Deletion of IRE1α in podocytes exacerbates diabetic nephropathy in mice.

Protein misfolding in the endoplasmic reticulum (ER) of podocytes contributes to the pathogenesis of glomerular diseases. Protein misfolding activates the unfolded protein response (UPR), a compensatory signaling network. We address the role of the UPR and the UPR transducer, inositol-requiring enzyme 1α (IRE1α), in streptozotocin-induced diabetic nephropathy in mice. Diabetes caused progressive albuminuria in control mice that was exacerbated in podocyte-specific IRE1α knockout (KO) mice. Compared to diabetic controls, diabetic IRE1α KO mice showed reductions in podocyte number and synaptopodin. Glomerular ultrastructure was altered only in diabetic IRE1α KO mice; the major changes included widening of podocyte foot processes and glomerular basement membrane. Activation of the UPR and autophagy was evident in diabetic control, but not diabetic IRE1α KO mice. Analysis of human glomerular gene expression in the JuCKD-Glom database demonstrated induction of genes associated with the ER, UPR and autophagy in diabetic nephropathy. Thus, mice with podocyte-specific deletion of IRE1α demonstrate more severe diabetic nephropathy and attenuation of the glomerular UPR and autophagy, implying a protective effect of IRE1α. These results are consistent with data in human diabetic nephropathy and highlight the potential for therapeutically targeting these pathways.

Analysis of gene expression and use of connectivity mapping to identify drugs for treatment of human glomerulopathies.

Background: Human glomerulonephritis (GN)-membranous nephropathy (MN), focal segmental glomerulosclerosis (FSGS) and IgA nephropathy (IgAN), as well as diabetic nephropathy (DN) are leading causes of chronic kidney disease. In these glomerulopathies, distinct stimuli disrupt metabolic pathways in glomerular cells. Other pathways, including the endoplasmic reticulum (ER) unfolded protein response (UPR) and autophagy, are activated in parallel to attenuate cell injury or promote repair. Methods: We used publicly available datasets to examine gene transcriptional pathways in glomeruli of human GN and DN and to identify drugs. Results: We demonstrate that there are many common genes upregulated in MN, FSGS, IgAN, and DN. Furthermore, these glomerulopathies were associated with increased expression of ER/UPR and autophagy genes, a significant number of which were shared. Several candidate drugs for treatment of glomerulopathies were identified by relating gene expression signatures of distinct drugs in cell culture with the ER/UPR and autophagy genes upregulated in the glomerulopathies ("connectivity mapping"). Using a glomerular cell culture assay that correlates with glomerular damage in vivo, we showed that one candidate drug - neratinib (an epidermal growth factor receptor inhibitor) is cytoprotective. Conclusion: The UPR and autophagy are activated in multiple types of glomerular injury. Connectivity mapping identified candidate drugs that shared common signatures with ER/UPR and autophagy genes upregulated in glomerulopathies, and one of these drugs attenuated injury of glomerular cells. The present study opens the possibility for modulating the UPR or autophagy pharmacologically as therapy for GN.

The unfolded protein response transducer IRE1α promotes reticulophagy in podocytes.

Glomerular diseases involving podocyte/glomerular epithelial cell (GEC) injury feature protein misfolding and endoplasmic reticulum (ER) stress. Inositol-requiring enzyme 1α (IRE1α) mediates chaperone production and autophagy during ER stress. We examined the role of IRE1α in selective autophagy of the ER (reticulophagy). Control and IRE1α knockout (KO) GECs were incubated with tunicamycin to induce ER stress and subjected to proteomic analysis. This showed IRE1α-dependent upregulation of secretory pathway mediators, including the coat protein complex II component Sec23B. Tunicamycin enhanced expression of Sec23B and the reticulophagy adaptor reticulon-3-long (RTN3L) in control, but not IRE1α KO GECs. Knockdown of Sec23B reduced autophagosome formation in response to ER stress. Tunicamycin stimulated colocalization of autophagosomes with Sec23B and RTN3L in an IRE1α-dependent manner. Similarly, during ER stress, glomerular α5 collagen IV colocalized with RTN3L and autophagosomes. Degradation of RTN3L and collagen IV increased in response to tunicamycin, and the turnover was blocked by deletion of IRE1α; thus, the IRE1α pathway promotes RTN3L-mediated reticulophagy and collagen IV may be an IRE1α-dependent reticulophagy substrate. In experimental glomerulonephritis, expression of Sec23B, RTN3L, and LC3-II increased in glomeruli of control mice, but not in podocyte-specific IRE1α KO littermates. In conclusion, during ER stress, IRE1α redirects a subset of Sec23B-positive vesicles to deliver RTN3L-coated ER fragments to autophagosomes. Reticulophagy is a novel outcome of the IRE1α pathway in podocytes and may play a cytoprotective role in glomerular diseases.

