Definition of fatty acid transport protein-2 (FATP2) structure facilitates identification of small molecule inhibitors for the treatment of diabetic complications.

Diabetes is a major public health problem due to morbidity and mortality associated with end organ complications. Uptake of fatty acids by Fatty Acid Transport Protein-2 (FATP2) contributes to hyperglycemia, diabetic kidney and liver disease pathogenesis. Because FATP2 structure is unknown, a homology model was constructed, validated by AlphaFold2 prediction and site-directed mutagenesis, and then used to conduct a virtual drug discovery screen. In silico similarity searches to two low-micromolar IC50 FATP2 inhibitors, followed by docking and pharmacokinetics predictions, narrowed a diverse 800,000 compound library to 23 hits. These candidates were further evaluated for inhibition of FATP2-dependent fatty acid uptake and apoptosis in cells. Two compounds demonstrated nanomolar IC50, and were further characterized by molecular dynamic simulations. The results highlight the feasibility of combining a homology model with in silico and in vitro screening, to economically identify high affinity inhibitors of FATP2, as potential treatment for diabetes and its complications.

Ventricular arrhythmias in mouse models of diabetic kidney disease.

Chronic kidney disease (CKD) affects more than 20 million people in the US, and it is associated with a significantly increased risk of sudden cardiac death (SCD). Despite the significance, the mechanistic relationship between SCD and CKD is not clear and there are few effective therapies. Using optical mapping techniques, we tested the hypothesis that mouse models of progressive diabetic kidney disease (DKD) exhibit enhanced ventricular arrhythmia incidence and underlying arrhythmia substrates. Compared to wild-type mice, both Leprdb/db eNOS-/- (2KO) and high fat diet plus low dose streptozotocin (HFD + STZ) mouse models of DKD experienced sudden death and greater arrhythmia inducibility, which was more common with isoproterenol than programmed electrical stimulation. 2KO mice demonstrated slowed conduction velocity, prolonged action potential duration (APD), and myocardial fibrosis; both 2KO and HFD + STZ mice exhibited arrhythmias and calcium dysregulation with isoproterenol challenge. Finally, circulating concentrations of the uremic toxin asymmetric dimethylarginine (ADMA) were elevated in 2KO mice. Incubation of human cardiac myocytes with ADMA prolonged APD, as also observed in 2KO mice hearts ex vivo. The present study elucidates an arrhythmia-associated mechanism of sudden death associated with DKD, which may lead to more effective treatments in the vulnerable DKD patient population.

Fatty acid transport protein-2 regulates glycemic control and diabetic kidney disease progression.

Kidney disease is one of the most devastating complications of diabetes, and tubular atrophy predicts diabetic kidney disease (DKD) progression to end-stage renal disease. We have proposed that fatty acids bound to albumin contribute to tubular atrophy by inducing lipotoxicity, after filtration across damaged glomeruli, and subsequent proximal tubule reabsorption by a fatty acid transport protein-2-dependent (FATP2-dependent) mechanism. To address this possibility, genetic (Leprdb/db eNOS-/-) and induced (high-fat diet plus low-dose streptozotocin) mouse models of obesity and DKD were bred with global FATP2 gene-deleted mice (Slc27a2) and then phenotyped. DKD-prone mice with the Slc27a2-/- genotype demonstrated normalization of glomerular filtration rate, reduced albuminuria, improved kidney histopathology, and longer life span compared with diabetic Slc27a2+/+ mice. Genetic and induced DKD-prone Slc27a2-/- mice also exhibited markedly reduced fasting plasma glucose, with mean values approaching euglycemia, despite increased obesity and decreased physical activity. Glucose lowering in DKD-prone Slc27a2-/- mice was accompanied by ß cell hyperplasia and sustained insulin secretion. Together, our data indicate that FATP2 regulates DKD pathogenesis by a combined lipotoxicity and glucotoxicity (glucolipotoxicity) mechanism.

Kidney Proximal Tubule Lipoapoptosis Is Regulated by Fatty Acid Transporter-2 (FATP2).

