Sphingomyelin Phosphodiesterase Acid-like 3b Research Articles

Podocytopathy and Nephrotic Syndrome in Smpdl3b Gene Knockout Mice

Recent clinical studies have shown that downregulation of sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) in podocytes is a hallmark of idiopathic nephrotic syndrome in children. Meanwhile, there is increasing evidence suggesting that changes of SMPDL3b level in podocytes determine the phenotypes of podocyte injury in glomerular diseases. However, it remains unknown whether SMPDL3b exclusively plays a crucial role in the pathogenesis of podocytopathy and nephrotic syndrome without any other pathogenic stimuli. In the present study, we tested whether Smpdl3b gene knockout induces podocyte injury and nephrotic syndrome in mice. By immunohistochemistry and confocal microscopy, we first confirmed that SMPDL3b was highly expressed in podocytes of wild type (WT) mice but undetectable in these cells of Smpdl3b knockout (KO) mice. Also, Western blot analysis showed that SMPDL3b was not detected in podocytes isolated from Smpdl3b KO mice compared to podocytes of WT mice. Although no significant morphologic changes in glomeruli were observed in Smpdl3b KO mice under light microscope, severe proteinuria and albuminuria were found in Smpdl3b KO mice compared to control littermates. Transmission electron microscopy showed that podocytes of Smpdl3b KO mice had distinctive foot process effacement and moderate microvillus formation and vacuolation. These functional and morphologic changes indicate the development of nephrotic syndrome in mice lacking Smpdl3b gene. In vitro, Smpdl3b gene silencing induced remarkable decrease in podocin levels in podocytes compared to control cells. Phalloidin staining of F-actin showed elevation of cortical actin and reduction of stress fiber in podocytes transfected with Smpdl3b shRNA. Such actin cytoskeleton rearrangement induced by Smpdl3b gene silencing was significantly attenuated by choline but exaggerated by CDP-choline, which are product and substrate of SMPDL3b, respectively. Furthermore, atom force microscopy was used to study mechanical properties of podocytes lacking Smpdl3b gene as the outcome of actin cytoskeleton reorganization. Smpdl3b gene deletion was found to increase podocyte height but decrease cortical elasticity in these cells. Moreover, these pathological changes were markedly inhibited by choline. Taken together, our findings suggest that SMPDL3b is essential for the maintenance of functional and structural integrity of podocytes and that Smpdl3b gene knockout may induce podocytopathy and nephrotic syndrome in mice. This study was supported by NIH grants DK054927 and DK120491. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Contribution of sphingomyelin phosphodiesterase acid-like 3B to the proliferation, migration, and invasion of ovarian cancer cells.

Cancer has the highest mortality rate among gynecological cancers and poses a serious threat to women's lives. However, the treatment options for ovarian cancer are still limited, and exploring effective targeted biomarkers is particularly important for predicting and treating ovarian cancer. Therefore, it is necessary to explore the molecular mechanisms of the occurrence and development of ovarian cancer. This investigation encompassed the analysis of gene expression profiles, measurement of transcription levels of potential target genes in peripheral blood samples from ovarian cancer patients and characterization of the ovarian cancer-related secretory protein sphingomyelin phosphodiesterase acid-like 3B (SMPDL3B). Through bioinformatics analysis, potential target genes were identified, and their association with overall survival (OS) and progression-free survival (PFS) in ovarian cancer patients was assessed utilizing relevant databases. Subsequently, differences in target gene expression in ovarian cancer tissue samples were validated through protein blotting and quantitative real-time PCR (qRT-qPCR). Cell proliferation assays using the cell count kit-8 (CCK-8) method, as well as transwell chamber assay and pre coated matrix gel chamber assay were employed to elucidate the role of SMPDL3B in ovarian cancer cell migration and invasion. This study revealed a substantial upregulation of SMPDL3B in the serum of ovarian cancer patients, correlating with an unfavorable prognosis. High SMPDL3B expression was linked not only to increased proliferation of ovarian cancer cells, but also enhanced migration and invasion. Remarkably, the knockdown the human alkaline ceramidase 2 (ACER2) gene in cancer cells with heightened SMPDL3B expression significantly inhibited cell proliferation, migration, and invasion induced by SMPDL3B activation (P<0.05), highlighting the functional interplay between ACER2 and SMPDL3B in ovarian cancer. In summary, this study proposes SMPDL3B as a prognostic marker for ovarian cancer, with implications for potential therapeutic intervention targeting the ACER2-SMPDL3B axis.

