochemical studies were performed. RESULTS: One isolated cDNA from the library encoded humanin, a recently identified antiapoptotic peptide. In vitro affinity assays showed specific interactions of recombinant actinin-4 R1-R4, R1, R2, R3, and R4 proteins with humanin-Sepharose. PAN had no effect on these interactions. Green fluorescent protein-fused humanin and endogenous actinin colocalized mainly in the perinuclear cytoplasm of HEK293 cells. Altered colocalization was not observed by the addition of PAN. In situ hybridization and immunohistochemistry showed the expression of humanin in podocytes. CONCLUSIONS: Our results suggest that humanin is a novel binding partner of the actinin-4 R1-R4 domain in podocytes. Humanin and PAN are unlikely to compete for the same binding surface in actinin-4.

Renal alpha-actinin-4: purification and puromycin aminonucleoside-binding property.

Mutations in the gene encoding nonmuscle alpha-actinin-4 (actinin-4), an actin cross-linking protein, lead to congenital nephrosis. This suggests that actinin-4 is an essential component of the glomerular filtration barrier. In the present study, we attempted to purify actinin-4 from the mammalian kidney. We also examined an interaction of the protein with puromycin aminonucleoside (PAN), which can induce nephrosis in animals. A 100-kD protein reactive with antibody against muscle alpha-actinin was purified from the Triton-insoluble cytoskeleton of porcine kidney, by MgCl2 treatment, ammonium sulfate fractionation, and subsequent DEAE-cellulose chromatography and hydroxyapatite chromatography. Its partial amino acid sequence was then determined. A filamentous actin (F-actin)-binding activity of the purified protein was examined by a cosedimentation assay. Interactions of the purified protein and its fragments with PAN were analyzed by an affinity assay using PAN-Sepharose. Determined 134 amino acid sequences of the purified porcine renal 100-kD protein were completely identical with those deduced from nucleotide sequence of the cDNA encoding human actinin-4. The purified protein possessed the known function of alpha-actinin, the F-actin-binding activity, and was tightly bound to PAN. The PAN-binding site was mapped within a central rod domain of the protein, which is a possible interaction site for various cytoskeletal and transmembrane proteins. We have established an efficient purification method for renal actinin-4. Moreover, our findings indicate that the central rod domain of actinin-4 has a high affinity to PAN. In the PAN nephrosis animal model, actinin-4 might be a target protein from PAN nephrotoxicity.

Identification of a set of genes involved in the biosynthesis of the aminonucleoside moiety of antibiotic A201A from Streptomyces capreolus.

A novel cosmid (pABC6.5) whose DNA insert from Streptomyces capreolus, the A201A antibiotic producer, overlaps the inserts of the previously reported pCAR11 and pCAR13 cosmids, has been isolated. These two latter cosmids were known to contain the aminonucleoside antibiotic A201A resistance determinants ard2 and ard1, respectively. Together, these three cosmids have permitted the identification of a DNA stretch of 19 kb between ard1 and ard2, which should comprise a large region of a putative A201A biosynthetic (ata) gene cluster. The sequence of the 7 kb upstream of ard1 towards ard2 reveals seven consecutive open reading frames: ataP3, ataP5, ataP4, ataP10, ataP7, ata12 and ataPKS1. Except for the last two, their deduced products present high similarities to an identical number of counterparts from the pur cluster of Streptomyces alboniger that were either known or proposed to be implicated in the biosynthesis of the N6,N6-dimethyl-3'-amino-3'-deoxyadenosine moiety of puromycin. Because A201A contains this chemical moiety, these ataP genes are most likely implicated in its biosynthesis. Accordingly, the ataP4, ataP5 and ataP10 genes complemented specific puromycin nonproducing Deltapur4, Deltapur5 and Deltapur10 mutants of S. alboniger, respectively. Amino acid sequence comparisons suggest that ata12 and ataPKS1 could be implicated in the biosynthesis of the d-rhamnose and alpha-p-coumaric acid moieties of A201A. Further sequencing of 2 kb of DNA downstream of ard1 has disclosed a region which might contain one end of the ata cluster.

Puromycin aminonucleoside metabolism by glomeruli and glomerular epithelial cells in vitro.

Two puromycin aminonucleoside (PAN) excretion products were purified by HPLC from urine of PAN-treated rats and characterized by nuclear magnetic resonance as N6-dimethyl-3'amino-3'deoxyadenosine (DA-Ado) and N6-methyl-3'amino-3'deoxyadenosine (MA-Ado), respectively, the former corresponding to unmodified PAN. DA-Ado was not a substrate for adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) or xanthine oxidase (XO), while MA-Ado was consecutively converted into hypoxanthine by a mixture of ADA and PNP. A different rate of transformation of DA-Ado and MA-Ado into hypoxanthine by isolated glomeruli was observed and was higher for the monomethylated analogue by a factor of 3 (79% vs. 21%); this was ascribed to the rate-limiting level of a demethylase activity acting on DA-Ado. Furthermore, DA-Ado was not transformed by glomerular epithelial cells in culture, while a little amount of MA-Ado was converted into hypoxanthine after six hours of incubation. In spite of this different metabolic behavior, the same order of cytotoxicity on glomerular epithelial cells in culture was observed for MA-Ado, DA-Ado and commercial PAN. All these molecules induced a dose response inhibition of [3H]thymidine incorporation into DNA after exposure for two hours and a marked alteration of cell viability which was not inhibited by free radical scavengers and deferoxamine. This study provides the first evidence for a glomerular metabolism of PAN and its urinary metabolite MA-Ado involving their transformation via the purine cycle enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

On the structural specificity of puromycin binding to Escherichia coli ribosomes.