Albuminuria and tubular atrophy are among the highest risks for CKD progression to ESRD. A parsimonious mechanism involves leakage of albumin-bound nonesterified fatty acids (NEFAs) across the damaged glomerular filtration barrier and subsequent reabsorption by the downstream proximal tubule, causing lipoapoptosis. We sought to identify the apical proximal tubule transporter that mediates NEFA uptake and cytotoxicity. We observed transporter-mediated uptake of fluorescently labeled NEFA in cultured proximal tubule cells and microperfused rat proximal tubules, with greater uptake from the apical surface than from the basolateral surface. Protein and mRNA expression analyses revealed that kidney proximal tubules express transmembrane fatty acid transporter-2 (FATP2), encoded by Slc27a2, but not the other candidate transporters CD36 and free fatty acid receptor 1. Kidney FATP2 localized exclusively to proximal tubule epithelial cells along the apical but not the basolateral membrane. Treatment of mice with lipidated albumin to induce proteinuria caused a decrease in the proportion of tubular epithelial cells and an increase in the proportion of interstitial space in kidneys from wild-type but not Slc27a2-/- mice. Ex vivo microperfusion and in vitro experiments with NEFA-bound albumin at concentrations that mimic apical proximal tubule exposure during glomerular injury revealed significantly reduced NEFA uptake and palmitate-induced apoptosis in microperfused Slc27a2-/- proximal tubules and Slc27a2-/- or FATP2 shRNA-treated proximal tubule cell lines compared with wild-type or scrambled oligonucleotide-treated cells, respectively. We conclude that FATP2 is a major apical proximal tubule NEFA transporter that regulates lipoapoptosis and may be an amenable target for the prevention of CKD progression.

Mesangial cell αvß8-integrin regulates glomerular capillary integrity and repair.

αvß8-Integrin is most abundantly expressed in the kidney, brain, and female reproductive organs, and its cognate ligand is latent transforming growth factor (LTGF)-ß. Kidney αvß8-integrin localizes to mesangial cells, and global ß8-integrin gene (Itgb8) deletion results in embryonic lethality due to impaired placentation and cerebral hemorrhage. To circumvent the lethality and better define kidney αvß8-integrin function, Cre-lox technology was used to generate mesangial-specific Itgb8-null mice. Platelet-derived growth factor-ß receptor (PDGFBR)-Cre mice crossed with a reporter strain revealed functional Cre recombinase activity in a predicted mesangial pattern. However, mating between two different PDGFBR-Cre or Ren1(d)-Cre strains with Itgb8 (flox/-) mice consistently resulted in incomplete recombination, with no renal phenotype in mosaic offspring. Induction of a renal phenotype with Habu snake venom, a reversible mesangiolytic agent, caused exaggerated glomerular capillary microaneurysms and delayed recovery in Cre(+/-) PDGFRB (flox/-) mice compared with Cre(+/-) PDGFRB (flox/+) control mice. To establish the mechanism, in vitro experiments were conducted in Itgb8-null versus Itgb8-expressing mesangial cells and fibroblasts, which revealed ß8-integrin-regulated adhesion to Arg-Gly-Asp (RGD) peptides within a mesangial-conditioned matrix as well as ß8-integrin-dependent migration on RGD-containing LTGF-ß or vitronectin matrices. We speculate that kidney αvß8-integrin indirectly controls glomerular capillary integrity through mechanical tension generated by binding RGD peptides in the mesangial matrix, and healing after glomerular injury may be facilitated by mesangial cell migration, which is guided by transient ß8-integrin interactions with RGD ligands.

Lipotoxic disruption of NHE1 interaction with PI(4,5)P2 expedites proximal tubule apoptosis.

