Disruption of the Rab7-Dependent Final Common Pathway of Endosomal and Autophagic Processing Results in a Severe Podocytopathy.

SIGNIFICANCE STATEMENT: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates and complex morphology, such as podocytes. To improve our understanding on how disturbances of these trafficking pathways are linked to podocyte depletion and slit diaphragm (SD) injury, the authors explored the role of the small GTPase Rab7, which is linked to endosomal, lysosomal, and autophagic pathways, using as model systems mice and Drosophila with podocyte-specific or nephrocyte-specific loss of Rab7, and a human podocyte cell line depleted for Rab7. Their findings point to maturation and fusion events during endolysosomal and autophagic maturation as key processes for podocyte homeostasis and function and identify altered lysosomal pH values as a putative novel mechanism for podocytopathies. BACKGROUND: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates, such as podocytes. How disturbances within these trafficking pathways may act as factors in proteinuric glomerular diseases is poorly understood. METHODS: To explore how disturbances in trafficking pathways may act as factors in proteinuric glomerular diseases, we focused on Rab7, a highly conserved GTPase that controls the homeostasis of late endolysosomal and autophagic processes. We generated mouse and Drosophila in vivo models lacking Rab7 exclusively in podocytes or nephrocytes, and performed histologic and ultrastructural analyses. To further investigate Rab7 function on lysosomal and autophagic structures, we used immortalized human cell lines depleted for Rab7. RESULTS: Depletion of Rab7 in mice, Drosophila , and immortalized human cell lines resulted in an accumulation of diverse vesicular structures resembling multivesicular bodies, autophagosomes, and autoendolysosomes. Mice lacking Rab7 developed a severe and lethal renal phenotype with early-onset proteinuria and global or focal segmental glomerulosclerosis, accompanied by an altered distribution of slit diaphragm proteins. Remarkably, structures resembling multivesicular bodies began forming within 2 weeks after birth, prior to the glomerular injuries. In Drosophila nephrocytes, Rab7 knockdown resulted in the accumulation of vesicles and reduced slit diaphragms. In vitro , Rab7 knockout led to similar enlarged vesicles and altered lysosomal pH values, accompanied by an accumulation of lysosomal marker proteins. CONCLUSIONS: Disruption within the final common pathway of endocytic and autophagic processes may be a novel and insufficiently understood mechanism regulating podocyte health and disease.

Activation of Hippo Pathway Damages Slit Diaphragm by Deprivation of Ajuba Proteins.

SIGNIFICANCE STATEMENT: Nuclear exclusion of the cotranscription factor YAP, which is a consequence of activation of the Hippo signaling pathway, leads to FSGS and podocyte apoptosis. Ajuba proteins play an important role in the glomerular filtration barrier by keeping the Hippo pathway inactive. In nephrocytes from Drosophila melanogaster , a well-established model system for podocyte research, Ajuba proteins ensure slit diaphragm (SD) formation and function. Hippo pathway activation leads to mislocalization of Ajuba proteins, decreased SD formation, rearrangement of the actin cytoskeleton, and increased SD permeability. Targeting the kinases of the Hippo pathway with specific inhibitors in the glomerulus could, therefore, be a promising strategy for therapy of FSGS. BACKGROUND: The highly conserved Hippo pathway, which regulates organ growth and cell proliferation by inhibiting transcriptional cofactors YAP/TAZ, plays a special role in podocytes, where activation of the pathway leads to apoptosis. The Ajuba family proteins (Ajuba, LIM domain-containing protein 1 (LIMD1) and Wilms tumor protein 1-interacting protein [WTIP]) can bind and inactivate large tumor suppressor kinases 1 and 2, (LATS1/2) two of the Hippo pathway key kinases. WTIP, furthermore, connects the slit diaphragm (SD), the specialized cell-cell junction between podocytes, with the actin cytoskeleton. METHODS: We used garland cell nephrocytes of Drosophila melanogaster to monitor the role of Ajuba proteins in Hippo pathway regulation and structural integrity of the SD. Microscopy and functional assays analyzed the interplay between Ajuba proteins and LATS2 regarding expression, localization, interaction, and effects on the functionality of the SD. RESULTS: In nephrocytes, the Ajuba homolog Djub recruited Warts (LATS2 homolog) to the SD. Knockdown of Djub activated the Hippo pathway. Reciprocally, Hippo activation reduced the Djub level. Both Djub knockdown and Hippo activation led to morphological changes in the SD, rearrangement of the cortical actin cytoskeleton, and increased SD permeability. Knockdown of Warts or overexpression of constitutively active Yki prevented these effects. In podocytes, Hippo pathway activation or knockdown of YAP also decreased the level of Ajuba proteins. CONCLUSIONS: Ajuba proteins regulate the structure and function of the SD in nephrocytes, connecting the SD protein complex to the actin cytoskeleton and maintaining the Hippo pathway in an inactive state. Hippo pathway activation directly influencing Djub expression suggests a self-amplifying feedback mechanism.