Role of IRE1α in podocyte proteostasis and mitochondrial health.

Glomerular epithelial cell (GEC)/podocyte proteostasis is dysregulated in glomerular diseases. The unfolded protein response (UPR) is an adaptive pathway in the endoplasmic reticulum (ER) that upregulates proteostasis resources. This study characterizes mechanisms by which inositol requiring enzyme-1α (IRE1α), a UPR transducer, regulates proteostasis in GECs. Mice with podocyte-specific deletion of IRE1α (IRE1α KO) were produced and nephrosis was induced with adriamycin. Compared with control, IRE1α KO mice had greater albuminuria. Adriamycin increased glomerular ER chaperones in control mice, but this upregulation was impaired in IRE1α KO mice. Likewise, autophagy was blunted in adriamycin-treated IRE1α KO animals, evidenced by reduced LC3-II and increased p62. Mitochondrial ultrastructure was markedly disrupted in podocytes of adriamycin-treated IRE1α KO mice. To pursue mechanistic studies, GECs were cultured from glomeruli of IRE1α flox/flox mice and IRE1α was deleted by Cre-lox recombination. In GECs incubated with tunicamycin, deletion of IRE1α attenuated upregulation of ER chaperones, LC3 lipidation, and LC3 transcription, compared with control GECs. Deletion of IRE1α decreased maximal and ATP-linked oxygen consumption, as well as mitochondrial membrane potential. In summary, stress-induced chaperone production, autophagy, and mitochondrial health are compromised by deletion of IRE1α. The IRE1α pathway is cytoprotective in glomerular disease associated with podocyte injury and ER stress.

Genetic ablation of SLK exacerbates glomerular injury in adriamycin nephrosis in mice.

Ste20-like kinase SLK is critical for embryonic development and may play an important role in wound healing, muscle homeostasis, cell migration, and tumor growth. Mice with podocyte-specific deletion of SLK show albuminuria and damage to podocytes as they age. The present study addressed the role of SLK in glomerular injury. We induced adriamycin nephrosis in 3- to 4-mo-old control and podocyte SLK knockout (KO) mice. Compared with control, SLK deletion exacerbated albuminuria and loss of podocytes, synaptopodin, and podocalyxin. Glomeruli of adriamycin-treated SLK KO mice showed diffuse increases in the matrix and sclerosis as well as collapse of the actin cytoskeleton. SLK can phosphorylate ezrin. The complex of phospho-ezrin, Na+/H+ exchanger regulatory factor 2, and podocalyxin in the apical domain of the podocyte is a key determinant of normal podocyte architecture. Deletion of SLK reduced glomerular ezrin and ezrin phosphorylation in adriamycin nephrosis. Also, deletion of SLK reduced the colocalization of ezrin and podocalyxin in the glomerulus. Cultured glomerular epithelial cells with KO of SLK showed reduced ezrin phosphorylation and podocalyxin expression as well as reduced F-actin. Thus, SLK deletion leads to podocyte injury as mice age and exacerbates injury in adriamycin nephrosis. The mechanism may at least in part involve ezrin phosphorylation as well as disruption of the cytoskeleton and podocyte apical membrane structure.

Urinary ERdj3 and mesencephalic astrocyte-derived neutrophic factor identify endoplasmic reticulum stress in glomerular disease.