Chronic kidney disease progression can be predicted based on the degree of tubular atrophy, which is the result of proximal tubule apoptosis. The Na+/H+ exchanger NHE1 regulates proximal tubule cell survival through interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], but pathophysiologic triggers for NHE1 inactivation are unknown. Because glomerular injury permits proximal tubule luminal exposure and reabsorption of fatty acid/albumin complexes, we hypothesized that accumulation of amphipathic, long-chain acyl-CoA (LC-CoA) metabolites stimulates lipoapoptosis by competing with the structurally similar PI(4,5)P2 for NHE1 binding. Kidneys from mouse models of progressive, albuminuric kidney disease exhibited increased fatty acids, LC-CoAs, and caspase-2-dependent proximal tubule lipoapoptosis. LC-CoAs and the cytosolic domain of NHE1 directly interacted, with an affinity comparable to that of the PI(4,5)P2-NHE1 interaction, and competing LC-CoAs disrupted binding of the NHE1 cytosolic tail to PI(4,5)P2. Inhibition of LC-CoA catabolism reduced NHE1 activity and enhanced apoptosis, whereas inhibition of proximal tubule LC-CoA generation preserved NHE1 activity and protected against apoptosis. Our data indicate that albuminuria/lipiduria enhances lipotoxin delivery to the proximal tubule and accumulation of LC-CoAs contributes to tubular atrophy by severing the NHE1-PI(4,5)P2 interaction, thereby lowering the apoptotic threshold. Furthermore, these data suggest that NHE1 functions as a metabolic sensor for lipotoxicity.

Phosphoinositide binding differentially regulates NHE1 Na+/H+ exchanger-dependent proximal tubule cell survival.

Tubular atrophy predicts chronic kidney disease progression, and is caused by proximal tubular epithelial cellcaused by proximal tubular epithelial cell (PTC) apoptosis. The normally quiescent Na(+)/H(+) exchanger-1 (NHE1) defends against PTC apoptosis, and is regulated by PI(4,5)P(2) binding. Because of the vast array of plasma membrane lipids, we hypothesized that NHE1-mediated cell survival is dynamically regulated by multiple anionic inner leaflet phospholipids. In membrane overlay and surface plasmon resonance assays, the NHE1 C terminus bound phospholipids with low affinity and according to valence (PIP(3) > PIP(2) > PIP = PA > PS). NHE1-phosphoinositide binding was enhanced by acidic pH, and abolished by NHE1 Arg/Lys to Ala mutations within two juxtamembrane domains, consistent with electrostatic interactions. PI(4,5)P(2)-incorporated vesicles were distributed to apical and lateral PTC domains, increased NHE1-regulated Na(+)/H(+) exchange, and blunted apoptosis, whereas NHE1 activity was decreased in cells enriched with PI(3,4,5)P(3), which localized to basolateral membranes. Divergent PI(4,5)P(2) and PI(3,4,5)P(3) effects on NHE1-dependent Na(+)/H(+) exchange and apoptosis were confirmed by selective phosphoinositide sequestration with pleckstrin homology domain-containing phospholipase Cδ and Akt peptides, PI 3-kinase, and Akt inhibition in wild-type and NHE1-null PTCs. The results reveal an on-off switch model, whereby NHE1 toggles between weak interactions with PI(4,5)P(2) and PI(3,4,5)P(3). In response to apoptotic stress, NHE1 is stimulated by PI(4,5)P(2), which leads to PI 3-kinase activation, and PI(4,5)P(2) phosphorylation. The resulting PI(3,4,5)P(3) dually stimulates sustained, downstream Akt survival signaling, and dampens NHE1 activity through competitive inhibition and depletion of PI(4,5)P(2).

Mesangial cell integrin αvß8 provides glomerular endothelial cell cytoprotection by sequestering TGF-ß and regulating PECAM-1.

