Inhibition of mtor delayed but could not prevent experimental collapsing focal segmental glomerulosclerosis

ABSTRACT Anti-Thy1.1 transgenic mice develop glomerular lesions that mimic collapsing focal segmental glomerulosclerosis (FSGS) in humans with collapse of the glomerular tuft and marked

hyperplasia of the parietal epithelial cells (PECs). Immunostaining of phosphor-S6 ribosomal protein (pS6RP) revealed high mTOR activity in PECs of the FSGS lesions of these mice. In this

study we questioned whether the mTOR inhibitor rapamycin (sirolimus) could attenuate the development and progression of glomerulosclerotic lesions in the anti-Thy1.1 transgenic mice. We

observed reduced mTOR signalling and proliferation in human parietal epithelial cells after rapamycin treatment. Experiments with anti-Thy1.1. mice showed that early treatment with sirolimus

reduced the development of glomerular lesions and glomerular cell proliferation at day 4. Levels of albuminuria, podocyte injury and podocyte number were similar in the sirolimus and

vehicle treated groups. The initial beneficial effects of sirolimus treatment were not observed at day 7. Late sirolimus treatment did not reduce albuminuria or the progression of

glomerulosclerosis. Taken together, rapamycin attenuated PEC proliferation and the formation of early FSGS lesions in experimental FSGS and reduced human PEC proliferation _in vitro_.

However, the initial inhibition of PEC proliferation did not translate into a decline of albuminuria nor in a sustained reduction in sclerotic lesions. SIMILAR CONTENT BEING VIEWED BY OTHERS

MINNELIDE COMBINED WITH ANTI-ANGPTL3-FLD MONOCLONAL ANTIBODY COMPLETELY PROTECTS MICE WITH ADRIAMYCIN NEPHROPATHY BY PROMOTING AUTOPHAGY AND INHIBITING APOPTOSIS Article Open access 09

September 2023 SYSTEMIC GENE THERAPY WITH THYMOSIN Β4 ALLEVIATES GLOMERULAR INJURY IN MICE Article Open access 16 July 2022 ANTI-ANGPTL3-FLD MONOCLONAL ANTIBODY TREATMENT AMELIORATES

PODOCYTE LESIONS THROUGH ATTENUATING MITOCHONDRIAL DAMAGE Article Open access 13 October 2022 INTRODUCTION Focal segmental glomerulosclerosis (FSGS) is characterized by the formation of

sclerotic lesions in the glomeruli of the kidneys. FSGS is one of the most common glomerular disorders and the leading cause of end-stage renal disease (ESRD) in the United States1. Several

underlying conditions can lead to FSGS such as diabetes, hypertension and obesity. In addition, FSGS can be caused by genetic mutations affecting the function of essential glomerular cell

proteins or it can be idiopathic1,2. The diagnosis of FSGS is largely based on histopathological findings characterized by the adhesions of the Bowman’s capsule with the glomerular tuft, the

formation of focal and segmental sclerotic lesions, obliteration of glomerular capillaries and extracellular matrix accumulation3,4. Currently, the different histological patterns of FSGS

have been divided into five subvariants: the perihilar-, the tip -, the cellular-, the NOS (not otherwise specified)-, and the collapsing variant, latter is characterized by collapse of the

glomerular tuft and PEC hyperplasia2. The pathogenesis of FSGS is not completely unravelled. In the last decade we and others demonstrated that parietal epithelial cells (PECs) are crucially

involved in the formation of sclerotic lesions. Genetic tagging of podocytes or PECs in different models of FSGS showed that upon induction of glomerular injury PECs migrate to the

capillary tuft and induce the adhesion between Bowman’s capsule and the tuft5,6. These activated PECs invade the injured area and deposit extracellular matrix that results in the progression

of FSGS7,8. In addition, similar pathohistological patterns were observed in secondary FSGS that are independent of the underlying disease9. These findings strengthen the assumption that

common molecular pathways lead to PEC activation and subsequently to FSGS formation and progression. Hamatani and colleagues observed activation of the mammalian target of rapamycin (mTOR),

a main player in cell proliferation and survival, specifically in PECs in experimental models of glomerular diseases. They observed that a reduction of the stress-inducible protein sestrin-2

in PECs was associated with increased pS6RP expression as well as de _novo_ expression of the activation marker CD4410. The results from Hamatani _et al_. suggested that mTOR signalling

plays an important role in the switch from resting to activated PECs seen in progressive glomerular disease. Therefore, mTOR might be a pharmaceutical target to prevent or reduce PEC

activation and subsequently glomerulosclerosis formation. In the present study we investigated this hypothesis and studied the effects of mTOR inhibition on immortalized human parietal

epithelial cells and in anti-Thy1.1 transgenic mice, a model resembling collapsing FSGS in humans. RESULTS RAPAMYCIN REDUCES MTOR SIGNALLING AND CELL PROLIFERATION OF HUMAN PARIETAL