SMPDL3B is palmitoylated and stabilized by ZDHHC5, and its silencing aggravates diabetic retinopathy of db/db mice: Activation of NLRP3/NF-κB pathway

Abnormal inflammation of vascular endothelial cells occurs frequently in diabetic retinopathy (DR). Sphingomyelin phosphodiesterase acid-like 3B (SMPDL3B) is a lipid raft enzyme and plays an anti-inflammatory role in various diseases but its function in DR-related vascular endothelial dysfunction remains unknown. We first found that SMPDL3B expression was upregulated from week 10 to 18 in the retinal tissues of db/db mice. Particularly, the high expression of SMPDL3B was mainly observed in retinal vascular endothelium of DR mice. To interfere retinal SMPDL3B expression, adeno-associated viruses 2 (AAV-2) containing SMPDL3B specific shRNA (1233–1253 bp) were injected into the vitreous cavity of db/db mice. SMPDL3B silencing exacerbated the spontaneous DR by further activating the NF-κB/NLRP3 pro-inflammatory pathway. In vitro, human retinal microvascular endothelial cells (HRVECs) were infected with SMPDL3B-shRNA lentiviruses and then stimulated with 30 mM glucose (HG) for 24 h. SMPDL3B-silenced HRVECs secreted more interleukin-1β and had enhanced nuclear p65 translocation. Notably, HG treatment induced the palmitoylation of SMPDL3B. Zinc finger DHHC-type palmitoyltransferase 5 (ZDHHC5) is a palmitoyltransferase that catalyzes the palmitoylation of its substrates, HG exposure increased the interaction between ZDHHC5 and SMPDL3B in HRVECs. 2-BP, a palmitoylation inhibitor, accelerated the protein degradation of SMPDL3B, whereas palmostatin B, a depalmitoylation inhibitor, decreased its turnover rate in HRVECs. Collectively, the present study suggests a compensatory increase of SMPDL3B in HG-treated HRVECs and the retinal tissues of DR mice, indicating that SMPDL3B may be a potential target for DR treatment.

SMPDL3B contributes to gastric adenocarcinoma cells progression by promoting the infiltration of M2 macrophages.

The common disease gastric adenocarcinoma (GAC) has a high morbidity and mortality, so there is an urgent need for research to explore new diagnostic markers and therapeutic targets. This investigation was carried out to investigate the expression of sphingomyelin phosphodiesterase acid-like 3b (SMPDL3B) in GAC and its effects on tumor progression. Samples were collected from patients who underwent radical gastrectomy from January 2021 to December 2022. Along with the normal gastric epithelial cell lines GES-1 and SGC-7901, the AGS, MGC-803, and MSN-45 human gastric cancer cell lines were used to confirm SMPDL3B expression. RT-qPCR, Western blot, immunohistochemical, cell proliferation, assay of wound healing, transwell migration assay, invasion assay, flow cytometry, and immune evaluation experiments were carried out. SMPDL3B was found to be substantially expressed in GAC, and this condition has a bad prognosis. By establishing SMPDL3B knockdown and overexpression of GAC cell lines, this study confirmed that SMPDL3B promoted tumor cell proliferation, migration, and invasion. Additional bioinformatics research revealed a connection between SMPDL3B and immune cell infiltration in the GAC immunological microenvironment, which enhanced tumor cell proliferation by promoting the infiltration content of M2 macrophages. This study determined the function of SMPDL3B for the clinical diagnosis, prediction, and novel management of GAC.

Porcine reproductive and respiratory syndrome virus upregulates SMPDL3B to promote viral replication by modulating lipid metabolism

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a severe threat to the health of pigs globally. Host factors play a critical role in PRRSV replication. Using PRRSV as a model for genome-scale CRISPR knockout (KO) screening, we identified a host factor critical to PRRSV infection: sphingomyelin phosphodiesterase acid-like 3B (SMPDL3B). Our findings show that SMPDL3B restricted PRRSV attachment, entry, replication, and secretion and that its depletion significantly inhibited PRRSV proliferation, indicating that SMPDL3B plays a positive role in PRRSV replication. Our data also show that SMPDL3B deficiency resulted in an accumulation of intracellular lipid droplets (LDs). The expression level of key genes (ACC, SCD-1, and FASN) involved in lipogenesis was increased, whereas the fundamental lipolysis gene, ATGL, was inhibited when SMPDL3B was knocked down. Overall, our findings suggest that SMPDL3B deficiency can effectively inhibit viral infection through the modulation of lipid metabolism.

SMPDL3b modulates radiation-induced DNA damage response in renal podocytes.