Podocyte injury and endoplasmic reticulum (ER) stress have been implicated in the pathogenesis of various glomerular diseases. ERdj3 (DNAJB11) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are ER chaperones lacking the KDEL motif, and may be secreted extracellularly. Since podocytes reside in the urinary space, we examined if podocyte injury is associated with secretion of KDEL-free ER chaperones from these cells into the urine, and if chaperones in the urine reflect ER stress in glomerulonephritis. In cultured podocytes, ER stress increased ERdj3 and MANF intracellularly and in culture medium, whereas GRP94 (KDEL chaperone) increased only intracellularly. ERdj3 and MANF secretion was blocked by the secretory trafficking inhibitor, brefeldin A. Urinary ERdj3 and MANF increased in rats injected with tunicamycin (in the absence of proteinuria). After induction of passive Heymann nephritis (PHN) and puromycin aminonucleoside nephrosis (PAN), there was an increase in glomerular ER stress, and appearance of ERdj3 and MANF in the urine, coinciding with the onset of proteinuria. Rats with PHN were treated with the chemical chaperone, 4-phenyl butyrate (PBA), starting at the time of disease induction, or after disease was established. In both protocols, 4-PBA reduced proteinuria and urinary ER chaperone secretion, compared with PHN rats treated with saline (control). In conclusion, urinary ERdj3 and MANF reflect glomerular ER stress. 4-PBA protected against complement-mediated podocyte injury and the therapeutic response could be monitored by urinary ERdj3 and MANF.

Genetic Ablation of Calcium-independent Phospholipase A2γ Exacerbates Glomerular Injury in Adriamycin Nephrosis in Mice.

Genetic ablation of calcium-independent phospholipase A2γ (iPLA2γ) in mice results in marked damage of mitochondria and enhanced autophagy in glomerular visceral epithelial cells (GECs) or podocytes. The present study addresses the role of iPLA2γ in glomerular injury. In adriamycin nephrosis, deletion of iPLA2γ exacerbated albuminuria and reduced podocyte number. Glomerular LC3-II increased and p62 decreased in adriamycin-treated iPLA2γ knockout (KO) mice, compared with treated control, in keeping with increased autophagy in KO. iPLA2γ KO GECs in culture also demonstrated increased autophagy, compared with control GECs. iPLA2γ KO GECs showed a reduced oxygen consumption rate and increased phosphorylation of AMP kinase (pAMPK), consistent with mitochondrial dysfunction. Adriamycin further stimulated pAMPK and autophagy. After co-transfection of GECs with mito-YFP (to label mitochondria) and RFP-LC3 (to label autophagosomes), or RFP-LAMP1 (to label lysosomes), there was greater colocalization of mito-YFP with RFP-LC3-II and with RFP-LAMP1 in iPLA2γ KO GECs, compared with WT, indicating enhanced mitophagy in KO. Adriamycin increased mitophagy in WT cells. Thus, iPLA2γ has a cytoprotective function in the normal glomerulus and in glomerulopathy, as deletion of iPLA2γ leads to mitochondrial damage and impaired energy homeostasis, as well as autophagy and mitophagy.

Proteostasis as a therapeutic target in glomerular injury associated with mutant α-actinin-4.

Mutations in α-actinin-4 (actinin-4) result in hereditary focal segmental glomerulosclerosis (FSGS) in humans. Actinin-4 mutants induce podocyte injury because of dysregulation of the cytoskeleton and proteotoxicity. Injury may be associated with endoplasmic reticulum (ER) stress and polyubiquitination of proteins. We assessed if the chemical chaperone 4-phenylbutyrate (4-PBA) can ameliorate the proteotoxicity of an actinin-4 mutant. Actinin-4 K255E, which causes FSGS in humans (K256E in the mouse), showed enhanced ubiquitination, accelerated degradation, aggregate formation, and enhanced association with filamentous (F)-actin in glomerular epithelial cells (GECs). The mutant disrupted ER function and stimulated autophagy. 4-PBA reduced actinin-4 K256E aggregation and its tight association with F-actin. Transgenic mice that express actinin-4 K256E in podocytes develop podocyte injury, proteinuria, and FSGS in association with glomerular ER stress. Treatment of these mice with 4-PBA in the drinking water over a 10-wk period significantly reduced albuminuria and ER stress. Another drug, celastrol, which enhanced expression of ER and cytosolic chaperones in GECs, tended to reduce actinin-4 aggregation but did not decrease the tight association of actinin-4 K256E with F-actin and did not reduce albuminuria in actinin-4 K256E transgenic mice. Thus, chemical chaperones, such as 4-PBA, may represent a novel therapeutic approach to certain hereditary glomerular diseases.

