Promoting mitochondrial dynamics by inhibiting the PINK1–PRKN pathway to relieve diabetic nephropathy

ABSTRACT Diabetes is a metabolic disorder characterized by high blood glucose levels and is a leading cause of kidney disease. Diabetic nephropathy has been attributed to dysfunctional mitochondria. However, many questions remain about the exact mechanism. The structure, function and molecular pathways are highly conserved between mammalian podocytes and Drosophila nephrocytes; therefore, we used flies on a high-sucrose diet to model type 2 diabetic nephropathy. The nephrocytes from flies on a high-sucrose diet showed a significant functional decline and decreased cell size, associated with a shortened lifespan. Structurally, the nephrocyte filtration structure, known as the slit diaphragm, was disorganized. At the cellular level, we found altered mitochondrial dynamics and dysfunctional mitochondria. Regulating mitochondrial dynamics by either genetic modification of the Pink1–Park (mammalian PINK1–PRKN) pathway or treatment with BGP-15, mitigated the mitochondrial defects and nephrocyte functional decline. These findings support a role for Pink1–Park-mediated mitophagy and associated control of mitochondrial dynamics in diabetic nephropathy, and demonstrate that targeting this pathway might provide therapeutic benefits for type 2 diabetic nephropathy.


FR-PO374 Poster Friday
Diabetic Kidney Disease: Basic -I Insulin and Glucose Tolerance Assessment in Guanylyl Cyclase/ Natriuretic Peptide Receptor-A Gene-Targeted Mutant Mice Kandasamy Neelamegam, Kailash N. Pandey.Tulane University School of Medicine, New Orleans, LA.
Background: Atrial natriuretic peptide (ANP), acting through the guanylyl cyclase/ natriuretic peptide receptor-A (GC-A/NPRA), is pivotal in regulating blood pressure and cardiac homeostasis.Ablation of Npr1 (encoding NPRA) in mice exhibits hypertension and provokes congestive heart failure; however, the underlying mechanisms are not yet precisely determined.The objective of this study was to investigate whether Npr1 plays a critical role in regulating glucose homeostasis.
Conclusions: NPRA markedly prevented a steep rise in blood glucose levels after glucose challenge and ameliorated glucose intolerance and insulin resistance in 2-copy and 4-copy mice than 1-copy mice.The results suggest that NPRA signaling predisposes to arterial pressure, hyperglycemia, and insulin resistance, thus Npr1 gene might regulate blood glucose homeostasis.Supported by NIH grant DK133833.
Funding: NIDDK Support

FR-PO375 Poster Friday
Diabetic Kidney Disease: Basic -I
Methods: C57BL6 WT male mice received adenine containing water or control water for 4 weeks.db/db male mice with type 2 diabetes received methylthioadenosine phosphorylase (MTAP) inhibitor or vehicle by drinking water for 8 weeks.Kidney metabolites were measured by ZipChip mass spectrometry.Mouse proximal tubule (MCT) cells were employed for in vitro experiments.
Results: Adenine increased albuminuria, blood urea nitrogen (BUN), kidney hypertrophy, kidney KIM-1 expression, kidney matrix protein accumulation, and kidney mTORC1 activity in WT mice.Adenine stimulated senescence-associated secretory phenotype along with reduction in Klotho expression in the kidney.Rapamycin (a selective inhibitor of mTORC1) inhibited adenine-induced mTORC1 activation and matrix protein accumulation in the MCT cells.MTAP inhibitor decreased kidney adenine content, serum cystatin C, albuminuria, and urine KIM-1 excretion in diabetic db/db mice.

FR-PO377 Poster Friday
Diabetic Kidney Disease: Basic -I

Novel Molecular Therapy of Diabetic Nephropathy by Repurposing Niclosamide to Modulate Renal RNA-Binding Protein HuR
Lili Zhuang, Xiao-Qing E. Tsai, Zhou Wang, Yufeng Huang.University of Utah Health, Salt Lake City, UT.
Background: Hu antigen R (HuR) influences the expression of genes involved in pathways critical to the pathogenesis of diabetic nephropathy (DN).We identified a FADapproved anthelmintic drug, niclosamide (NCS), as a novel inhibitor of HuR.This study sought to determine whether HuR-targeted therapeutics with NCS are therapeutic for DN.
Methods: Four groups of uninephrectomized mice with normal control and diabetic db/db mice without treatment but being terminated at 14 and 22 wks respectively, or treated with NCS (20mg/kg daily via i.p.) from wks 18 to 22 were included.
Results: A significantly increased HuR expression was observed in diabetic kidneys from both patients and db/db mice, while the latter was inhibited by NCS treatment.Immunofluorescent staining for HuR confirmed the Western blot measurement.Untreated db/db mice developed progressive albuminuria and glomerular mesangial matrix expansion between age of weeks 14 and 22, associated with increased renal production of fibronectin and a-smooth muscle actin but decreased glomerular WT-1 + -podocytes and nephrin expression.NCS treatment did not affect mouse body weight but reduced blood HbA1c levels (10.8±1.0 in treated db/db vs. 13.4±0.93% in db/db, P<0.05), arrested the increases in albuminuria, markers of glomerulosclerosis and podocyte injury seen in db/ db mice.Renal expressions of NF-κBp65, TNF-a, MCP-1, Nox2, and urine TBARS levels, the markers of inflammation and oxidative stress, were increased during disease progression in db/db mice, which were halted by NCS treatment (P<0.05).In addition, a downstream factor of the Wnt signaling pathway, known as WNT1-inducible signaling pathway protein 1 (WISP1), has been identified as one of key downstream mediators of HuR-dependent action and found to be markedly increased by 3.2 fold in db/db mouse kidneys at 22 wks, compared with non-diabetic controls, which was abrogated by NCS treatment, approaching to normal levels.

Conclusions:
These results indicate that inhibition of HuR with NCS is therapeutic for DN through improving hyperglycemia and renal inflammation and oxidative stress.The efficacy of NCS in reducing renal WISP1 expression and action may also contribute to its reno-protective effect.Our study provides a proof-of-concept for re-purposing HuR inhibitor as a novel intervention therapy for progressive DN.
Funding: NIDDK Support

FR-PO378 Poster Friday
Diabetic Kidney Disease: Basic -I
Conclusions: Decreased renal PON2 may be involved in the pathogenesis of T2DN.
Background: Measurement of RSNA via bipolar electrodes from multi-fiber preparations in various species has been used for decades in the context of autonomic regulation research.Traditionally, raw neurograms are usually integrated over short time intervals and this approach works perfectly for the analysis of dynamic RSNA changes due to acute intervention.However, comparability of baseline activities between groups is limited.We present a novel analysis method, based on the raw neurogram sampled at rate of 25 kHz.
Methods: For RSNA-burst analysis a software based programmable algorithm was used.Continuous activities longer than three single spikes (i.e., >8ms) were defined as bursts, followed by silent periods with some single spikes.Approximately 10.000 bursts/ rat were analyzed and burst amplitude, burst duration, burst area (i.e., duration integral), as well as the burst frequencies were analyzed.
Conclusions: Our new method of RSNA baseline analysis was able to detect subtle differences between groups of animals that could not be detected by the traditional method of integrated RSNA analysis.Furthermore, this method has the potential to give further insights into RNSA patterns and synchronization to afferent regulatory input form the kidney itself or other organs.
1University of Texas Health San Antonio, San Antonio, TX; 2 Audie L Murphy Memorial Veterans' Hospital, San Antonio, TX.
1,2The Core Laboratory for Clinical Research.