The kidneys are radiosensitive and dose-limiting organs for radiotherapy (RT) targeting abdominal and paraspinal tumors. Excessive radiation doses to the kidneys ultimately lead to radiation nephropathy. Our prior work unmasked a novel role for the lipid-modifying enzyme, sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b), in regulating the response of renal podocytes to radiation injury. In this study, we investigated the role of SMPDL3b in DNA double-strand breaks (DSBs) repair in vitro and in vivo. We assessed the kinetics of DSBs recognition and repair along with the ATM pathway and nuclear sphingolipid metabolism in wild-type (WT) and SMPDL3b overexpressing (OE) human podocytes. We also assessed the extent of DNA damage repair in SMPDL3b knock-down (KD) human podocytes, and C57BL6 WT and podocyte-specific SMPDL3b-knock out (KO) mice after radiation injury. We found that SMPDL3b overexpression enhanced DSBs recognition and repair through modulating ATM nuclear shuttling. OE podocytes were protected against radiation-induced apoptosis by increasing the phosphorylation of p53 at serine 15 and attenuating subsequent caspase-3 cleavage. SMPDL3b overexpression prevented radiation-induced alterations in nuclear ceramide-1-phosphate (C1P) and ceramide levels. Interestingly, exogenous C1P pretreatment radiosensitized OE podocytes by delaying ATM nuclear foci formation and DSBs repair. On the other hand, SMPDL3b knock-down, in vitro and in vivo, induced a significant delay in DSBs repair. Additionally, increased activation of apoptosis was induced in podocytes of SMPDL3b-KO mice compared to WT mice at 24 h post-irradiation. Together, our results unravel a novel role for SMPDL3b in radiation-induced DNA damage response. The current work suggests that SMPDL3b modulates nuclear sphingolipid metabolism, ATM nuclear shuttling, and DSBs repair.

Sphingomyelin Phosphodiesterase Acid-Like 3b is Essential for Toll-Like Receptor 3 Signaling in Human Podocytes.

Recent studies have revealed the importance of cell membrane stability in normal cell function. Sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b), a lipid modifying enzyme that converts sphingomyelin to ceramide in the cell membrane, is expressed in macrophages and regulates Toll-like receptor (TLR) 4 signaling by altering cell membrane fluidity. SMPDL3b is also expressed in human podocytes, which are involved in the pathogenesis of several glomerular diseases such as diabetic kidney disease, focal segmental glomerulosclerosis, and idiopathic nephrotic syndrome in children; however, the role of SMPDL3b in podocyte innate immunity is unclear. As podocytes are equipped with innate immune systems including TLR3, and viral infections often exacerbate proteinuria in children with idiopathic nephrotic syndrome, we hypothesized that changes in SMPDL3b expression levels could affect anti-viral responses via TLR3 signaling in podocytes, consequently impairing normal podocyte function. To examine the role of SMPDL3b in TLR3 signaling in podocytes, we treated conditionally immortalized human podocytes with polyinosinic-polycytidylic acid (poly IC), to activate TLR3 signaling. The cells were then transfected with small interfering RNA against SMPDL3b. Poly IC activated the TLR3 pathway, whereas knockdown of SMPDL3b attenuated poly IC-induced interferon-β/chemokine C-X-C ligand 10 expression in podocytes. To our knowledge, this is the first report demonstrating SMPDL3b involvement in podocyte innate immunity; these results suggest that SMPDL3b is essential for adequate anti-viral responses in podocytes, possibly by modulating lipid metabolism in the cell membrane.

SMPDL3B Predicts Poor Prognosis and Contributes to Development of Acute Myeloid Leukemia.

Background: Acute myeloid leukemia (AML), characterized by the low cure rate and high relapse, urgently needs novel diagnostic or prognostic biomarkers and potential therapeutic targets. Sphingomyelin Phosphodiesterase Acid Like 3B (SMPDL3B) is a negative regulator of Toll-like receptor signaling that plays important roles in the interface of membrane biology and innate immunity. However, the potential role of SMPDL3B in human cancer, especially in AML, is still unknown.Methods: The expression of SMPDL3B in AML samples was investigated through data collected from Gene Expression Omnibus (GEO). Association between SMPDL3B expression and clinicopathologic characteristics was analyzed with the chi-square test. Survival curves were calculated by the Kaplan–Meier method. Cox univariate and multivariate analyses were used to detect risk factors for overall survival. The biological functions of SMPDL3B in human AML were investigated both in vitro and in vivo.Results: Expression of SMPDL3B mRNA was significantly upregulated in human AML samples and closely correlated to cytogenetics risk and karyotypes. Elevated expression of SMPDL3B was associated with poor overall survival and emerged as an independent predictor for poor overall survival in human AML. Blocked SMPDL3B expression inhibited AML cells growth both in vitro and in vivo via promoting cell apoptosis.Conclusion: Taken together, our results demonstrate that SMPDL3B could be used as an efficient prognostic biomarker and represent a potential therapeutic target for human AML.