Ste20-like kinase, SLK, a novel mediator of podocyte integrity.

SLK is essential for embryonic development and may play a key role in wound healing, tumor growth, and metastasis. Expression and activation of SLK are increased in kidney development and during recovery from ischemic acute kidney injury. Overexpression of SLK in glomerular epithelial cells/podocytes in vivo induces injury and proteinuria. Conversely, reduced SLK expression leads to abnormalities in cell adhesion, spreading, and motility. Tight regulation of SLK expression thus may be critical for normal renal structure and function. We produced podocyte-specific SLK-knockout mice to address the functional role of SLK in podocytes. Mice with podocyte-specific deletion of SLK showed reduced glomerular SLK expression and activity compared with control. Podocyte-specific deletion of SLK resulted in albuminuria at 4-5 mo of age in male mice and 8-9 mo in female mice, which persisted for up to 13 mo. At 11-12 mo, knockout mice showed ultrastructural changes, including focal foot process effacement and microvillous transformation of podocyte plasma membranes. Mean foot process width was approximately twofold greater in knockout mice compared with control. Podocyte number was reduced by 35% in knockout mice compared with control, and expression of nephrin, synaptopodin, and podocalyxin was reduced in knockout mice by 20-30%. In summary, podocyte-specific deletion of SLK leads to albuminuria, loss of podocytes, and morphological evidence of podocyte injury. Thus, SLK is essential to the maintenance of podocyte integrity as mice age.

Regulation of Ste20-like kinase, SLK, activity: Dimerization and activation segment phosphorylation.

The Ste20-like kinase, SLK, has diverse cellular functions. SLK mediates organ development, cell cycle progression, cytoskeletal remodeling, cytokinesis, and cell survival. Expression and activity of SLK are enhanced in renal ischemia-reperfusion injury, and overexpression of SLK was shown to induce apoptosis in cultured glomerular epithelial cells (GECs) and renal tubular cells, as well as GEC/podocyte injury in vivo. The SLK protein consists of a N-terminal catalytic domain and an extensive C-terminal domain, which contains coiled-coils. The present study addresses the regulation of SLK activity. Controlled dimerization of the SLK catalytic domain enhanced autophosphorylation of SLK at T183 and S189, which are located in the activation segment. The full-length ectopically- and endogenously-expressed SLK was also autophosphorylated at T183 and S189. Using ezrin as a model SLK substrate (to address exogenous kinase activity), we demonstrate that dimerized SLK 1-373 or full-length SLK can effectively induce activation-specific phosphorylation of ezrin. Mutations in SLK, including T183A, S189A or T193A reduced T183 or S189 autophosphorylation, and showed a greater reduction in ezrin phosphorylation. Mutations in the coiled-coil region of full-length SLK that impair dimerization, in particular I848G, significantly reduced ezrin phosphorylation and tended to reduce autophosphorylation of SLK at T183. In experimental membranous nephropathy in rats, proteinuria and GEC/podocyte injury were associated with increased glomerular SLK activity and ezrin phosphorylation. In conclusion, dimerization via coiled-coils and phosphorylation of T183, S189 and T193 play key roles in the activation and signaling of SLK, and provide targets for novel therapeutic approaches.

Ste20-like kinase, SLK, activates the heat shock factor 1 - Hsp70 pathway.

