Inhibition of the lncRNA 585189 Prevents Podocyte Injury and Mitochondria Dysfunction by Promoting hnRNP A1 and SIRT1 in Diabetic Nephropathy

Background: Dysfunction of podocytes has been identified as a crucial pathologic characteristic of diabetic nephropathy (DN), while the regulatory effect of long noncoding RNAs (lncRNAs) in this process has not been fully elucidated. Methods: We conducted RNA-seq on renal tissues and identified a significantly upregulated lncRNA ENST00000585189.1 (lncRNA 585189) in patients with DN. Subsequently, we assessed its correlation with clinical indicators and demonstrated its localization with RNA-FISH. In high-glucose-induced human podocytes, the expression of lncRNA 585189 was assessed via real-time PCR and RNA-FISH. Subsequently, gain-and loss-of-function experiments detected the effect of lncRNA 585189. The expression of Desmin, ZO-1, hnRNP A1 and SIRT1 were evaluated using real-time PCR, western blot and immunofluorescence assays. Mitochondrial morphologies were evaluated using MitoTracker, while mitochondrial ROS and membrane potential were assessed using MitoSOX Red and TMRM staining. Mechanistically, bioinformatics analysis predicted the interaction between lncRNA 585189 and hnRNP A1, which was confirmed by RIP, pull-down, and EMSA assays. Moreover, the binding of hnRNP A1 to SIRT1 mRNA was validated through RIP and pull-down assays. Additionally, the stability of hnRNPA1 and SIRT1 was assessed by treatment with Cloheximide, MG-132, and Actinomycin D. Results: lncRNA 585189 displayed a positive correlation with renal insufficiency and was found to be upregulated in both DN patients and high-glucose-induced human podocytes. Silence of lncRNA 585189 decreased the production of ROS, rescued the aberrant mitochondrial morphology and membrane potential, restored the podocyte damage caused by high glucose. Mechanistically, lncRNA 585189 binds to hnRNP A1, inducing destabilization of hnRNP A1 protein and downregulating its expression. Conversely, hnRNP A1 promotes the expression of lncRNA 585189. Furthermore, the interaction between hnRNP A1 and SIRT1 mRNA promotes the stability of SIRT1 mRNA and enhances its expression. Finally, our findings suggested that lncRNA 585189 inhibited SIRT1 expression through hnRNP A1, hindering the recovery of mitochondrial abnormalities and podocyte damage. Conclusions: In summary, targeting lncRNA 585189 may represent a promising strategy for reversing mitochondrial dysfunction and treating DN.


FR-PO340 Poster Friday
Diabetic Kidney Disease: Basic -I
Background: The stimulator of interferon genes (STING) is an adaptor protein that can drive noninfectious inflammation and pyroptosis.NLRP3-mediated inflammasome activation is an important type of inflammatory programmed death with strong proinflammatory activity and has been confirmed to promote a chronic inflammatory response in DKD.However, the mechanism of STING regulating immune inflammation and the interaction with NLRP3-dependent pyroptosis in the high glucose (HG) state remains unknown.The present study aims to evaluate whether STING contributes to HG-induced podocyte inflammation response through modulating NLRP3-dependent pyroptosis.
Methods: In vivo study, co-localization expression of STING and synaptopodin was evaluated in glomeruli by double immunolabeling.Western blot was performed to assess STING and inflammatory-related protein expression in glomeruli.q-PCR was used to detect inflammatory molecule expression.Transmission electron microscope (TEM) was used to identify ultrastructure changes of podocytes in diabetic mice.Pathological changes were evaluated by periodic acid-Schiff stain (PAS) or hematoxylin and eosin (HE) staining.In vitro study, podocytes were exposed to HG (40mM) for 24 h.Immunofluorescence assay and western blot were performed to evaluate STING expression.Pyroptosis and inflammatory-related molecule expression were detected by western blot and q-PCR.STING siRNA was transfected to podocytes to evaluate the effect of STING inhibition on podocyte inflammatory response under HG stimulation.
Results: Double immunolabeling of STING and synaptopodin in glomeruli was obviously increased in diabetic animals.In addition, STING protein expression was increased in diabetic mice compared with controls.Deletion of STING alleviated podocyte injury, renal dysfunction inflammation, NLRP3 inflammasome activation, and pyroptosis in diabetic mice.In cultured podocytes, STING protein expression was increased in HGtreated podocytes.Modulated STING expression by STING siRNA alleviated pyroptosis and NLRP3 inflammasome activation in HG-treated podocytes.
Conclusions: These results indicate that STING deletion suppresses podocytes inflammation response by targeting NLRP3 and provide evidence that STING may be a potential target for podocyte injury in DKD.Background: Rho GTPase activity plays a key role in cytoskeletal rearrangement and apoptosis of podocytes in diabetic nephropathy (DN).However, the upstream regulatory mechanism directing Rho GTPases in podocytes of DN remains unclear.This study aimed to verify the expression of SrGAP3, a Rho GTPase activating protein, in kidney and its role and mechanism in podocyte injury of DN.