Loss of surface transport is a main cellular pathomechanism of CRB2 variants causing podocytopathies.

Crumbs2 (CRB2) is a central component of the renal filtration barrier and part of the slit diaphragm, a unique cell contact formed by glomerular podocytes. Some CRB2 variants cause recessive inherited forms of steroid-resistant nephrotic syndrome. However, the disease-causing potential of numerous CRB2 variants remains unknown. Here, we report the establishment of a live-cell imaging-based assay, allowing a quantitative evaluation of the pathogenic potential of so far non-categorized CRB2 variants. Based on in silico data analysis and protein prediction software, putative disease-associated CRB2 missense variants were selected, expressed as CRB2-GFP fusion proteins, and analyzed in reporter cell lines with BFP-labeled plasma membrane. We found that in comparison with PM-localized WT, disease-associated CRB2 variants remained predominantly at the ER. Accumulation at the ER was also present for several non-characterized CRB2 variants and variants in which putative disulfide bridge-forming cysteines were replaced. Strikingly, WT CRB2 retained inside the ER in cells lacking protein disulfide isomerase A3, indicating that posttranslational modification, especially the formation of disulfide bridges, is a crucial step for the CRB2 PM transport.

Rap1 Activity Is Essential for Focal Adhesion and Slit Diaphragm Integrity.

Glomerular podocytes build, with their intercellular junctions, part of the kidney filter. The podocyte cell adhesion protein, nephrin, is essential for developing and maintaining slit diaphragms as functional loss in humans results in heavy proteinuria. Nephrin expression and function are also altered in many adult-onset glomerulopathies. Nephrin signals from the slit diaphragm to the actin cytoskeleton and integrin ß1 at focal adhesions by recruiting Crk family proteins, which can interact with the Rap guanine nucleotide exchange factor 1 C3G. As Rap1 activity affects focal adhesion formation, we hypothesize that nephrin signals via Rap1 to integrin ß. To address this issue, we combined Drosophila in vivo and mammalian cell culture experiments. We find that Rap1 is necessary for correct targeting of integrin ß to focal adhesions in Drosophila nephrocytes, which also form slit diaphragm-like structures. In the fly, the Rap1 activity is important for signaling of the nephrin ortholog to integrin ß, as well as for nephrin-dependent slit diaphragm integrity. We show by genetic interaction experiments that Rap1 functions downstream of nephrin signaling to integrin ß and downstream of nephrin signaling necessary for slit diaphragm integrity. Similarly, in human podocyte culture, nephrin activation results in increased activation of Rap1. Thus, Rap1 is necessary for downstream signal transduction of nephrin to integrin ß.

A Novel Homozygous Mutation in the COL4A4 Gene (Gly1436del) Causing Alport Syndrome Exposed by Pregnancy: A Case Report and Review of the Literature.

BACKGROUND: Alport syndrome results from a hereditary defect of collagen IV synthesis. This causes progressive glomerular disease, ocular abnormalities, and inner ear impairment. Case Presentation. Herein, we present a case of Alport syndrome in a 28-year-old woman caused by a novel mutation (Gly1436del) in the COL4A4 gene that was not unveiled until her first pregnancy. Within the 29th pregnancy week, our patient presented with massive proteinuria and nephrotic syndrome. Light microscopic examination of a kidney biopsy showed typical histological features of segmental sclerosis, and electron microscopy revealed extensive podocyte alterations as well as thickness of glomerular basement membranes with splitting of the lamina densa. One and a half years after childbirth, renal function deteriorated to a preterminal stage, whereas nephrotic syndrome subsided quickly after delivery. CONCLUSION: This case report highlights the awareness of atypical AS courses and emphasizes the importance of genetic testing in such cases.

TrkC Is Essential for Nephron Function and Trans-Activates Igf1R Signaling.