Integrins are heterodimeric receptors that regulate cell adhesion, migration, and apoptosis. Integrin αvß8 is most abundantly expressed in kidney and brain, and its major ligand is latent transforming growth factor-ß (TGF-ß). Kidney αvß8 localizes to mesangial cells, which appose glomerular endothelial cells and maintain glomerular capillary structure by mechanical and poorly understood paracrine mechanisms. To establish kidney αvß8 function, mice with homozygous Itgb8 deletion (Itgb8(-/-)) were generated on outbred and C57BL/6 congenic backgrounds. Most Itgb8(-/-) mice died in utero, and surviving Itgb8(-/-) mice failed to gain weight, and rarely survived beyond 6 weeks. A renal glomerular phenotype included azotemia and albuminuria, as well as increased platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, which was surprisingly not associated with conventional functions, such as endothelial cell hyperplasia, hypertrophy, or perivascular inflammation. Itgb8(-/-) mesangial cells demonstrated reduced latent TGF-ß binding, resulting in bioactive TGF-ß release, which stimulated glomerular endothelial cell apoptosis. Using PECAM-1 gain and loss of function strategies, we show that PECAM-1 provides endothelial cytoprotection against mesangial cell TGF-ß. These results clarify a singular mechanism of mesangial-to-endothelial cell cross-talk, whereby mesangial cell αvß8 homeostatically arbitrates glomerular microvascular integrity by sequestering TGF-ß in its latent conformation. Under pathological conditions associated with decreased mesangial cell αvß8 expression and TGF-ß secretion, compensatory PECAM-1 modulation facilitates glomerular endothelial cell survival.

Podocyte injury induces nuclear translocation of WTIP via microtubule-dependent transport.

Podocyte structural and transcriptional phenotype plasticity characterizes glomerular injury. Transcriptional activity of WT1 (Wilm's tumor 1) is required for normal podocyte structure and is repressed by the podocyte adherens junction protein, WTIP (WT1 interacting protein). Here we show that WTIP translocated into podocyte nuclei in lipopolysaccharide (LPS)-treated mice, a model of transient nephrotic syndrome. Cultured podocytes, which stably expressed an epitope-tagged WTIP, were treated with LPS. Imaging and cellular fractionation studies demonstrated that WTIP translocated from podocyte cell contacts into nuclei within 6 h and relocalized to cell contacts within 24 h after LPS treatment. LPS-stimulated WTIP nuclear translocation required JNK activity, which assembled a multiprotein complex of the scaffolding protein JNK-interacting protein 3 and the molecular motor dynein. Intact microtubule networks and dynein activity were necessary for LPS-stimulated WTIP translocation. Podocytes expressing sh-Wtip change morphology and demonstrate altered actin assembly in cell spreading assays. Stress signaling pathways initiate WTIP nuclear translocation, and the concomitant loss of WTIP from cell contacts changes podocyte morphology and dynamic actin assembly, suggesting a mechanism that transmits changes in podocyte morphology to the nucleus.

JAK kinases promote invasiveness in VHL-mediated renal cell carcinoma by a suppressor of cytokine signaling-regulated, HIF-independent mechanism.

von Hippel-Lindau (VHL) disease is a cancer syndrome, which includes renal cell carcinoma (RCC), and is caused by VHL mutations. Most, but not all VHL phenotypes are due to failure of mutant VHL to regulate constitutive proteolysis of hypoxia-inducible factors (HIFs). Janus kinases (JAK1, 2, 3, and TYK2) promote cell survival and proliferation, processes tightly controlled by SOCS proteins, which have sequence and structural homology to VHL. We hypothesized that in VHL disease, RCC pathogenesis results from enhanced SOCS1 degradation, leading to upregulated JAK activity. We find that baseline JAK2, JAK3, and TYK2 activities are increased in RCC cell lines, even after serum deprivation or coincubation with cytokine inhibitors. Furthermore, JAK activity is sustained in RCC stably expressing HIF2alpha shRNA. Invasion through Matrigel and migration in wound-healing assays, in vitro correlates of metastasis, are significantly greater in VHL mutant RCC compared with wild-type cells, and blocked by dominant-negative JAK expression or JAK inhibitors. Finally, we observe enhanced SOCS2/SOCS1 coprecipitation and reduced SOCS1 expression due to proteasomal degradation in VHL-null RCC compared with wild-type cells. The data support a new HIF-independent mechanism of RCC metastasis, whereby SOCS2 recruits SOCS1 for ubiquitination and proteasome degradation, which lead to unrestricted JAK-dependent RCC invasion. In addition to commonly proposed RCC treatment strategies that target HIFs, our data suggest that JAK inhibition represents an alternative therapeutic approach.

Beta8 integrin binds Rho GDP dissociation inhibitor-1 and activates Rac1 to inhibit mesangial cell myofibroblast differentiation.