EPITHELIAL CELLS Since we hypothesized that mTOR signalling is important for PEC proliferation in FSGS, we investigated the effect of the mTOR inhibitor rapamycin on cultured human

immortalized PECs. The expression of pS6RP as readout for mTOR signalling and PEC proliferation was studied, when exposed to different concentrations of rapamycin or DMSO as control. Already

at a concentration of 5 nM rapamycin the expression of the pS6RP was reduced (Fig. 1A, p < 0.001). The non-phosphorylated protein was not significantly affected (Fig. 1B). In addition,

higher concentrations of rapamycin (≥30 µM) reduced PEC proliferation (Fig. 1C, p < 0.001). These findings show that rapamycin can target PECs directly _in vitro_. As PEC proliferation is

described to be related to CD44 expression11, the effect of rapamycin on CD44 gene and protein expression was studied (Supplementary Fig. S1). The results indicate that the inhibition of

PEC proliferation after rapamycin treatment is independent of the expression level of CD44. MTOR ACTIVITY IS INCREASED IN SCLEROTIC LESIONS To study the effect of mTOR inhibition in PECs _in

vivo_ we used the transgenic anti-Thy1.1 mouse, an experimental model for collapsing FSGS. Injection of the anti-Thy1.1 antibody in the transgenic Thy1.1 mice results in an almost immediate

development of albuminuria that peaks at day 1 after injection12. Within 7 days the FSGS lesions, that resemble collapsing FSGS and are associated with hyperplasia of the PECs5,12,13, are

formed. Early stages of the lesions can already be observed after 4 days13. Immunostaining of the podocyte marker synaptopodin, the PEC marker SSeCKS14 and pS6RP as marker for mTOR

signalling revealed an increased pS6RP expression in PECs in the affected glomeruli of the anti-Thy1.1 mice, while in morphologically normal glomeruli only few cells showed mTOR signalling

(Fig. 2). These results suggest that increased mTOR activity may play a role in PEC activation and disease progression in the anti-Thy1.1. mice. SIROLIMUS TREATMENT DOES NOT REDUCE

ALBUMINURIA AND PODOCYTE DAMAGE We evaluated in the anti-Thy1.1 mouse model the effect of sirolimus on the development of the first visible glomerular lesions at day 4 and the fully

developed lesions at day 7. Following sirolimus treatment, albuminuria was not reduced at day 4 (Fig. 3A) and day 7 (Fig. 3B), while binding of the anti-Thy1.1 antibody was equal in the

sirolimus and phosal (vehicle) treated mice (data not shown). Immunofluorescent staining for synaptopodin and the podocyte injury marker desmin (Fig. 3C–E) showed no differences in the

amount of podocyte injury between the phosal and sirolimus treated mice at day 4 (Fig. 3F). In addition, immunofluorescent staining for DACH1 in combination with synaptopodin (Fig. 3G–I)

revealed that the number of podocytes was similar in sirolimus and phosal treated mice (Fig. 3J). These results indicate that early mTOR inhibition did not have an effect on the degree of

podocyte injury, podocyte number and proteinuria in the anti-Thy1.1 mouse model. SIROLIMUS REDUCES GLOMERULAR DAMAGE AND CELL PROLIFERATION AT DAY 4 Despite similar levels of albuminuria and

podocyte injury between the phosal and sirolimus treated mice, we observed less affected glomeruli in mice treated with sirolimus compared to the control mice at day 4 (Fig. 4A). Glomeruli

were considered affected when they showed one of the following histological changes: vacuolization of the epithelial cells, hyalinosis or epithelial cell hyperplasia (Supplementary Fig. S2).

The majority of phosal treated mice had a more severe histological phenotype showing more protein casts and affected glomeruli (Fig. 4C). The affected glomeruli of sirolimus treated mice

mainly appeared with vacuolization and mild cell proliferation. However, the greatest part of glomeruli was unaffected (Fig. 4E). In addition, we detected fewer proliferating (ki-67

positive) cells in the Bowman’s space in sirolimus treated mice, indicating less PEC proliferation (Fig. 4B, D, F). The sirolimus treated mice also showed fewer proliferating cells in the

glomerular tuft (Fig. 4B). As proliferating endothelial cells were identified in the mice (Supplementary Fig. S3), their proliferation was probably also reduced due to sirolimus treatment.