BACKGROUND: Injury to kidney podocytes often results in chronic glomerular disease and consecutive nephron malfunction. For most glomerular diseases, targeted therapies are lacking. Thus, it is important to identify novel signaling pathways contributing to glomerular disease. Neurotrophic tyrosine kinase receptor 3 (TrkC) is expressed in podocytes and the protein transmits signals to the podocyte actin cytoskeleton. METHODS: Nephron-specific TrkC knockout (TrkC-KO) and nephron-specific TrkC-overexpressing (TrkC-OE) mice were generated to dissect the role of TrkC in nephron development and maintenance. RESULTS: Both TrkC-KO and TrkC-OE mice exhibited enlarged glomeruli, mesangial proliferation, basement membrane thickening, albuminuria, podocyte loss, and aspects of FSGS during aging. Igf1 receptor (Igf1R)-associated gene expression was dysregulated in TrkC-KO mouse glomeruli. Phosphoproteins associated with insulin, erb-b2 receptor tyrosine kinase (Erbb), and Toll-like receptor signaling were enriched in lysates of podocytes treated with the TrkC ligand neurotrophin-3 (Nt-3). Activation of TrkC by Nt-3 resulted in phosphorylation of the Igf1R on activating tyrosine residues in podocytes. Igf1R phosphorylation was increased in TrkC-OE mouse kidneys while it was decreased in TrkC-KO kidneys. Furthermore, TrkC expression was elevated in glomerular tissue of patients with diabetic kidney disease compared with control glomerular tissue. CONCLUSIONS: Our results show that TrkC is essential for maintaining glomerular integrity. Furthermore, TrkC modulates Igf-related signaling in podocytes.

LIM and SH3 protein 1 (LASP-1): A novel link between the slit membrane and actin cytoskeleton dynamics in podocytes.

The foot processes of podocytes exhibit a dynamic actin cytoskeleton, which maintains their complex cell structure and antagonizes the elastic forces of the glomerular capillary. Interdigitating secondary foot processes form a highly selective filter for proteins in the kidney, the slit membrane. Knockdown of slit membrane components such as Nephrin or Neph1 and cytoskeletal adaptor proteins such as CD2AP in mice leads to breakdown of the filtration barrier with foot process effacement, proteinuria, and early death of the mice. Less is known about the crosstalk between the slit membrane-associated proteins and cytoskeletal components inside the podocyte foot processes. Our study shows that LASP-1, an actin-binding protein, is highly expressed in podocytes. Electron microscopy studies demonstrate that LASP-1 is found at the slit membrane suggesting a role in anchoring slit membrane components to the actin cytoskeleton. Live cell imaging experiments with transfected podocytes reveal that LASP-1 is either part of a highly dynamic granular complex or a static, actin cytoskeleton-bound protein. We identify CD2AP as a novel LASP-1 binding partner that regulates its association with the actin cytoskeleton. Activation of the renin-angiotensin-aldosterone system, which is crucial for podocyte function, leads to phosphorylation and altered localization of LASP-1. In vivo studies using the Drosophila nephrocyte model indicate that Lasp is necessary for the slit membrane integrity and functional filtration.

Nephrin Signaling Results in Integrin ß1 Activation.

BACKGROUND: Patients with certain mutations in the gene encoding the slit diaphragm protein Nephrin fail to develop functional slit diaphragms and display severe proteinuria. Many adult-onset glomerulopathies also feature alterations in Nephrin expression and function. Nephrin signals from the podocyte slit diaphragm to the Actin cytoskeleton by recruiting proteins that can interact with C3G, a guanine nucleotide exchange factor of the small GTPase Rap1. Because Rap activity affects formation of focal adhesions, we hypothesized that Nephrin transmits signals to the Integrin receptor complex, which mediates podocyte adhesion to the extracellular matrix. METHODS: To investigate Nephrin's role in transmitting signals to the Integrin receptor complex, we conducted genetic studies in Drosophila nephrocytes and validated findings from Drosophila in a cultured human podocyte model. RESULTS: Drosophila nephrocytes form a slit diaphragm-like filtration barrier and express the Nephrin ortholog Sticks and stones (Sns). A genetic screen identified c3g as necessary for nephrocyte function. In vivo, nephrocyte-specific gene silencing of sns or c3g compromised nephrocyte filtration and caused nephrocyte diaphragm defects. Nephrocytes with impaired Sns or C3G expression displayed an altered localization of Integrin and the Integrin-associated protein Talin. Furthermore, gene silencing of c3g partly rescued nephrocyte diaphragm defects of an sns overexpression phenotype, pointing to genetic interaction of sns and c3g in nephrocytes. We also found that activated Nephrin recruited phosphorylated C3G and resulted in activation of Integrin ß1 in cultured podocytes. CONCLUSIONS: Our findings suggest that Nephrin can mediate a signaling pathway that results in activation of Integrin ß1 at focal adhesions, which may affect podocyte attachment to the extracellular matrix.