Expression and activation of SLK increases during renal ischemia-reperfusion injury. When highly expressed, SLK signals via c-Jun N-terminal kinase and p38 to induce apoptosis, and it exacerbates apoptosis induced by ischemia-reperfusion injury. Overexpression of SLK in glomerular epithelial cells (GECs)/podocytes in vivo induces injury and proteinuria. In response to various stresses, cells enhance expression of chaperones or heat shock proteins (e.g. Hsp70), which are involved in the folding and maturation of newly synthesized proteins, and can refold denatured or misfolded proteins. We address the interaction of SLK with the heat shock factor 1 (HSF1)-Hsp70 pathway. Increased expression of SLK in GECs (following transfection) induced HSF1 transcriptional activity. Moreover, HSF1 transcriptional activity was increased by in vitro ischemia-reperfusion injury (chemical anoxia/recovery) and heat shock, and in both instances was amplified further by SLK overexpression. HSF1 binds to promoters of target genes, such as Hsp70 and induces their transcription. By analogy to HSF1, SLK stimulated Hsp70 expression. Hsp70 was also enhanced by anoxia/recovery and was further amplified by SLK overexpression. Induction of HSF1 and Hsp70 was dependent on the kinase activity of SLK, and was mediated via polo-like kinase-1. Transfection of constitutively active HSF1 enhanced Hsp70 expression and inhibited SLK-induced apoptosis. Conversely, the proapoptotic action of SLK was augmented by HSF1 shRNA, or the Hsp70 inhibitor, pifithrin-µ. In conclusion, increased expression/activity of SLK activates the HSF1-Hsp70 pathway. Hsp70 attenuates the primary proapoptotic effect of SLK. Modulation of chaperone expression may potentially be harnessed as cytoprotective therapy in renal cell injury.

Genetic Ablation of Calcium-independent Phospholipase A2γ Induces Glomerular Injury in Mice.

Glomerular visceral epithelial cells (podocytes) play a critical role in the maintenance of glomerular permselectivity. Podocyte injury, manifesting as proteinuria, is the cause of many glomerular diseases. We reported previously that calcium-independent phospholipase A2γ (iPLA2γ) is cytoprotective against complement-mediated glomerular epithelial cell injury. Studies in iPLA2γ KO mice have demonstrated an important role for iPLA2γ in mitochondrial lipid turnover, membrane structure, and metabolism. The aim of the present study was to employ iPLA2γ KO mice to better understand the role of iPLA2γ in normal glomerular and podocyte function as well as in glomerular injury. We show that deletion of iPLA2γ did not cause detectable albuminuria; however, it resulted in mitochondrial structural abnormalities and enhanced autophagy in podocytes as well as loss of podocytes in aging KO mice. Moreover, after induction of anti-glomerular basement membrane nephritis in young mice, iPLA2γ KO mice exhibited significantly increased levels of albuminuria, podocyte injury, and loss of podocytes compared with wild type. Thus, iPLA2γ has a protective functional role in the normal glomerulus and in glomerulonephritis. Understanding the role of iPLA2γ in glomerular pathophysiology provides opportunities for the development of novel therapeutic approaches to glomerular injury and proteinuria.

Identification of Tpr and α-actinin-4 as two novel SLK-interacting proteins.

Expression and activity of the Ste20-like kinase, SLK, are increased during kidney development and recovery from ischemia-reperfusion injury. SLK mediates apoptosis in various cells, and can regulate cell cycle progression and cytoskeletal remodeling. In cells, SLK is detected in a high molecular mass complex, suggesting that SLK is a dimer/oligomer, or is in tight association with other proteins. To better understand the regulation, localization and function of SLK, we sought to identify proteins in this high molecular mass complex. Analysis by mass spectroscopy identified the nucleoporin, translocated promoter region (Tpr), and the cytoskeletal protein, α-actinin-4, as potential SLK-interacting proteins. Using a protein complementation assay, we showed that the 350 amino acid C-terminal, coiled-coil domain of SLK was responsible for homodimerization, as well as interaction with Tpr and α-actinin-4. The association of SLK with Tpr and α-actinin-4, respectively, was confirmed by co-immunoprecipitation. Subsets of total cellular SLK colocalized with Tpr at the nuclear envelope, and α-actinin-4 in the cytoplasm. Expression of Tpr attenuated activation-specific autophosphorylation of SLK, and blocked SLK-induced apoptosis and AP-1 activity. In contrast to the effect of Tpr, autophosphorylation of SLK was not affected by α-actinin-4. Thus, SLK interacts with Tpr and α-actinin-4 in cells, and these protein-protein interactions may control the subcellular localization and the biological activity of SLK.