_In vitro_ we showed that rapamycin indeed has a direct effect on the glomerular endothelial cell proliferation (Supplementary Fig. S4). SIROLIMUS DOES NOT REDUCE GLOMERULAR DAMAGE AND CELL

PROLIFERATION AT DAY 7 As sirolimus reduced PEC proliferation and the formation of lesions during the early proliferative phases of FSGS development, we next investigated whether early

sirolimus treatment sustained reduction of sclerotic lesions. However, the number of affected glomeruli at day 7 in the phosal and sirolimus treated groups was similar (Fig. 5A).

Histological analysis revealed that hyalinosis was clearly visible in the glomeruli and that tubular protein casts could be observed in both the phosal and sirolimus treated mice. In

addition, the majority of affected glomeruli showed extensive PEC proliferation (Fig. 5C–F). Notably, the sirolimus treated mice showed the highest number of ki-67 positive cells in Bowman’s

space (Fig. 5B). SIROLIMUS TREATMENT AFTER GLOMERULOSCLEROSIS DEVELOPMENT DOES NOT REDUCE ALBUMINURIA AND SCLEROSIS To evaluate whether mTOR inhibition can attenuate the progression of

glomerulosclerosis in a later phase of disease development, we performed an experiment treating anti-Thy1.1 mice daily with sirolimus or phosal for a period of 20 days. Knowing that

sclerotic lesions are formed within the first 7 days after anti-Thy1.1 antibody injection13, we started sirolimus and phosal treatment at day 11. Comparing the albuminuria levels of the

sirolimus and phosal treated mice, no significant differences could be observed (Fig. 6A). Furthermore, the number of sclerotic glomeruli was similar (Fig. 6B). In general, the kidney tissue

of all mice showed influx of immune cells in the glomeruli and the interstitium. Hyalinosis and collapsing tufts could be detected in the glomeruli (Fig. 6C, D). Some glomeruli still

displayed some PEC proliferation. The results indicate that mTOR inhibition did not have an effect on the glomerular pathology once the glomerular lesions are formed. In general, our

findings indicate that sirolimus reduces PEC proliferation and formation of lesions in the early proliferative phase of FSGS development but cannot prevent the further development of

glomerulosclerosis. DISCUSSION In this study, we tested the hypothesis that mTOR signalling regulates PEC activation and that mTOR inhibition may prevent PEC proliferation and subsequently

glomerulosclerosis in the anti-Thy1.1 mouse model, an experimental model of collapsing FSGS. We observed an increased mTOR activity, expressed as the phosphorylation of S6RP, in affected

glomeruli of the anti-Thy1.1 mice. Especially PECs showed a striking increase in pS6RP expression. Sirolimus treatment reduced the number of affected glomeruli, which was associated with

less PEC proliferation, in the early lesions seen at day 4. The reduced PEC proliferation could be a direct effect of rapamycin on PECs, since also rapamycin treated PECs in culture showed

reduced pS6RP levels and reduced proliferation. In addition, the effects on PECs were independent of podocytes as the sirolimus treatment did not have an effect on albuminuria, the number of

podocytes or podocyte injury. However, despite the marked effects of rapamycin in culture and in the development of the early lesions in the mice, continuation of the treatment with

sirolimus could not prevent glomerulosclerosis at day 7. Also sirolimus treatment after sclerosis development could not reduce lesion formation. Recent studies indicated that in the

formation of lesions in FSGS and crescentic glomerulonephritis the PECs become activated5,9. The normal quiescent flat cells become enlarged and show different marker expression, i.e. a _de

novo_ expression of CD448,11,15. Although, we and others have established the involvement of PECs in the development of glomerular lesions, still little is known about the molecular

processes driving PEC activation16. Several studies have described a role for mTOR in glomerular disease models. Although, most of these studies focused on mTOR signalling in

podocytes17,18,19,20,21, a few studies have studied the pathway in PECs10,22,23. These studies indicated that mTOR signalling might be involved in PEC activation and therefore the

development of sclerotic lesions. This study shows a link between mTOR signalling and PEC activation as well. Inhibiting mTOR in the anti-Thy1.1 mice reduced PEC proliferation and sclerosis

formation at day 4. However, it was striking that the beneficial effects of mTOR signalling were not seen in later phases of the disease. These contradictory findings can also be seen in

other studies that investigated mTOR signalling in PECs. In general, all studies, including this work, showed that the effects of mTOR inhibition are dependent on the dose of the mTOR

inhibitor, the animal model used, the route of administration and on the onset and duration of treatment. Similar to our observations, Kurayama _et al_. showed that the outcome of mTOR