Alpha(v)beta8 integrin expression is restricted primarily to kidney, brain, and placenta. Targeted alpha(v) or beta8 deletion is embryonic lethal due to defective placenta and brain angiogenesis, precluding investigation of kidney alpha(v)beta8 function. We find that kidney beta8 is localized to glomerular mesangial cells, and expression is decreased in mouse models of glomerulosclerosis, suggesting that beta8 regulates normal mesangial cell differentiation. To interrogate beta8 signaling pathways, yeast two-hybrid and co-precipitation studies demonstrated beta8 interaction with Rho guanine nucleotide dissociation inhibitor-1 (GDI). Selective beta8 stimulation enhanced beta8-GDI interaction as well as Rac1 (but not RhoA) activation and lamellipodia formation. Mesangial cells from itgb8-/- mice backcrossed to a genetic background that permitted survival, or gdi-/- mice, which develop glomerulosclerosis, demonstrated RhoA (but not Rac1) activity and alpha-smooth muscle actin assembly, which characterizes mesangial cell myofibroblast transformation in renal disease. To determine whether Rac1 directly modulates RhoA-associated myofibroblast differentiation, mesangial cells were transduced with inhibitory Rac peptide fused to human immunodeficiency virus-Tat, resulting in enhanced alpha-smooth muscle actin organization. We conclude that the beta8 cytosolic tail in mesangial cells organizes a signaling complex that culminates in Rac1 activation to mediate wild-type differentiation, whereas decreased beta8 activation shifts mesangial cells toward a RhoA-dependent myofibroblast phenotype.

WT1-interacting protein and ZO-1 translocate into podocyte nuclei after puromycin aminonucleoside treatment.

Podocyte differentiation is required for normal glomerular filtration barrier function and is regulated by the transcription factor WT1. We identified WT1-interacting protein (WTIP) and hypothesized that it functions as both a scaffold for slit diaphragm proteins and a corepressor of WT1 transcriptional activity by shuttling from cell-cell junctions to the nucleus after injury. Endogenous WTIP colocalizes with zonula occludens-1 (ZO-1) in cultured mouse podocyte adherens junctions. To model podocyte injury in vitro, we incubated differentiated podocytes with puromycin aminonucleoside (PAN; 100 microg/ml) for 24 h, which disassembled cell-cell contacts, rearranged actin cytoskeleton, and caused process retraction. Podocyte synaptopodin expression diminished after PAN treatment, consistent with podocyte dedifferentiation in some human glomerular diseases. To assess podocyte function, we measured albumin flux across differentiated podocytes cultured on collagen-coated Transwell filters. Albumin transit across PAN-treated cells increased to levels observed with undifferentiated podocytes. Consistent with our hypothesis, WTIP, as well as ZO-1, translocated from podocyte adherens junctions to nuclei in PAN-treated cells. Because WTIP is a transcriptional corepressor for WT1, we examined the effect of PAN on expression of retinoblastoma binding protein Rbbp7 (also known as RbAp46), a WT1 target gene expressed in S-shaped bodies during nephrogenesis. Rbbp7 expression in PAN-treated podocytes was reduced compared with untreated cells. In conclusion, WTIP translocates from cell-cell junctions to the nucleus in PAN-treated podocytes. We suggest that WTIP monitors slit diaphragm protein assembly and shuttles into the nucleus after podocyte injury, translating changes in slit diaphragm structure into altered gene expression and a less differentiated phenotype.

Renal phenotype is exacerbated in Os and lpr double mutant mice.