Tropomyosin-related kinase C (TrkC) enhances podocyte migration by ERK-mediated WAVE2 activation.

Podocyte malfunction is central to glomerular diseases and is marked by defective podocyte intercellular junctions and actin cytoskeletal dynamics. Podocytes share many morphologic features with neurons, so that similar sets of proteins appear to regulate cell process formation. One such protein is the tropomyosin-related kinase C (TrkC). TrkC deficiency in mice leads to proteinuria as a surrogate of defective kidney filter function. Activation of endogenous TrkC by its ligand neurotrophin-3 resulted in increased podocyte migration-a surrogate of podocyte actin dynamics in vivo. Employing a mutagenesis approach, we found that the Src homologous and collagen-like (Shc) binding site Tyr516 within the TrkC cytoplasmic domain was necessary for TrkC-induced migration of podocytes. TrkC activation led to a mobility shift of Wiskott-Aldrich syndrome family verprolin-homologous protein (WAVE)-2 which is known to orchestrate Arp2/3 activation and actin polymerization. Chemical inactivation of Erk or mutagenesis of 2 of 4 known Erk target sites within WAVE2, Thr346 and Ser351, abolished the TrkC-induced WAVE2 mobility shift. Knockdown of WAVE2 by shRNA abolished TrkC-induced podocyte migration. In summary, TrkC signals to the podocyte actin cytoskeleton to induce migration by phosphorylating WAVE2 Erk dependently. This signaling mechanism may be important for TrkC-mediated cytoskeletal dynamics in podocyte disease.-Gromnitza, S., Lepa, C., Weide, T., Schwab, A., Pavenstädt, H., George, B. Tropomyosin-related kinase C (TrkC) enhances podocyte migration by ERK-mediated WAVE2 activation.

Pals1 Haploinsufficiency Results in Proteinuria and Cyst Formation.

The nephron is the basic physiologic subunit of the mammalian kidney and is made up of several apicobasally polarized epithelial cell types. The process of apicobasal polarization in animal cells is controlled by the evolutionarily conserved Crumbs (CRB), Partitioning-defective, and Scribble protein complexes. Here, we investigated the role of protein associated with LIN-7 1 (Pals1, also known as Mpp5), a core component of the apical membrane-determining CRB complex in the nephron. Pals1 interacting proteins, including Crb3 and Wwtr1/Taz, have been linked to renal cyst formation in mice before. Immunohistologic analysis revealed Pals1 expression in renal tubular cells and podocytes of human kidneys. Mice lacking one Pals1 allele (functionally haploid for Pals1) in nephrons developed a fully penetrant phenotype, characterized by cyst formation and severe defects in renal barrier function, which led to death within 6-8 weeks. In Drosophila nephrocytes, deficiency of the Pals1 ortholog caused alterations in slit-diaphragm-like structures. Additional studies in epithelial cell culture models revealed that Pals1 functions as a dose-dependent upstream regulator of the crosstalk between Hippo- and TGF-ß-mediated signaling. Furthermore, Pals1 haploinsufficiency in mouse kidneys associated with the upregulation of Hippo pathway target genes and marker genes of TGF-ß signaling, including biomarkers of renal diseases. These findings support a link between apical polarity proteins and renal diseases, especially renal cyst diseases. Further investigation of the Pals1-linked networks is required to decipher the mechanisms underlying the pathogenesis of these diseases.

Crk1/2 and CrkL form a hetero-oligomer and functionally complement each other during podocyte morphogenesis.