inhibition is dependent on the onset and duration of the mTOR inhibition. The study demonstrated that in a rat model of crescentic nephritis early treatment (day of disease induction) with

everolimus led to increased cellular necrosis. However, later treatment (7 days after disease induction) reduced glomerular crescent formation22. Since the anti-Thy1.1 model and the

nephritis model are different models of treatment strategies and time, the observed effects are difficult to compare to our findings. Nevertheless, both studies show that the timing of

treatment is critical. Besides the timing, also the dose of rapamycin is of importance for the treatment results. From seminal studies performed by Gödel and co-workers it has become clear

that mTOR signalling is tightly regulated and that an imbalance in mTOR activity may lead to adverse effects, facilitating glomerular disease17. In their study Gödel and colleagues deleted

the regulatory-associated protein (Raptor), an essential adaptor molecule of mTOR, in podocytes of diabetic mice. They demonstrated that complete reduction of mTORC1 in RaptorΔpodocyte mice

resulted in proteinuria and the progression of diabetic nephropathy, while deletion of only one allele of Raptor (RaptorHet podocyte) inhibited mTOR activation, leading to decreased

proteinuria and glomerulosclerosis17. Hence, it was demonstrated that complete reduction of mTOR activity can be responsible for the progression of glomerular injury. This means that the

reduction of mTOR activity to prevent disease progression should be done with care, as a too strong reduction can have deleterious effects. This conclusion can also be drawn from a similar

study performed by Zschiedrich _et al_. They showed that a homozygous podocyte specific knockout (KO) of Raptor in an adriamycin induced nephropathy mouse model caused massive proteinuria

and sclerosis, while a heterozygous KO resulted in a reduction of proteinuria and sclerosis21. They also showed that a high dose of administered rapamycin has a similar effect as a complete

Raptor KO, while low rapamycin concentrations resembled the beneficial outcome of the heterozygous KO, again stressing the tight regulation of mTOR in the glomerulus. The possible decrease

in endothelial proliferation due to mTOR inhibition might have contributed to the recurrent sclerosis formation observed in our experiment, as reduced endothelial cell proliferation

presumably impairs endothelial repair of capillaries that normally is required after glomerular damage due to anti-Thy1.1 injection24. Due to the narrow therapeutic window of sirolimus, and

in the murine studies described problems of treatment onset and duration, the implementation of sirolimus into the clinic to treat glomerulosclerosis will be challenging. Sirolimus and other

rapamycin analogues (rapalogous) are approved by the Food and Drug administration (FDA) for the treatment of cancers, transplant rejection, lymphangioleiomyomatosis and tuberous sclerosis

complex25. Therapeutic effects of sirolimus or rapalogous are often hampered due to recurrence of the disease assumably caused by negative feedback loops. To prevent this, treatments

combining sirolimus with drugs targeting the pathways involved in the negative feedback (e.g. Akt pathway), seem more promising25. Also, ATP-competitive mTOR inhibitors are developed that

prevent the feedback-mediated Akt activation26. Although the treatment with mTOR inhibitors can have beneficial effects, it is often accompanied with severe side effects such as

haematological abnormalities, nephrotoxicity, dermatological diseases, impaired wound healing and metabolic disorders27, which makes long lasting treatment difficult. In conclusion, this

study shows that rapamycin can have a direct effect on parietal epithelial cells, which _in vivo_ can lead to a short-lasting reduction of PEC proliferation and sclerotic lesions. Our

findings point out that an elevated mTOR expression in parietal epithelial cells might contribute to PEC activation and the formation of glomerulosclerosis. However, literature and this

study show that within the glomerulus mTOR signalling is tightly regulated, resulting in a narrow therapeutic window of rapamycin. When not used in optimal concentrations, as well as in the

favourable time window for treatment, mTOR inhibition can have no beneficial or even deleterious effects. In addition, systemic administration of the inhibitor may have non-specific effects,

decreasing mTOR inhibition in other cells resulting in for instance prevention of normal kidney repair functions. Whether ATP-competitive mTOR inhibitors or combination treatments of

sirolimus and drugs targeting pathways involved in negative feedback loops could decrease sclerosis formation permanently evoking tolerating side effects has to be investigated. MATERIAL AND

METHODS CELL CULTURE Immortalized polyclonal human parietal epithelial cells (hPECs) were cultured as described in Kietzmann _et al_.28. HPECs were differentiated for 2 weeks at 37 °C, 5%

(v/v) CO2. Conditionally immortalized glomerular endothelial cells (ciGENCs, clone 1D4) were kindly provided by Simon C. Satchell29. Details about the culture conditions can be found online

in the supplementary materials. ANIMAL STUDIES All experiments were performed using 6–8 weeks old male Thy1.1 transgenic mice from own breeding. These mice have been backcrossed into the