We have examined the structural specificity of the puromycin binding sites on the Escherichia coli ribosome that we have previously identified [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry 19, 3809-3817, and references cited therein] by examining the interactions of a series of adenine-containing compounds with these sites. We have used as measures of such interactions the inhibition of [3H]puromycin photoincorporation into ribosomal proteins from these sites, the site-specific photoincorporation of the 3H-labeled compounds themselves, and the inhibition of peptidyl transferase activity. For the first two of these measures we have made extensive use of a recently developed high-performance liquid chromatography (HPLC) method for ribosomal protein separation [Kerlavage, A. R., Weitzmann, C., Hasan, T., & Cooperman, B.S. (1983) J. Chromatogr. 266, 225-237]. We find that puromycin aminonucleoside (PANS) contains all of the structural elements necessary for specific binding to the three major puromycin binding sites, those of higher affinity leading to photoincorporation into L23 and S14 and that of lower affinity leading to photoincorporation into S7. Although tight binding to the L23 and S7 sites requires both the N6,N6-dimethyl and 3'-amino groups within PANS, only the N6,N6-dimethyl group and not the 3'-amino group is required for binding to the S14 site. Our current results reinforce our previous conclusion that photoincorporation into L23 takes place from the A' site within the peptidyl transferase center and lead us to speculate that the S14 site might be specific for the binding of modified nucleosides. They also force the conclusion that puromycin photoincorporation proceeds through its adenosyl moiety.

Basis for the differential action of aminonucleoside on normal and transformed human fibroblasts.

Acid-soluble extracts of normal human fibroblasts (IMR 90 cells) exposed to [3H]aminonucleoside of puromycin ([3H]AMS) contained larger amounts of unchanged AMS than did similar extracts of their transformed counterparts (AG 2804 cells). The radioactive compounds present in IMR 90 cells were further analyzed by sequential high-voltage paper electrophoresis, enzyme digestion, and paper chromatography. In addition to unchanged [3H]AMS, only [3H]adenosine, and [3H]inosine, and [3H]adenosine monophosphate could be detected, apparently derived from [3H]adenosine present in the [3H]AMS samples added to the cultures. Consistent with the absence of metabolism of AMS in IMR 90 cells was the failure to find AMS derivatives in the RNA or DNA of these cells. The content of ribonucleoside triphosphates (rNTPs) in acid-soluble extracts in IMR 90 cells was significantly reduced by AMS treatment, and nuclei or broken cell preparations obtained from AMS-treated IMR 90 cells incorporated [3H]UTP into macromolecules at approximately control rates, when supplemented with rNTPs. These findings indicated that the reduced level of rNTPs may be responsible for the AMS-induced inhibition of RNA synthesis in normal cells.

Nascent RNA chain termination and ultrastructural changes in nucleoli isolated from SV40-transformed fibroblasts treated with aminonucleoside of puromycin.

The mechanism for the selective inhibition of preribosomal RNA synthesis by puromycin aminonucleoside (AMS) was investigated in cultured AG 2804 cells, which are human embryonic lung fibroblasts transformed by SV40 virus. Sequential high voltage electrophoresis and paper chromatography of the RNA of fractionated nuclei isolated from fibroblasts exposed to tritiated AMS (340 microM, 5 microCi. per ml., for 18 hours) demonstrated that the inhibitor is demethylated to 3'-amino-3'-deoxyadenosine and incorporated into terminal positions in both nucleolar and nonnucleolar nuclear RNA chains in the same relative amounts. 3'-Amino-3'-deoxyadenosine was not detected in the RNA obtained from the cytoplasm of AMS-treated fibroblasts. Electron microscopic observation of isolated nucleoli showed that neighboring intranucleolar fibrils have a tendency to coalesce after AMS treatment. It is suggested that, when the modified AMS molecule has reached the growing ends of nascent polyribonucleotide chains elongation stops, the chain remains attached to its template, and the strong positive charge of the aminosugar portion of AMS interacts with negatively charged components (e.g., phosphate groups) of adjacent nascent RNA chains. The high density of the components of the nucleolus produces frequent interactions of this type and leads to secondary interference with nucleolar RNA synthesis, thus accounting for the selective effect of AMS on preribosomal RNA synthesis.