A Murine Model Of Kidney Injury: The role of SMPDL3b In Radiation And Cisplatin-Induced Nephrotoxicity

Cisplatin is widely used as a chemotherapeutic agent and a known radiosensitizer. The current study aimed to investigate the role of lipid-modulating enzyme sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) in cisplatin toxicity. Nephrotoxicity is dose-limiting toxicity. Cultured oocytes were treated with rituximab mAb (100 μg/ml) or IgG (100 μg/ml) 30 minutes before irradiation with 0-8 Gy, with or without Cisplatin (2μM). In other experiments, 10–14-week old C57BL/6 male and female mice were given bilateral kidney X-irradiation (4Gyx1, 6 Gyx5, and 2Gyx24) with or without Cisplatin (6mg/kg, IP). Animals received rituximab mAb (RTX) or IgG (50mg/kg; IV) 30 minutes before radiation treatment. Functional kidney parameters (serum BUN, hematocrit, and creatinine), histopathological assessments (oil red staining, H&E, Periodic Acid-Schiff, and TEM) and biochemical changes were studied at baseline, 10- and 20-weeks post-chemo-radiotherapy. Glomerular filtration rate (GFR) was estimated at 10- and 20-weeks post-radiation using FITC-sinistrin based transdermal monitor and iSMAART dynamic contrast-enhanced (DCE) imaging. RT and concurrent Cisplatin (2 μM) produced time-dependent apoptosis in wild-type human podocytes, causing an 85% increase in caspase 3 activity 2h post-RT (8Gy). Different doses of RT, 1x4Gy, 1x10Gy, and 1x14Gy increased glomerular base membrane (GBM) thickness in irradiated mice significantly (100%, 150%, and 200% respectively). Cisplatin and RT (1x4Gy) increased GBM thickness from 225nm to 425nm, respectively (p = 0.01). The eGFR values changed significantly from 1010±214 to 718±205, 687±175, 508±56, 596±180, and 639±221 mL/min after x4Gy, 1x10Gy, 1x14Gy, 5x6Gy, and 24x2Gy, respectively. RTX pretreatment improved eGFR and other functional parameters (serum BUN, hematocrit, serum creatinine, and urine albumin/creatinine ratio (ACR) significantly post chemoradiotherapy. RTX pretreatment protected podocytes cytoskeletal remodeling in vitro and foot process effacement in vivo. SMPDL3b expression and podocytes count significantly decreased post chemoradiotherapy. RTX ameliorated SMPDL3b down-regulation, and improved podocytes count significantly. This study shows that RTX pretreatment protects podocytopathy in vitro and chemoradiation induced kidney injury in vivo. SMPDL3b may be an important therapeutic target for radiation-induced kidney injuries in cancer patients.

Sphingomyelin Phosphodiesterase Acid-like 3B (SMPDL3b) Affects Podocyte Lipid Metabolism and Lipid Droplets Formation

Sphingomyelin Phosphodiesterase Acid-like 3B (SMPDL3b) Affects Podocyte Lipid Metabolism and Lipid Droplets Formation

Podocyte sphingomyelin phosphodiesterase acid-like 3b decreases among children with idiopathic nephrotic syndrome.

Sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b), a regulator of the cytoskeleton, is expressed on podocytes. Recent reports present evidence that it is directly targeted by rituximab in the treatment of intractable nephrotic syndrome. However, the implications of SMPDL-3b for treatment of paediatric-onset idiopathic nephrotic syndrome (INS) remain unclear. This study aimed to investigate the level of expression of SMPDL-3b in urine, serum, and biopsy specimens and explore its implications in treatment of patients with INS. Levels of urinary SMPDL-3b among 31 patients (20 in remission and 11 in relapse) with INS were analysed by dot blotting. For reference of precise quantitative analysis, we examined urinary excretion of SMPDL-3b from 10 patients with INS by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in both remitted and relapsed status. The levels of serum SMPDL-3b among 20 patients (13 in remission and 7 in relapse or onset) with INS were also measured using enzyme-linked immunosorbent assay. Further, the immunoreactivity of SMPDL-3b in the biopsy specimens obtained from patients with INS was compared with those from patients with proteinuric IgA nephropathy, lupus nephritis, and non-proteinuric controls. Urinary excretion of SMPDL-3b in patients with INS was significantly decreased in relapse cases compared with cases of remission and other types of proteinuric glomerular disease or controls by both dot blotting and LC-MS/MS method. On the other hand, serum SMPDL-3b level in INS was not different between cases of remission and relapse. Glomerular immunoreactivity of SMPDL-3b in patient with INS in remission was almost the same level to that of control. The expression of SMPDL-3b on podocytes is specifically decreased in paediatric-onset INS and its urinary excretion level reflects such conditions.