C57BL/6 J for 10 generations. The animal experiments were approved by the Animal Ethics Committee of the Radboud University Nijmegen and performed in accordance with the European Communities

Council Directive (86/609/EEC). The setup of the animal experiments is shown in Fig. 7. More details can be found online in the supplementary materials. ALBUMIN/CREATININE RATIO (ACR) Urine

albumin of the mice was determined using the Mancini immunodiffusion method. The creatinine values were determined at the Radboudumc laboratory of diagnostics (RLD), Radboudumc, Nijmegen,

the Netherlands, using a modulation analyser Cobas C8000-1/-2 (Roche Diagnostics). WESTERN BLOTTING RIPA buffer (Cell signaling, #9806) supplemented with 1 mM protease inhibitor

phenylmethylsulfonyl fluoride (PMSF) was used for protein extraction of hPECs and ciGENCs. Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Fischer

Scientific). Samples were prepared using 4x Laemmli buffer (Bio-Rad) supplemented with beta-mercaptoethanol. Per sample, 25 µg protein was loaded on a 12% (v/v) Sodium Dodecyl

Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel and transferred to a nitrocellulose membrane. Antibodies used to detect the pS6RP, the S6RP and the housekeeping gene γ-tubulin are

shown in Table 1. These antibodies are well established and commonly used18,19,21,30,31,32,33. The fluorescent signal was analysed with the Odyssey Imaging System (LI-COR Biosciences) and

Odyssey Image studio Software (version 4.0). BROMODEOXYURIDINE (BRDU) ASSAY We performed the BrdU proliferation ELISA, according to manufacturer’s protocol (BrdU Cell Proliferation ELISA

Kit, Abcam, ab126556). Cells were treated with different rapamycin concentrations and their respective DMSO controls for 24 hours. The BrdU antibody was incubated for 15 hours. The hPECs

were seeded in a density of 20,000 cells/cm2 two days prior to rapamycin or DMSO exposure, ciGENCs were seeded in a density of 20,000 cells/cm2 4 days before starting the experiment. TISSUE

STAINING All stainings were performed on 4 µm formalin-fixed paraffin-embedded (FFPE) kidney sections. Antigen retrieval was performed in citrate buffer (pH 6) or Tris-EDTA buffer (pH 9) at

boiling point for 13 minutes. Details about primary- and matching secondary antibodies used are described in Table 1. Fluorescent stained slices were mounted in DAPI Fluoromount-G®

(SouthernBiotech). Co-staining of synaptopodin and desmin was performed to detect injured podocytes. The synaptopodin antibody used was detected with Alexa Fluor donkey anti-goat 546

followed by a 10% (v/v) goat serum in PBS blocking step. Next, the secondary antibody against desmin was applied. To assess pS6RP in podocytes and PECs a triple staining was performed. The

P-S6 Ribosomal Protein (S235/236) antibody was visualized using the Tyramide Signal Amplification (TSA) kit #16 (Molecular probes, life technologies, T20926). Subsequently podocytes and PECs

were stained with antibodies against synaptopodin and SSeCKS, respectively. To detect proliferating endothelial cells, sections were incubated with CD34 and ki-67 antibodies. Secondary

antibodies were used as shown in Table 1. For the detection of podocyte nuclei, sections were incubated with the nuclear marker DACH1 combined with the podocyte maker synaptopodin. The tuft

area was measured using CaseViewer software (3DHISTECH Ltd.) and DACH1 positive cells in the glomeruli were counted. A minimum of 48 glomeruli and a maximum of 110 glomeruli per mouse were

scored. From each scored glomerulus we calculated the ratio of DACH1 positive podocytes per tuft area (mm2) and took the average of these ratios for each mouse. For detecting ki-67 positive

cells immunohistochemically, endogenous peroxidases as well as endogenous biotin, biotin receptors, and avidin were blocked using 3% (v/v) hydrogen peroxide and the Avidin/Biotin blocking

Kit (Vector laboratories SP-2001). As primary antibody anti-ki-67 antibody (Table 1) was used, which was detected using a biotinylated Goat Anti-Rabbit IgG Antibody (Vector Laboratories,