FR-PO341
Methods: The level of SrGAP3 expression was analyzed using immunofluorescence in the glomerular podocytes of renal tissue from DN patients and diabetic mice with albuminuria.Albuminuria, podocyte injury and glomerular pathology were evaluated in SrGAP3 overexpression of type 2 diabetic db/db mice and STZ-induced type 1 diabetic mice.The expressions and activities of Cdc42 and Rac1 in the glomerular podocytes in vivo in diabetic mice and in vitro in podocytes exposed to either SrGAP3 knockdown or high glucose treated podocytes with SrGAP3 overexpression were detected by immunofluorescence, western blot and enzyme activity detection.Podocyte marker proteins, cytoskeleton, cell motility and apoptosis were observed in in vitro podocytes under different conditions.
Results: SrGAP3 was markedly reduced in glomerular podocytes from renal tissues of DN patients, db/db mice and STZ-induced diabetic mice with albuminuria.The albuminuria in db/db and STZ-induced diabetic mouse model was markedly attenuated after overexpression of SrGAP3 established by adeno-associated virus.This was accompanied by alleviation of basement membrane thickening, mesangial matrix expansion, and podocyte injury.The decreased expression of SrGAP3 in podocytes cultured with high glucose was upregulated after overexpression of SrGAP3, accompanied by restored decreased podocyte marker proteins, and improved actin cytoskeleton rearrangement, podocyte motility and apoptosis.Further studies found that increased activity of Cdc42 and Rac1 in podocytes in vivo and in vitro under the diabetic state were significantly inhibited after overexpression of SrGAP3.
Conclusions: SrGAP3 deficiency mediates podocyte injury of DN by promoting Cdc42 and Rac1 activation, which may provide a clinical therapeutic target for protecting podocyte injury of DN.

FR-PO342 Poster Friday
Diabetic Kidney Disease: Basic -I

Podocyte-Specific Clusterin Triggers the Activation of Proximal
Tubule-Specific CAMK1D Signaling to Attenuate Kidney Injury in Diabetic Kidney Disease Nehaben A. Gujarati, 1 Bismark Owusu Frimpong, 1,2 Malaika Zaidi, 1 Sandeep K. Mallipattu.Background: The role of podocyte-proximal tubule (PT) crosstalk in the progression of Diabetic Kidney Disease (DKD) remains understudied.Podocytespecific overexpression of Krüppel-like factor 6 (KLF6), a zinc finger transcription factor, attenuated proximal tubule (PT) injury in murine DKD model.Initial snRNAseq studies demonstrated a potential interaction between podocyte Clusterin (CLU), a secretory ligand, and proximal tubule CAMK1D.The aim of this study is to investigate the mechanism by which the podocyte primes the PT to attenuate kidney injury in DKD.
Methods: Conditioned media (CM) was collected from GFP-labeled podocytes obtained from podocyte-specific induction of KLF6 (KLF6 PODTA ) and control mice to carry out proteomic analysis and in vitro assays in primary (1°) PT. 1° PT cells were treated with control CM as well as CM blocked with CLU antibody.CAMK1D signaling was pharmacologically inhibited using STO-609, an inhibitor of the upstream kinase CAMKK.Oxygen consumption rate (OCR) was measured with seahorse analyzer.Immunofluorescence staining, western blot analysis, and pulldown assay for calmodulin were performed.Single nuclei ATAC (SnATAC) and snRNA sequencing were conducted on the kidney cortex of all mice.
Results: Immunostaining of experimental mice as well as proteomic analysis of the podocyte CM and urine, led to identification of CLU as a potential ligand for PT signaling.KLF6 PODTA CM-treated 1° PT cells demonstrated an increase in calmodulin binding affinity as well as an increase in OCR compared to control CM-treated cells in high glucose (HG) conditions, and this OCR change was abated by the addition of CLU blocking antibody.CAMK1D expression was unique to the first segment of PT in mouse as well as human tissue.1° PT cells treated with STO-609 had a decrease in cell viability and OCR in HG conditions, a decrease in p-DRP1, and an increase in fragmented mitochondria.SnATACseq in combination with snRNAseq also validated pathways related to calcium signaling and podocyte-PT communication.
Conclusions: These data suggest that CLU secreted from podocytes attenuates mitochondrial fission in the PT by inducing CAMK1D signaling, thereby priming the PT against injury under diabetic conditions.
Funding: NIDDK Support, Veterans Affairs Support

SrGAP3 Deficiency Mediates Podocyte Injury by Promoting Cdc42 and Rac1 Activity in Diabetic Nephropathy
Yan Yang, Li Zhang, Xinling Liang.Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guang Zhou, China.