BACKGROUND: ROP-Os/+ mice are born with oligosyndactyly and oligonephronia and develop renal dysfunction, which includes renal tubular epithelial cell (RTC) Fas-dependent apoptosis and tubular atrophy. MRL/lpr mice harbor a Fas-inactivating mutation and develop glomerulonephritis, whereas mice expressing lpr on a C3H background demonstrate no renal phenotype. We hypothesized that crossing ROP-Os/+ with CH3-lpr/lpr mice would rescue the Os/+ renal phenotype by reducing Fas-dependent RTC apoptosis. METHODS: ROP-Os/+ mice were intercrossed with C3H-lpr/lpr mice and F(2) generation animals were phenotyped by kidney weight, serum creatinine, and albuminuria. Kidney sections were scored for histopathology and apoptosis. Univariate and multivariate analyses were used to examine additive effects of Os and lpr on renal phenotype. RESULTS: By 16 weeks, F(2)Os/+ lpr/lpr mice developed significantly more albuminuria, glomerulosclerosis, and interstitial inflammation compared to Os/++/+ mice. Glomerular cell apoptosis was increased in Os/+ lpr/lpr compared to Os/++/+ mice, with no significant difference in RTC apoptosis. A statistically significant Os-lpr effect on renal phenotype was demonstrated by multivariate analysis, which exceeded the combined independent effects if Os and lpr, indicating a biologic interaction exists between Os and lpr. CONCLUSION: Os/+ mice with a superimposed lpr mutation displayed a more severe renal phenotype, rather than phenotype rescue, suggesting that Fas pathway activation is necessary to delete cells resulting from Os-dependent injury. We further propose that an Os-lpr gene interaction and/or mixed ROP-C3H genetic background regulated the renal phenotype, consistent with the concept that chronic renal disease pathogenesis reflects effects of multiple nephropathy susceptibility alleles.

The NHE1 Na+/H+ exchanger recruits ezrin/radixin/moesin proteins to regulate Akt-dependent cell survival.

Apoptosis results in cell shrinkage and intracellular acidification, processes opposed by the ubiquitously expressed NHE1 Na(+)/H(+) exchanger. In addition to mediating Na(+)/H(+) transport, NHE1 interacts with ezrin/radixin/moesin (ERM), which tethers NHE1 to cortical actin cytoskeleton to regulate cell shape, adhesion, motility, and resistance to apoptosis. We hypothesize that apoptotic stress activates NHE1-dependent Na(+)/H(+) exchange, and NHE1-ERM interaction is required for cell survival signaling. Apoptotic stimuli induced NHE1-regulated Na(+)/H(+) transport, as demonstrated by ethyl-N-isopropyl-amiloride-inhibitable, intracellular alkalinization. Ectopic NHE1, but not NHE3, expression rescued NHE1-null cells from apoptosis induced by staurosporine or N-ethylmaleimide-stimulated KCl efflux. When cells were subjected to apoptotic stress, NHE1 and phosphorylated ERM physically associated within the cytoskeleton-enriched fraction, resulting in activation of the pro-survival kinase, Akt. NHE1-associated Akt activity and cell survival were inhibited in cells expressing ERM binding-deficient NHE1, dominant negative ezrin constructs, or ezrin mutants with defective binding to phosphoinositide 3-kinase, an upstream regulator of Akt. We conclude that NHE1 promotes cell survival by dual mechanisms: by defending cell volume and pH(i) through Na(+)/H(+) exchange and by functioning as a scaffold for recruitment of a signalplex that includes ERM, phosphoinositide 3-kinase, and Akt.

A WT1 co-regulator controls podocyte phenotype by shuttling between adhesion structures and nucleus.

Glomerular podocyte differentiation state is critical for filtration barrier function and is regulated by WT1, a zinc finger transcription factor. A yeast two-hybrid assay identified a novel, WT1-interacting protein (WTIP) that maps to human chromosome 19q13.1, a region with genes linked to familial focal segmental glomerulosclerosis. The domain structure of WTIP is similar to the zyxin subfamily of cytosolic LIM domain-containing proteins, which contain three carboxyl-terminal LIM protein-protein interaction domains and a proline-rich, pre-LIM region with a nuclear export signal. Other LIM domain-containing proteins (zyxin and mouse muscle LIM protein) did not interact with WT1 in two-hybrid assays, and WTIP did not interact with an unrelated transcription factor, LMX1B. WTIP mRNA was detected in cultured podocytes and was developmentally regulated, with expression peaking in mouse kidney at embryonic day 15-16 (E15-E16) in kidney but persisting into adulthood. In situ hybridization demonstrated WTIP expression in developing E15 glomeruli and in cultured podocytes. The partial WTIP clone, which interacted with WTIP in the two-hybrid assay, co-localized with WT1 in nuclei, co-precipitated with WT1, and inhibited WT1-dependent transcriptional activation of the amphiregulin promoter. In contrast, full-length WTIP was excluded from cell nuclei, but after the addition of leptomycin B, an inhibitor of Crm1-mediated nuclear export, it accumulated in the nucleus and co-precipitated with WT1 in whole cell lysates. Epitope-tagged WTIP co-localized with the adaptor protein CD2AP (CMS) in podocyte actin spots and with Mena at cell-cell junctions. We propose that WTIP monitors slit diaphragm protein assembly as part of a multiple protein complex, linking this specialized adhesion junction to the actin cytoskeleton, and shuttles into the nucleus after podocyte injury, providing a mechanism whereby changes in slit diaphragm structure modulate gene expression.