Activation of the slit diaphragm protein nephrin induces actin cytoskeletal remodeling, resulting in lamellipodia formation in podocytes in vitro in a phosphatidylinositol-3 kinase-, focal adhesion kinase-, Cas-, and Crk1/2-dependent fashion. In mice, podocyte-specific deletion of Crk1/2 prevents or attenuates foot process effacement in two models of podocyte injury. This suggests that cellular mechanisms governing lamellipodial protrusion in vitro are similar to those in vivo during foot process effacement. As Crk1/2-null mice developed and aged normally, we tested whether the Crk1/2 paralog, CrkL, functionally complements Crk1/2 in a podocyte-specific context. Podocyte-specific CrkL-null mice, like podocyte-specific Crk1/2-null mice, developed and aged normally but were protected from protamine sulfate-induced foot process effacement. Simultaneous podocyte-specific deletion of Crk1/2 and CrkL resulted in albuminuria detected by 6 weeks postpartum and associated with altered podocyte process architecture. Nephrin-induced lamellipodia formation in podocytes in vitro was CrkL-dependent. CrkL formed a hetero-oligomer with Crk2 and, like Crk2, was recruited to tyrosine phosphorylated nephrin. Thus, Crk1/2 and CrkL are physically linked, functionally complement each other during podocyte foot process spreading, and together are required for developing typical foot process architecture.

Signaling from the podocyte intercellular junction to the actin cytoskeleton.

Observations of hereditary glomerular disease support the contention that podocyte intercellular junction proteins are essential for junction formation and maintenance. Genetic deletion of most of these podocyte intercellular junction proteins results in foot process effacement and proteinuria. This review focuses on the current understanding of molecular mechanisms by which podocyte intercellular junction proteins such as the nephrin-neph1-podocin-receptor complex coordinate cytoskeletal dynamics and thus intercellular junction formation, maintenance, and injury-dependent remodeling.

Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease.

The morphology of healthy podocyte foot processes is necessary for maintaining the characteristics of the kidney filtration barrier. In most forms of glomerular disease, abnormal filter barrier function results when podocytes undergo foot process spreading and retraction by remodeling their cytoskeletal architecture and intercellular junctions during a process known as effacement. The cell adhesion protein nephrin is necessary for establishing the morphology of the kidney podocyte in development by transducing from the specialized podocyte intercellular junction phosphorylation-mediated signals that regulate cytoskeletal dynamics. The present studies extend our understanding of nephrin function by showing that nephrin activation in cultured podocytes induced actin dynamics necessary for lamellipodial protrusion. This process required a PI3K-, Cas-, and Crk1/2-dependent signaling mechanism distinct from the previously described nephrin-Nck1/2 pathway necessary for assembly and polymerization of actin filaments. Our present findings also support the hypothesis that mechanisms governing lamellipodial protrusion in culture are similar to those used in vivo during foot process effacement in a subset of glomerular diseases. In mice, podocyte-specific deletion of Crk1/2 prevented foot process effacement in one model of podocyte injury and attenuated foot process effacement and associated proteinuria in a delayed fashion in a second model. In humans, focal adhesion kinase and Cas phosphorylation - markers of focal adhesion complex-mediated Crk-dependent signaling - was induced in minimal change disease and membranous nephropathy, but not focal segmental glomerulosclerosis. Together, these observations suggest that activation of a Cas-Crk1/2-dependent complex is necessary for foot process effacement observed in distinct subsets of human glomerular diseases.

Podocyte-specific deletion of Myh9 encoding nonmuscle myosin heavy chain 2A predisposes mice to glomerulopathy.

Genome-wide association studies linked single-nucleotide polymorphisms (SNPs) at the MYH9 locus to chronic kidney disease among African-Americans, particularly glomerular diseases such as HIV nephropathy and idiopathic focal and segmental glomerulosclerosis (FSGS). However, these MYH9 SNPs are intronic, and despite extensive sequencing, a causal variant remains elusive. To investigate the role of MYH9 in kidney disease, we selectively deleted Myh9 from mouse podocytes and found that mutant C57BL/6 mice did not develop renal insufficiency or proteinuria compared to control littermates, even when the mice were aged for 9 months. To explain the surprisingly normal phenotype, we considered genetic redundancy with the paralog Myh10 in podocytes, but we found that Myh10 was not expressed in podocytes in Myh9-deficient or control mice. We tested whether Myh9 podocyte deletion predisposed mice to glomerulopathy in response to injury by doxorubicin hydrochloride (Adriamycin), and we found that Myh9 podocyte-deleted mice developed proteinuria and glomerulosclerosis, while control mice were resistant. In summary, Myh9 podocyte deletion in C57BL/6 mice results in susceptibility to experimental doxorubicin hydrochloride glomerulopathy. We review evidence that MYH9 dysfunction in humans results in similar susceptibility and place our data, the first examination of Myh9 kidney disease in experimental animals, in the context of recent findings in human kidney disease, including the role of APOL1.