BA-1000) and the VECTASTAIN® Elite®ABC-HRP Kit (Vector laboratories, PK-6100). The 3,3′-diaminobenzidine (DAB) horseradish peroxidase was used as substrate. Periodic acid–Schiff (PAS)

staining without haematoxylin stain was performed afterwards. Per mouse, the ki-67 positive cells were counted in a minimum of 57 and a maximum of 114 glomeruli. A PAS staining was performed

to identify affected glomeruli. A minimum of 58 glomeruli and a maximum of 122 glomeruli per mouse were scored. STATISTICS The differences between sirolimus and phosal treated mice

concerning podocyte count, glomerular proliferation, the percentage of affected glomeruli and desmin expression was statistically tested using a two-tailed Mann-Whitney U test with a

confidence interval of 95%. Differences in the albumin/creatinine ratio was tested using a 2-way ANOVA including a Bonferroni post-test for the first experiment and for the second experiment

a two-tailed Mann-Whitney U test was used. For the _in vitro_ results we used a One-way ANOVA with a Bonferroni’s Multiple Comparison Test. A P value below 0.05 was considered as

significant. DATA AVAILABILITY The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Additional

information and data is available from the corresponding author on reasonable request. REFERENCES * Rosenberg, A. Z. & Kopp, J. B. Focal Segmental Glomerulosclerosis. _Clin J Am Soc

Nephrol_ 12, 502–517, https://doi.org/10.2215/CJN.05960616 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * D’Agati, V. D., Fogo, A. B., Bruijn, J. A. & Jennette, J. C.

Pathologic classification of focal segmental glomerulosclerosis: A working proposal. _American Journal of Kidney Diseases_ 43, 368–382, https://doi.org/10.1053/j.ajkd.2003.10.024 (2004).

Article  PubMed  Google Scholar  * Fogo, A. B. Causes and pathogenesis of focal segmental glomerulosclerosis. _Nat Rev Nephrol_ 11, 76–87, https://doi.org/10.1038/nrneph.2014.216 (2015).

Article  CAS  PubMed  Google Scholar  * Meliambro, K., Schwartzman, M., Cravedi, P. & Campbell, K. N. The Impact of Histologic Variants on FSGS Outcomes. _Int Sch Res Notices_ 2014,

913690, https://doi.org/10.1155/2014/913690 (2014). Article  PubMed  PubMed Central  Google Scholar  * Smeets, B. _et al_. Tracing the origin of glomerular extracapillary lesions from

parietal epithelial cells. _J Am Soc Nephrol_ 20, 2604–2615, https://doi.org/10.1681/ASN.2009010122 (2009). Article  PubMed  PubMed Central  Google Scholar  * Suzuki, T. _et al_. Genetic

podocyte lineage reveals progressive podocytopenia with parietal cell hyperplasia in a murine model of cellular/collapsing focal segmental glomerulosclerosis. _Am J Pathol_ 174, 1675–1682,

https://doi.org/10.2353/ajpath.2009.080789 (2009). Article  PubMed  PubMed Central  Google Scholar  * Dijkman, H., Smeets, B., van der Laak, J., Steenbergen, E. & Wetzels, J. The

parietal epithelial cell is crucially involved in human idiopathic focal segmental glomerulosclerosis. _Kidney Int_ 68, 1562–1572, https://doi.org/10.1111/j.1523-1755.2005.00568.x (2005).

Article  PubMed  Google Scholar  * Smeets, B. _et al_. Parietal epithelial cells participate in the formation of sclerotic lesions in focal segmental glomerulosclerosis. _J Am Soc Nephrol_

22, 1262–1274, https://doi.org/10.1681/ASN.2010090970 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kuppe, C. _et al_. Common histological patterns in glomerular epithelial

cells in secondary focal segmental glomerulosclerosis. _Kidney Int_ 88, 990–998, https://doi.org/10.1038/ki.2015.116 (2015). Article  CAS  PubMed  Google Scholar  * Hamatani, H. _et al_.

Expression of a novel stress-inducible protein, sestrin 2, in rat glomerular parietal epithelial cells. _Am J Physiol Renal Physiol_ 307, F708–717,

https://doi.org/10.1152/ajprenal.00625.2013 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Eymael, J. _et al_. CD44 is required for the pathogenesis of experimental

crescentic glomerulonephritis and collapsing focal segmental glomerulosclerosis. _Kidney Int_ 93, 626–642, https://doi.org/10.1016/j.kint.2017.09.020 (2018). Article  CAS  PubMed  Google

Scholar  * Smeets, B. _et al_. Podocyte changes upon induction of albuminuria in Thy-1.1 transgenic mice. _Nephrol Dial Transplant_ 18, 2524–2533 (2003). Article  CAS  Google Scholar  *

Smeets, B. _et al_. The parietal epithelial cell: a key player in the pathogenesis of focal segmental glomerulosclerosis in Thy-1.1 transgenic mice. _J Am Soc Nephrol_ 15, 928–939 (2004).