Phosphodiesterase SMPDL3B Gene Expression as Independent Outcome Prediction Marker in Localized Prostate Cancer.

Current outcome prediction markers for localized prostate cancer (PCa) are insufficient. The impact of the lipid-modifying Sphingomyelin Phosphodiesterase Acid Like 3B (SMPDL3B) in PCa is unknown. Two cohorts of patients with PCa who underwent radical prostatectomy (n = 40, n = 56) and benign prostate hyperplasia (BPH) controls (n = 8, n = 11) were profiled for SMPDL3B expression with qRT-PCR. Publicly available PCa cohorts (Memorial Sloane Kettering Cancer Centre (MSKCC; n = 131, n = 29 controls) and The Cancer Genome Atlas (TCGA; n = 497, n = 53 controls)) served for validation. SMPDL3B’s impact on proliferation and migration was analyzed in PC3 cells by siRNA knockdown. In both cohorts, a Gleason score and T stage independent significant overexpression of SMPDL3B was seen in PCa compared to BPH (p < 0.001 each). A lower expression of SMPDL3B was associated with a shorter overall survival (OS) (p = 0.005) in long term follow-up. A SMPDL3B overexpression in PCa tissue was confirmed in the validation cohorts (p < 0.001 each). In the TCGA patients with low SMPDL3B expression, biochemical recurrence-free survival (p = 0.011) and progression-free interval (p < 0.001) were shorter. Knockdown of SMPDL3B impaired PC3 cell migration but not proliferation (p = 0.0081). In summary, SMPLD3B is highly overexpressed in PCa tissue, is inversely associated with localized PCa prognosis, and impairs PCa cell migration.

Human alkaline ceramidase 2 promotes the growth, invasion, and migration of hepatocellular carcinoma cells via sphingomyelin phosphodiesterase acid-like 3B.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. It has a poor prognosis because it is often diagnosed at the advanced stage when treatments are limited. In addition, HCC pathogenesis is not fully understood, and this has affected early diagnosis and treatment of this disease. Human alkaline ceramidase 2 (ACER2), a key enzyme that regulates hydrolysis of cellular ceramides, affects cancer cell survival, however its role in HCC has not been well characterized. Our results showed that ACER2 is overexpressed in HCC tissues and cell lines. In addition, high ACER2 protein expression was associated with tumor growth; ACER2 knockdown resulted in decreased cell growth and migration. Sphingomyelin phosphodiesterase acid‐like 3B (SMPDL3B) promoted HCC cell growth, invasion, and migration; SMPDL3B knockdown had a significant inhibitory effect on HCC tumor growth in vivo. Moreover, ACER2 positively regulated the protein level of SMPDL3B. Of note, ACER2/SMPDL3B promoted ceramide hydrolysis and S1P production. This axis induced HCC survival and could be blocked by inhibition of S1P formation. In conclusion, ACER2 promoted HCC cell survival and migration, possibly via SMPDL3B. Thus, inhibition of ACER2/SMPDL3B may be a novel therapeutic target for HCC treatment.

Modulation of radiation-induced damage of human glomerular endothelial cells by SMPDL3B.