Macrophage migration inhibitory factor antagonist blocks the development of endometriosis in vivo.

Endometriosis, a disease of reproductive age women, is a major cause of infertility, menstrual disorders and pelvic pain. Little is known about its etiopathology, but chronic pelvic inflammation is a common feature in affected women. Beside symptomatic treatment of endometriosis-associated pain, only two main suboptimal therapeutic approaches (hormonal and invasive surgery) are generally recommended to patients and no specific targeted treatment is available. Our studies led to the detection of a marked increase in the expression of macrophage migration inhibitory factor (MIF) in the eutopic endometrium, the peripheral blood and the peritoneal fluid of women with endometriosis, and in early, vascularized and active endometriotic lesions. Herein, we developed a treatment model of endometriosis, where human endometrial tissue was first allowed to implant into the peritoneal cavity of nude mice, to assess in vivo the effect of a specific antagonist of MIF (ISO-1) on the progression of endometriosis and evaluate its efficacy as a potential therapeutic tool. Administration of ISO-1 led to a significant decline of the number, size and in situ dissemination of endometriotic lesions. We further showed that ISO-1 may act by significantly inhibiting cell adhesion, tissue remodeling, angiogenesis and inflammation as well as by altering the balance of pro- and anti-apoptotic factors. Actually, mice treatment with ISO-1 significantly reduced the expression of cell adhesion receptors αv and ß3 integrins (P<0.05), matrix metalloproteinases (MMP) 2 and 9 (P<0.05), vascular endothelial cell growth factor (VEGF) (P<0.01), interleukin 8 (IL8) (P<0.05), cyclooxygenease (COX)2 (P<0.001) and the anti-apoptotic protein Bcl2 (P<0.01), but significantly induced the expression of Bax (P<0.05), a potent pro-apoptotic protein. These data provide evidence that specific inhibition of MIF alters endometriotic tissue growth and progression in vivo and may represent a promising potential therapeutic avenue.

Complement modulates the function of the ubiquitin-proteasome system and endoplasmic reticulum-associated degradation in glomerular epithelial cells.

In experimental membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 also activates mechanisms that restrict injury or facilitate recovery. The ubiquitin-proteasome system (UPS) selectively degrades damaged or abnormal proteins, while misfolded proteins in the endoplasmic reticulum (ER) undergo ER-associated degradation (ERAD). In this study, we investigated the effect of complement on the UPS and ERAD. We monitored UPS function by transfection of rat GECs with a UPS reporter, GFP(u) (CL1 degron fused with green fluorescent protein). By analogy, CD3δ-yellow fluorescent protein (YFP) was employed as a reporter of ERAD. We demonstrated decreased GFP(u) levels in GECs after incubation with antibody and complement, compared with control. Using C8-deficient serum with or without purified C8, cycloheximide (an inhibitor of protein synthesis), and the proteasome inhibitor, MG132, we confirmed that the decrease of GFP(u) was mediated by C5b-9, and subsequent proteasomal degradation of the reporter. Inhibition of the c-Jun N-terminal kinase attenuated the effect of complement on GFP(u) degradation. Complement, however, increased the level of CD3δ-YFP in GECs, implying an impairment of ERAD, likely due to an overabundance of misfolded proteins in the ER. The overall ubiquitination of proteins was enhanced in complement-treated GECs and in glomeruli of rats with experimental membranous nephropathy, although ubiquitin mRNA was unchanged in GECs. Proteasome inhibition with MG132 increased the cytotoxic effect of complement in GECs. Complement-stimulated UPS function, by accelerating removal of damaged proteins, may be a novel mechanism to limit complement-induced injury.