Article  Google Scholar  * Burnworth, B. _et al_. SSeCKS sequesters cyclin D1 in glomerular parietal epithelial cells and influences proliferative injury in the glomerulus. _Lab Invest_ 92,

499–510, https://doi.org/10.1038/labinvest.2011.199 (2012). Article  CAS  PubMed  Google Scholar  * Fatima, H. _et al_. Parietal epithelial cell activation marker in early recurrence of FSGS

in the transplant. _Clin J Am Soc Nephrol_ 7, 1852–1858, https://doi.org/10.2215/CJN.10571011 (2012). Article  PubMed  PubMed Central  Google Scholar  * Miesen, L., Steenbergen, E. &

Smeets, B. Parietal cells-new perspectives in glomerular disease. _Cell Tissue Res_ 369, 237–244, https://doi.org/10.1007/s00441-017-2600-5 (2017). Article  PubMed  PubMed Central  Google

Scholar  * Godel, M. _et al_. Role of mTOR in podocyte function and diabetic nephropathy in humans and mice. _J Clin Invest_ 121, 2197–2209, https://doi.org/10.1172/JCI44774 (2011). Article

  CAS  PubMed  PubMed Central  Google Scholar  * Inoki, K. _et al_. mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice. _J Clin Invest_ 121,

2181–2196, https://doi.org/10.1172/JCI44771 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ito, N. _et al_. mTORC1 activation triggers the unfolded protein response in

podocytes and leads to nephrotic syndrome. _Lab Invest_ 91, 1584–1595, https://doi.org/10.1038/labinvest.2011.135 (2011). Article  CAS  PubMed  Google Scholar  * Mao, J. _et al_. Mammalian

target of rapamycin complex 1 activation in podocytes promotes cellular crescent formation. _Am J Physiol Renal Physiol_ 307, F1023–1032, https://doi.org/10.1152/ajprenal.00018.2014 (2014).

Article  CAS  PubMed  Google Scholar  * Zschiedrich, S. _et al_. Targeting mTOR Signaling Can Prevent the Progression of FSGS. _J Am Soc Nephrol_ 28, 2144–2157,

https://doi.org/10.1681/ASN.2016050519 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kurayama, R. _et al_. Role of amino acid transporter LAT2 in the activation of mTORC1

pathway and the pathogenesis of crescentic glomerulonephritis. _Lab Invest_ 91, 992–1006, https://doi.org/10.1038/labinvest.2011.43 (2011). Article  CAS  PubMed  Google Scholar  *

McNicholas, B. A. _et al_. Reducing mTOR augments parietal epithelial cell density in a model of acute podocyte depletion and in aged kidneys. _Am J Physiol Renal Physiol_ 311, F626–639,

https://doi.org/10.1152/ajprenal.00196.2016 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Iruela-Arispe, L. _et al_. Participation of glomerular endothelial cells in the

capillary repair of glomerulonephritis. _Am J Pathol_ 147, 1715–1727 (1995). CAS  PubMed  PubMed Central  Google Scholar  * Li, J., Kim, S. G. & Blenis, J. Rapamycin: one drug, many

effects. _Cell Metab_ 19, 373–379, https://doi.org/10.1016/j.cmet.2014.01.001 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Benjamin, D., Colombi, M., Moroni, C. &

Hall, M. N. Rapamycin passes the torch: a new generation of mTOR inhibitors. _Nat Rev Drug Discov_ 10, 868–880, https://doi.org/10.1038/nrd3531 (2011). Article  CAS  PubMed  Google Scholar 

* Pallet, N. & Legendre, C. Adverse events associated with mTOR inhibitors. _Expert Opin Drug Saf_ 12, 177–186, https://doi.org/10.1517/14740338.2013.752814 (2013). Article  CAS  PubMed

  Google Scholar  * Kietzmann, L. _et al_. MicroRNA-193a Regulates the Transdifferentiation of Human Parietal Epithelial Cells toward a Podocyte Phenotype. _J Am Soc Nephrol_ 26, 1389–1401,

https://doi.org/10.1681/ASN.2014020190 (2015). Article  CAS  PubMed  Google Scholar  * Satchell, S. C. _et al_. Conditionally immortalized human glomerular endothelial cells expressing

fenestrations in response to VEGF. _Kidney Int_ 69, 1633–1640, https://doi.org/10.1038/sj.ki.5000277 (2006). Article  CAS  PubMed  Google Scholar  * Daher, B. _et al_. Genetic Ablation of

the Cystine Transporter xCT in PDAC Cells Inhibits mTORC1, Growth, Survival, and Tumor Formation via Nutrient and Oxidative Stresses. _Cancer Res_ 79, 3877–3890,

https://doi.org/10.1158/0008-5472.CAN-18-3855 (2019). Article  CAS  PubMed  Google Scholar  * Mathieu, J. _et al_. Folliculin regulates mTORC1/2 and WNT pathways in early human pluripotency.