The intracellular molecular pathways involved in radiation-induced nephropathy are still poorly understood. Glomerular endothelial cells are key components of the structure and function of the glomerular filtration barrier but little is known about the mechanisms implicated in their injury and repair. The current study establishes the response of immortalized human glomerular endothelial cells (GEnC) to ionizing radiation (IR). We investigated the role of sphingolipids and the lipid-modifying enzyme sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) in radiation-induced GEnC damage. After delivering a single dose of radiation, long and very-long-chain ceramide species, and the expression levels of SMPDL3b were elevated. In contrast, levels of ceramide-1-phosphate (C1P)dropped in a time-dependent manner although mRNA and protein levels of ceramide kinase (CERK) remained stable. Treatment with C1P or knocking down SMPDL3b partially restored cell survival and conferred radioprotection. We also report a novel role for the NADPH oxidase enzymes (NOXs), namely NOX1, and NOX-derived reactive oxygen species (ROS) in radiation-induced GEnC damage. Subjecting cultured endothelial cells to radiation was associated with increased NOX activity and superoxide anion generation. Silencing NOX1 using NOX1-specific siRNA mitigated radiation-induced oxidative stress and cellular injury. In addition, we report a novel connection between NOX and SMPDL3b. Treatment with the NOX inhibitor, GKT, decreased radiation-induced cellular injury and restored SMPDL3b basal levels of expression. Our findings indicate the importance of SMPDL3b as a potential therapeutic target in radiation-induced kidney damage.

SMPDL3b modulates insulin receptor signaling in diabetic kidney disease

Sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is a lipid raft enzyme that regulates plasma membrane (PM) fluidity. Here we report that SMPDL3b excess, as observed in podocytes in diabetic kidney disease (DKD), impairs insulin receptor isoform B-dependent pro-survival insulin signaling by interfering with insulin receptor isoforms binding to caveolin-1 in the PM. SMPDL3b excess affects the production of active sphingolipids resulting in decreased ceramide-1-phosphate (C1P) content as observed in human podocytes in vitro and in kidney cortexes of diabetic db/db mice in vivo. Podocyte-specific Smpdl3b deficiency in db/db mice is sufficient to restore kidney cortex C1P content and to protect from DKD. Exogenous administration of C1P restores IR signaling in vitro and prevents established DKD progression in vivo. Taken together, we identify SMPDL3b as a modulator of insulin signaling and demonstrate that supplementation with exogenous C1P may represent a lipid therapeutic strategy to treat diabetic complications such as DKD.

The Role of SMPDL3b in Regulating Radiation Nephropathy

Radiation nephropathy (RN) is functionally characterized by an initial acute phase (proteinuria) followed by a chronic decline of glomerular filtration rate leading to renal failure. RN is irreversible and affects the quality of life of both short- and long-term cancer survivors. The molecular mechanisms responsible for RN remain largely unknown. The current study investigates the role of sphingolipids in radiation-induced podocytopathy and RN. Sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) expression was assessed in cultured podocytes after X-irradiation (1-8 Gy) by real-time PCR and Western blotting. Changes in podocyte morphology and radiation-induced DNA damage repair were assessed in wild-type, SMPDL3b-KO and SMPDL3b-over-expressing podocytes. Wild-type C57BL/6 male and female mice (10–14 weeks) were X-irradiated using CT image-guidance (SD 14 Gy, fractionated 24x2 Gy; and 4 Gy SD + 6 mg/kg cisplatin). 50 mg/kg Rituximab (or IgG) was administered IP 30 min before irradiation to protect SMPDL3b. Functional kidney parameters (proteinuria and albumin/creatinine urine ratio, along with serum BUN [blood urea nitrogen], creatinine and hematocrit) were analyzed 10–40 weeks post treatment. Kidney histology, podocyte number and SMPDL3b levels were assessed by immunohistochemistry and Western blotting. Following irradiation, SMPDL3b expression was significantly reduced in vitro and in vivo . In irradiated kidneys, podocyte number decreased significantly and H&E kidney sections demonstrated a multifocal increase in the number of pericytes, tubular atrophy and glomerular damage. Transmission electron microscopy indicated radiation-mediated changes to glomerular base membrane thickness and podocyte foot process effacement. Periodic Acid-Schiff (PAS) staining demonstrated increases in glomerular mesangial matrix accumulation post-irradiation, along with diffuse intertubular fibrosis as determined by Sirius red staining, especially in the renal cortex. Rituximab improved kidney functional parameters, vascular structure, normalization of pericyte coverage and suppressed the development of fibrosis and tubular damage post irradiation. SMPDL3b expression was identified as a molecular determinant of podocyte injury after single dose and fractionated X-irradiation. Rituximab pretreatment protected mice against radiation-induced nephrotoxicity, which may have therapeutic implications for radiation-induced injuries in cancer patients.