GSK3ß inactivation in podocytes results in decreased phosphorylation of p70S6K accompanied by cytoskeletal rearrangements and inhibited motility.

The inhibition of mTOR kinase after renal transplantation has been associated with podocyte injury and proteinuria; however, the signaling pathways regulating these effects are not well understood. We found that prolonged rapamycin treatment in podocytes leads to an increase in glycogen synthase kinase 3ß (GSK3ß) phosphorylation, resulting in inactivation of total GSK3ß kinase activity. To investigate the cellular consequences of the inactivation of GSK3ß, we used two inhibitors reducing kinase activity and studied the cross talk between GSK3 function and the Akt/mammalian target of rapamycin (mTOR) pathway. Both GSK3 inhibitors reduced the phosphorylation of the mTOR downstream target, p70(S6K), indicating that GSK3 inhibition in podocytes is able to cause similar effects as treatment with rapamycin. Moreover, GSK3 inhibition was accompanied by the reduced expression of slit diaphragm-associated proteins and resulted in an altered cytoskeletal structure and reduced motility of podocytes, suggesting that GSK3 kinase can modulate Akt/mTOR-dependent signaling in podocytes.

Actin-depolymerizing factor cofilin-1 is necessary in maintaining mature podocyte architecture.

Actin dynamics determines podocyte morphology during development and in response to podocyte injury and might be necessary for maintaining normal podocyte morphology. Because podocyte intercellular junction receptor Nephrin plays a role in regulating actin dynamics, and given the described role of cofilin in actin filament polymerization and severing, we hypothesized that cofilin-1 activity is regulated by Nephrin and is necessary in normal podocyte actin dynamics. Nephrin activation induced cofilin dephosphorylation via intermediaries that include phosphatidylinositol 3-kinase, SSH1, 14-3-3, and LIMK in a cell culture model. This Nephrin-induced cofilin activation required a direct interaction between Nephrin and the p85 subunit of phosphatidylinositol 3-kinase. In a similar fashion, cofilin-1 dephosphorylation was observed in a rat model of podocyte injury at a time when foot process spreading is initially observed. To investigate the necessity of cofilin-1 in the glomerulus, podocyte-specific Cfl1 null mice were generated. Cfl1 null podocytes developed normally. However, these mice developed persistent proteinuria by 3 months of age, although they did not exhibit foot process spreading until 8 months, when the rate of urinary protein excretion became more exaggerated. In a mouse model of podocyte injury, protamine sulfate perfusion of the Cfl1 mutant mouse induced a broadened and flattened foot process morphology that was distinct from that observed following perfusion of control kidneys, and mutant podocytes did not recover normal structure following additional perfusion with heparin sulfate. We conclude that cofilin-1 is necessary for maintenance of normal podocyte architecture and for actin structural changes that occur during induction and recovery from podocyte injury.

mTOR regulates expression of slit diaphragm proteins and cytoskeleton structure in podocytes.

The immunosuppressive mammalian target of rapamycin (mTOR) inhibitors can cause proteinuria, especially in kidney and heart transplanted patients. Podocytes play a major role in establishing the selective permeability of the blood-urine filtration barrier. Damage of these cells leads to proteinuria, a hallmark of most glomerular diseases. Interestingly, podocyte damage and focal segmental glomerulosclerosis can occur after treatment with an mTOR inhibitor in some transplant patients. To investigate the mechanisms of mTOR inhibitor-induced podocyte damage, we analyzed the effect of rapamycin on mTOR signaling and cellular function in human podocytes. We found that prolonged rapamycin treatment reduced the expression of total mTOR, which correlates with diminished levels of mTOR phosphorylation at Ser(2448) and Ser(2481). In addition, treatment with rapamycin reduced rictor expression and mTORC2 formation, resulting in a reduced phosphorylation of protein kinase B at Ser(473). The expression level of the slit-diaphragm proteins nephrin and transient receptor potential cation channel 6 as well as the cytoskeletal adaptor protein Nck significantly decreased. Moreover, rapamycin reduced cell adhesion and cell motility, which was accompanied by an enhanced formation of dot-like actin-rich structures. Our data provide new molecular insights explaining which pathways and molecules are affected in podocytes by an imbalanced mTOR function because of rapamycin treatment.