_Nat Commun_ 10, 632, https://doi.org/10.1038/s41467-018-08020-0 (2019). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Sunamura, N., Iwashita, S., Enomoto, K., Kadoshima, T.

& Isono, F. Loss of the fragile X mental retardation protein causes aberrant differentiation in human neural progenitor cells. _Sci Rep_ 8, 11585,

https://doi.org/10.1038/s41598-018-30025-4 (2018). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Ohashi, T., Yamamoto, T., Yamanashi, Y. & Ohsugi, M. Human TUBG2 gene is

expressed as two splice variant mRNA and involved in cell growth. _FEBS Lett_ 590, 1053–1063, https://doi.org/10.1002/1873-3468.12163 (2016). Article  CAS  PubMed  Google Scholar  Download

references ACKNOWLEDGEMENTS The authors wish to thank Rutger Maas, PhD, MD (Department of Nephrology, Radboudumc, Nijmegen) for the technical support with the ciGENC model. In addition we

kindly thank Simon C. Satchell, PhD (Academic Renal Unit, University of Bristol, UK) providing us with the conditionally immortalized glomerular endothelial cell line. The study was funded

by the Dutch Kidney foundation (grant 14A3D104) and The Netherlands Organization for Scientific Research (NWO VIDI grant: 016.156.363). AUTHOR INFORMATION Author notes * These authors

contributed equally: Johan van der Vlag and Bart Smeets. AUTHORS AND AFFILIATIONS * Department of pathology, Radboud Institute for Molecular Life Sciences, Radboud university medical center,

Nijmegen, The Netherlands Laura Miesen, Jennifer Eymael, Brigith Willemsen, Fieke Mooren, Henry Dijkman, Jitske Jansen & Bart Smeets * School of Biomedical Sciences, University of

Plymouth, Plymouth, UK Shagun Sharma * Department of nephrology, Radboud Institute for Molecular Life Sciences, Radboud Institute for Health Sciences, Radboud university medical center,

Nijmegen, The Netherlands Shagun Sharma, Markus A. Loeven, Marinka Bakker-van Bebber, Jack F. M. Wetzels & Johan van der Vlag * Institute of Cellular and Integrative Physiology, Center

for Experimental Medicine, University Medical Center Hamburg -Eppendorf, Hamburg, Germany Catherine Meyer-Schwesinger * Department of pediatric nephrology, Radboud Institute for Molecular

Life Sciences, Radboud university medical center, Amalia Children’s Hospital, Nijmegen, The Netherlands Jitske Jansen Authors * Laura Miesen View author publications You can also search for

this author inPubMed Google Scholar * Jennifer Eymael View author publications You can also search for this author inPubMed Google Scholar * Shagun Sharma View author publications You can

also search for this author inPubMed Google Scholar * Markus A. Loeven View author publications You can also search for this author inPubMed Google Scholar * Brigith Willemsen View author

publications You can also search for this author inPubMed Google Scholar * Marinka Bakker-van Bebber View author publications You can also search for this author inPubMed Google Scholar *

Fieke Mooren View author publications You can also search for this author inPubMed Google Scholar * Catherine Meyer-Schwesinger View author publications You can also search for this author

inPubMed Google Scholar * Henry Dijkman View author publications You can also search for this author inPubMed Google Scholar * Jack F. M. Wetzels View author publications You can also search

for this author inPubMed Google Scholar * Jitske Jansen View author publications You can also search for this author inPubMed Google Scholar * Johan van der Vlag View author publications

You can also search for this author inPubMed Google Scholar * Bart Smeets View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS L.M. carried out

the experiments and was supported by J.E., S.S., B.W., F.M., H.D., M.B.v.B. and M.A.L., C.M.S. provided the used PEC cell line. L.M wrote the manuscript with support of J.E., H.D., C.M.S,

J.F.M.W., J.J., J.v.d.V. and B.S. J.F.M.W, J.v.d.V and B.S. developed the theory and supervised the project. CORRESPONDING AUTHOR Correspondence to Bart Smeets. ETHICS DECLARATIONS COMPETING

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http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Miesen, L., Eymael, J., Sharma, S. _et al._ Inhibition of mTOR delayed but could

not prevent experimental collapsing focal segmental glomerulosclerosis. _Sci Rep_ 10, 8580 (2020). https://doi.org/10.1038/s41598-020-65352-y Download citation * Received: 20 September 2019

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