Renal tubular epithelial cell apoptosis is associated with caspase cleavage of the NHE1 Na+/H+ exchanger.

Renal tubular epithelial cell (RTC) apoptosis causes tubular atrophy, a hallmark of renal disease progression. Apoptosis is generally characterized by reduced cell volume and cytosolic pH, but epithelial cells are relatively resistant to shrinkage due to regulatory volume increase, which is mediated by Na(+)/H(+) exchanger (NHE) 1. We investigated whether RTC apoptosis requires caspase cleavage of NHE1. Staurosporine- and hypertonic NaCl-induced RTC apoptosis was associated with cell shrinkage and diminished cytosolic pH, and apoptosis was potentiated by amiloride analogs, suggesting NHE1 activity opposes apoptosis. NHE1-deficient fibroblasts demonstrated increased susceptibility to apoptosis, which was reversed by NHE1 reconstitution. NHE1 expression was markedly decreased in apoptotic RTC due to degradation, and preincubation with peptide caspase antagonists restored NHE1 expression, indicating that NHE1 is degraded by caspases. Recombinant caspase-3 cleaved the in vitro-translated NHE1 cytoplasmic domain into five distinct peptides, identical in molecular weight to NHE1 degradation products derived from staurosporine-stimulated RTC lysates. In vivo, NHE1 loss-of-function C57BL/6.SJL-swe/swe mice with adriamycin-induced nephropathy demonstrated increased RTC apoptosis compared with adriamycin-treated wild-type controls, thereby implicating NHE1 inactivation as a potential mechanism of tubular atrophy. We conclude that NHE1 activity is critical for RTC survival after injury and that caspase cleavage of RTC NHE1 may promote apoptosis and tubular atrophy by preventing compensatory intracellular volume and pH regulation.

Fas activation induces renal tubular epithelial cell beta 8 integrin expression and function in the absence of apoptosis.

Cell fate following Fas (CD95) ligand or agonistic anti-Fas antibody stimulation is determined by multiple factors, including Fas expression level, microdomain localization, and modulating cytokines. Highly expressed Fas clusters and activates a canonical apoptosis signaling pathway. In less susceptible cells, Fas transduces apoptosis-independent signals, which are not well defined, but have been linked to inflammation, angiogenesis, and fibrosis. To identify apoptosis-independent Fas pathways, cultured renal tubular epithelial cells were stimulated with agonistic anti-Fas antibodies under conditions that did not cause cell death. Analysis of filter cDNA microarrays revealed beta(8) integrin subunit mRNA induction in Fas-stimulated cells. beta(8) integrin mRNA expression increased within 3-6 h of Fas ligation due to enhanced mRNA stabilization, and mRNA increases were sustained for 48-72 h. Expression of plasma membrane beta(8) integrin, as well as its heterodimer partner alpha(v), was increased by Fas activation with a similar kinetic pattern. Fas-induced alpha(v)beta(8) expression correlated with increased migration to vitronectin, the ligand for alpha(v)beta(8). Results from studies with function-blocking antibodies against other alpha(v)beta integrins or suppression of beta(8) integrin expression by RNA interference demonstrated that induced beta(8) integrin expression mediated Fas-stimulated migration. We conclude that alpha(v)beta(8) integrin induction defines an unexpected role for Fas in cell migration, rather than as a cell death receptor.