Abstract 4161: Protecting Sphingomyelin Phosphodiesterase Acid Like 3B (SMPDL3b) enhances kidney function and reduces concurrent chemoradiotherapy-induced nephrotoxicity

Abstract Background: Cisplatin is a widely used radiosensitizer. Dose-related and cumulative renal insufficiency, including acute renal failure, are the primary dose-limiting toxicities of cisplatin. The lipid-modulating enzyme sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) and Sphingosine 1-Phosphate Receptor-1 (S1P1) are critical determinants of renal podocyte injury. In the current study, we investigated the functions of SMPDL3b and S1P in cisplatin and radiation-induced renal injury, and the use of rituximab (RTX) to attenuate nephrotoxicity. Material/Methods: Podocytes were incubated for 2 h with S1P (100 nM) or vehicle (2% dodecane/98% ethanol) and irradiated with 0-8 Gy, or podocytes were irradiated (8 Gy) in the presence of RTX (100 mg/ml) or IgG (100 mg/ml). In other experiments, 10-14-week old C57BL/6 mice were given bilateral kidney X-irradiation (4 Gy) using an image-guided small animal arc radiation treatment system (iSMAART), 6mg/kg cisplatin or combined cisplatin plus radiation. Some animals received a single intravenous (IV) injection of rituximab mAb or IgG (50 mg/kg) prior to treatment. Functional kidney parameters and immuno-histopathological assessments of renal damage (e.g. oil red staining, H &amp; E, Periodic Acid-Schiff (PAS) and SMPDL3B expression) were measured post-RT. iSMAART dynamic contrast-enhanced (DCE) imaging was used for estimating glomerular filtration rate 20 weeks post radiation. Results: Exogenous S1P rescued podocytes from concurrent chemoradiotherapy-induced cellular cytoskeletal remodeling, and RTX pretreatment mitigated podocytopathy in vitro. iSMAART dynamic contrast-enhanced (DCE) imaging analysis showed that rituximab pretreatment improved the GFR and kidney function parameters post concurrent chemoradiotherapy. SMPDL3b expression at the protein level and podocyte counts dropped significantly in irradiated kidney post-RT in cisplatin-treated mice. Rituximab pretreatment in cisplatin-treated mice, improved GFR, kidney functional parameters, vascular structure, normalization of pericyte coverage, suppress the development of fibrosis and tubular damage post-irradiation. Conclusion: This study shows that rituximab pretreatment protects mice against concurrent chemoradiation induced kidney injury. SMPDL3b and Sphingosine 1-Phosphate Receptor-1 may be important therapeutic targets for radiation-induced kidney injuries in cancer patients. Citation Format: Anis Ahmad, Alla Mitrofanova, Saba Ansari, Junwei Shi, Yidong Yang, Sandra Merscher, Alessia Fornoni, Youssef Zeidan, Brian Marples. Protecting Sphingomyelin Phosphodiesterase Acid Like 3B (SMPDL3b) enhances kidney function and reduces concurrent chemoradiotherapy-induced nephrotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4161.

Novel Role for Sphingolipid Metabolism in Radiation Nephropathy

The molecular mechanisms of radiation nephropathy remain largely unidentified. Sphingomyelin phosphodiesterase acid like 3b (SMPDL3b) is a lipid modulating enzyme that plays a major role in podocyte injury, and in the pathogenesis of proteinuria in glomerular diseases. The current study investigates the role of bioactive sphingolipids and SMPDL3b in radiation-induced podocytopathy, and identifies a monoclonal antibody targeting CD20, rituximab, as a radioprotector. Immortalized human podocytes were X-irradiated with 8 Gy ± rituximab (100 μg/mL) 30 minutes prior to radiation. Metabolic activity and viability were assessed. Transcriptional and translational levels of SMPDL3b were measured by RT-PCR and Western blot respectively. Cytoskeletal remodeling and morphological changes were detected using immunofluorescence microscopy. C57BL6 mice, 12-14 week-old, were subjected to bilateral kidney irradiation (14 Gy) using a CT-image guided X-irradiator and functional parameters of kidney damage measured every 10 weeks for 40 weeks (BUN, hematocrit, serum creatinine, urine albumin and creatinine ratio). Histological assessments (H&E, Picro-sirius red, and Periodic Acid Schiff), podocyte number, and SMPDL3B expression were measured in harvested kidneys post-RT. In culture, podocytes viability and metabolic activity decreased after X-irradiation. Cytoskeletal remodeling, manifesting as loss of lamellipodia and remodeling of cortical actin, was detected. Ezrin, an actin binding protein, relocated from the plasma membrane to the cytosol as early as 2h post radiation. Ceramide levels increased while sphingosine-1 phosphate (S1P) and sphingosine levels decreased. In contrast, overexpression of SMPDL3b and exogenous S1P rescued podocytes from radiation-induced cytoskeletal changes. Treatment with rituximab protected podocytes from SMPDL3b loss and caspase-3 cleavage. In irradiated animals, kidney weight and volume were reduced significantly, SMPDL3b expression and podocyte number decreased, changes that were not evident after rituximab administration. Loss of SMPDL3b expression and podocyte number were associated with reduction in functional and histological parameters. Radiation-injury induces downregulation of SMPDL3b, and elevation of ceramide levels as well as early cytoskeletal remodeling. Overexpression of SMPDL3b or pretreatment with rituximab is radioprotective in cultured podocytes and animals. These findings suggest a potential role for SMPDL3b in radiation-induced podocytopathy.