Regulation of the Ste20-like kinase, SLK: involvement of activation segment phosphorylation.

Expression and activation of the Ste20-like kinase, SLK, is increased during kidney development and recovery from ischemic acute kidney injury. SLK promotes apoptosis, and it may regulate cell survival during injury or repair. This study addresses the role of phosphorylation in the regulation of kinase activity. We mutated serine and threonine residues in the putative activation segment of the SLK catalytic domain and expressed wild type (WT) and mutant proteins in COS-1 or glomerular epithelial cells. Compared with SLK WT, the T183A, S189A, and T183A/S189A mutants showed reduced in vitro kinase activity. SLK WT, but not mutants, increased activation-specific phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase. Similarly, SLK WT stimulated activator protein-1 reporter activity, but activation of activator protein-1 by the three SLK mutants was ineffective. To test if homodimerization of SLK affects phosphorylation, the cDNA encoding SLK amino acids 1-373 (which include the catalytic domain) was fused with a cDNA for a modified FK506-binding protein, Fv (Fv-SLK 1-373). After transfection, the addition of AP20187 (an FK506 analog) induced regulated dimerization of Fv-SLK 1-373. AP20187-stimulated dimerization enhanced the kinase activity of Fv-SLK 1-373 WT. In contrast, kinase activity of Fv-SLK 1-373 T183A/S189A was weak and was not enhanced after dimerization. Finally, apoptosis was increased after expression of Fv-SLK 1-373 WT but not T183A/S189A. Thus, phosphorylation of Thr-183 and Ser-189 plays a key role in the activation and signaling of SLK and could represent a target for novel therapeutic approaches to renal injury.

Activity of the Ste20-like kinase, SLK, is enhanced by homodimerization.

The expression and activation of the Ste20-like kinase, SLK, is increased during renal development and recovery from ischemic acute renal failure. SLK promotes apoptosis, and during renal injury and repair, transcriptional induction or posttranscriptional control of SLK may, therefore, regulate cell survival. SLK contains protein interaction (coiled-coil) domains, suggesting that posttranslational homodimerization may also modulate SLK activity. We therefore expressed coiled-coil regions in the C-terminal domain of SLK as fusion proteins and demonstrated their homodimerization. By gel-filtration chromatography, endogenous and heterologously expressed SLK were detected in a macromolecular protein complex. To test the role of homodimerization in kinase activation, we constructed a fusion protein consisting of the SLK catalytic domain (amino acids 1-373) and a modified FK506 binding protein, Fv (Fv-SLK 1-373). Addition of AP20187 (an analog of FK506) enhanced the homodimerization of Fv-SLK 1-373. In an in vitro kinase assay, the dimeric Fv-SLK 1-373 displayed greater kinase activity than the monomeric form. In cells expressing Fv-SLK 1-373, homodimerization increased activation-specific phosphorylation of the proapoptotic kinases, c-Jun N-terminal kinase and p38 kinase. Compared with the monomer, dimeric Fv-SLK 1-373 enhanced the activation of a Bax promoter-luciferase reporter. Finally, expression of Fv-SLK 1-373 induced apoptosis, and the effect was increased by homodimerization. Thus the activity, downstream signaling, and functional effects of SLK are enhanced by dimerization of the kinase domain.

Podocyte injury and albuminuria in mice with podocyte-specific overexpression of the Ste20-like kinase, SLK.

SLK expression and activity are increased during kidney development and recovery from renal ischemia-reperfusion injury. In cultured cells, SLK promotes F-actin destabilization as well as apoptosis, partially via the p38 kinase pathway. To better understand the effects of SLK in vivo, a transgenic mouse model was developed where SLK was expressed in a podocyte-specific manner using the mouse nephrin promoter. Offspring of four founder mice carried the SLK transgene. Among male transgenic mice, 66% developed albuminuria at approximately 3 months of age, and the albuminuric mice originated from three of four founders. Overall, the male transgenic mice demonstrated about fivefold greater urinary albumin/creatinine compared with male non-transgenic mice. Transgenic and non-transgenic female mice did not develop albuminuria, suggesting that females were less susceptible to glomerular filtration barrier damage than their male counterparts. In transgenic mice, electron microscopy revealed striking podocyte injury, including poorly formed or effaced foot processes, and edematous and vacuolated cell bodies. By immunoblotting, nephrin expression was decreased in glomeruli of the albuminuric transgenic mice. Activation-specific phosphorylation of p38 was increased in transgenic mice compared with non-transgenic animals. Glomeruli of SLK transgenic mice showed around 30% fewer podocytes, and a reduction in F-actin compared with control glomeruli. Thus, podocyte SLK overexpression in vivo results in injury and podocyte loss, consistent with the effects of SLK in cultured cells.