Rituximab protects podocytes and exerts anti-proteinuric effects in rat adriamycin-induced nephropathy independent of B-lymphocytes.

This study was aimed at examining the effects of treatment with rituximab, a chimeric monoclonal antibody against the protein CD20, in a B-lymphocyte independent adriamycin-induced rat model of nephrotic syndrome. Rituximab is an emerging rescue therapy used in patients with complicated nephrotic syndrome and, therefore, we sought to elucidate the apparent B-lymphocyte independent mechanism underlying its anti-proteinuric effect. Adriamycin-induced nephropathy was established in Wistar rats by intravenously injecting 10 mg/kg of adriamycin, which were then treated with rituximab or purified human IgG weekly and euthanized on day 28. Proteinuria, glomerular expression of sphingomyelin phosphodiesterase acid-like 3b protein, and podocyte-related proteins were examined using immunofluorescence staining and a reverse transcription-polymerase chain reaction. Rituximab treatment of rats with adriamycin-induced nephropathy significantly reduced urinary protein excretion 14 to 28 days after induction of disease, compared with those treated with purified normal human IgG. Furthermore, rituximab treatment also prevented the reduction of glomerular nephrin and podocin expression on day 28. Double-immunofluorescence staining revealed that after in vivo administration, rituximab was bound to the glomeruli, which also expressed synaptopodin or sphingomyelin phosphodiesterase, acid-like 3b. Moreover, sphingomyelin phosphodiesterase, acid-like 3b expression was significantly decreased on day 28 of adriamycin-induced nephropathy, which was also prevented by rituximab. This study demonstrated that rituximab directly affected glomerular podocytes and ameliorated proteinuria in adriamycin-induced nephropathy in rats. Furthermore, protection of podocyte function by rituximab may be mediated by direct modulation of a sphingomyelin phosphodiesterase, acid-like 3b-dependent mechanism.

Sphingomyelinase-like phosphodiesterase 3b mediates radiation-induced damage of renal podocytes.

The molecular mechanisms responsible for the development of proteinuria and glomerulosclerosis in radiation nephropathy remain largely unknown. Podocytes are increasingly recognized as key players in the pathogenesis of proteinuria in primary and secondary glomerular disorders. The lipid-modulating enzyme sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) is a key determinant of podocyte injury and a known off target of the anti-CD20 antibody rituximab (RTX). The current study investigates the role of sphingolipids in radiation-induced podocytopathy. After a single dose of radiation (8 Gy), several ceramide species were significantly elevated. In particular, C16:00, C24:00, and C24:1 ceramides were the most abundant ceramide species detected. These changes were paralleled by a time-dependent drop in SMPDL3b protein, sphingosine, and sphingosine-1-phosphate levels. Interestingly, SMPDL3b-overexpressing podocytes had higher basal levels of sphingosine-1-phosphate and maintained basal ceramide levels after irradiation. Morphologically, irradiated podocytes demonstrated loss of filopodia and remodeling of cortical actin. Furthermore, the actin binding protein ezrin relocated from the plasma membrane to the cytosol as early as 2 h after radiation. In contrast, SMPDL3b overexpressing podocytes were protected from radiation-induced cytoskeletal remodeling. Treatment with RTX before radiation exposure partially protected podocytes from SMPDL3b loss, cytoskeletal remodeling, and caspase 3 cleavage. Our results demonstrate that radiation injury induces early cytoskeletal remodeling, down-regulation of SMPDL3b, and elevation of cellular ceramide levels. Overexpression of SMPDL3b and pretreatment with RTX confer a radioprotective effect in cultured podocytes. These findings indicate a potential role for SMPDL3b and RTX in radiation-induced podocytopathy.-Ahmad, A., Mitrofanova, A., Bielawski, J., Yang, Y., Marples, B., Fornoni, A., Zeidan, Y. H. Sphingomyelinase-like phosphodiesterase 3b mediates radiation-induced damage of renal podocytes.