The Ste20-like kinase SLK promotes p53 transactivation and apoptosis.

Expression and activity of the germinal center kinase [corrected] SLK are increased during kidney development and recovery from renal ischemia-reperfusion injury. SLK promotes apoptosis, in part, via pathway(s) involving apoptosis signal-regulating kinase-1 and p38 mitogen-activated protein kinase. This study addresses the role of p53 as a potential effector of SLK. p53 transactivation was measured after transient transfection of a luciferase reporter plasmid that contains a p53 cis-acting enhancer element. Overexpression of SLK in COS-1 cells and cotransfection of SLK and p53-wild type (wt) cDNAs in glomerular epithelial cells (GECs) stimulated p53 transactivational activity, as measured by a p53 response element-driven luciferase reporter. In GECs, chemical anoxia followed by glucose reexposure (in vitro ischemia-reperfusion) increased p53 reporter activity, and this increase was amplified by overexpression of SLK. Expression of SLK induced p53 phosphorylation on serine (S)-33 and S315. In GECs, cotransfection of SLK with p53-wt, p53-S33A, p53-S315A, or p53-S33A+S315A mutants showed that only the double mutation abolished the SLK-induced increase in p53 reporter activity. SLK-induced stimulation of p53 reporter activity was attenuated by inhibition of JNK. Overexpression of SLK amplified apoptosis induced by subjecting cells to in vitro ischemia-reperfusion injury, while ectopic expression of a dominant negative SLK mutant attenuated the ischemia-reperfusion-induced apoptosis. The p53 transactivation inhibitor pifithrin-alpha significantly attenuated the amount of apoptosis after ischemia-reperfusion and SLK overexpression. Thus SLK induces p53 phosphorylation and transactivation, which enhances apoptosis after in vitro ischemia-reperfusion injury.

Glomerular epithelial cell injury associated with mutant alpha-actinin-4.

Focal segmental glomerulosclerosis (FSGS) may be associated with glomerular epithelial cell (GEC; podocyte) apoptosis due to acquired injury or mutations in alpha-actinin-4. This study addresses how FSGS-associated mutant alpha-actinin-4 may induce GEC injury, focusing on endoplasmic reticulum (ER) stress and metabolism of mutant alpha-actinin-4 via the ubiquitin-proteasome system. In a model of experimental FSGS induced by expression of an alpha-actinin-4 K256E transgene in podocytes, we show induction of ER stress, including upregulation of ER chaperones (bip, grp94), phosphorylation of the eukaryotic translation initiation factor-2alpha subunit, and induction of the proapoptotic gene C/EBP homologous protein-10 (CHOP). To address mechanisms of ER stress, we studied signaling in cultured GEC and COS cells expressing alpha-actinin-4 K256E. Previously, we showed that expression of this alpha-actinin-4 mutant in GEC increased apoptosis. In the present study, we show that alpha-actinin-4 K256E upregulates grp94 and CHOP expression in COS cells and significantly exacerbates induction of bip and CHOP in GEC in the presence of tunicamycin. ER stress was associated with aggregation and ubiquitination of alpha-actinin-4 K256E and impairment of the ubiquitin-proteasome system. In addition, alpha-actinin-4 K256E exacerbated apoptosis in the context of mild proteasome inhibition. Thus alpha-actinin-4 K256E triggers several metabolic abnormalities, which may lead to GEC injury and glomerulosclerosis.