期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2020
卷号：117
期号：27
页码：15862-15873
DOI：10.1073/pnas.2004651117
出版社：The National Academy of Sciences of the United States of America
摘要：Albuminuria is an independent risk factor for the progression to end-stage kidney failure, cardiovascular morbidity, and premature death. As such, discovering signaling pathways that modulate albuminuria is desirable. Here, we studied the transcriptomes of podocytes, key cells in the prevention of albuminuria, under diabetic conditions. We found that Neuropeptide Y (NPY) was significantly down-regulated in insulin-resistant vs. insulin-sensitive mouse podocytes and in human glomeruli of patients with early and late-stage diabetic nephropathy, as well as other nondiabetic glomerular diseases. This contrasts with the increased plasma and urinary levels of NPY that are observed in such conditions. Studying NPY-knockout mice, we found that NPY deficiency in vivo surprisingly reduced the level of albuminuria and podocyte injury in models of both diabetic and nondiabetic kidney disease. In vitro, podocyte NPY signaling occurred via the NPY2 receptor (NPY2R), stimulating PI3K, MAPK, and NFAT activation. Additional unbiased proteomic analysis revealed that glomerular NPY-NPY2R signaling predicted nephrotoxicity, modulated RNA processing, and inhibited cell migration. Furthermore, pharmacologically inhibiting the NPY2R in vivo significantly reduced albuminuria in adriamycin-treated glomerulosclerotic mice. Our findings suggest a pathogenic role of excessive NPY-NPY2R signaling in the glomerulus and that inhibiting NPY-NPY2R signaling in albuminuric kidney disease has therapeutic potential. Chronic kidney disease (CKD) is a major global healthcare concern, affecting over 10% of the general population, and frequently occurs secondary to other systemic disorders including diabetes, obesity, hypertension, and the metabolic syndrome. A common early hallmark of CKD is albuminuria, which not only reflects damage to the glomerular filtration barrier (GFB) in the kidney but also is an important independent risk factor for the progression to end-stage renal failure and cardiovascular disease ( 1 ⇓ – 3 ). Thus, strategies to prevent albuminuria have important therapeutic potential, particularly in the early stages of CKD progression. Podocytes are highly specialized epithelial cells of the glomerulus, lining the urinary side of the filtration barrier. Owing to their complex, dynamic structures and their ability to secrete (and adapt to) a number of growth factors, these cells have a central role in filtration barrier maintenance ( 4 ). As such, podocyte damage is a key driver of albuminuria and glomerular disease in numerous settings and occurs early in the pathogenesis of many albuminuric conditions ( 5 ⇓ ⇓ ⇓ – 9 ). While it is well-established that podocyte damage is a major cause of albuminuria ( 8 ), the pathways and molecules involved in podocyte injury are incompletely understood. We ( 10 , 11 ) and others ( 12 , 13 ) have highlighted the importance of podocyte insulin responses in maintaining glomerular function, and it is now evident that circulating factors associated with common systemic disorders, including diabetes, obesity, and the metabolic syndrome, can directly induce podocyte insulin resistance ( 14 ⇓ ⇓ – 17 ) and associated damage ( 15 , 18 ). In this study, we analyzed the transcriptomes of insulin-sensitive and insulin-resistant podocytes with the aim of identifying molecules that are differentially regulated in podocyte damage, which may play a role in albuminuric kidney disease. This unbiased transcriptome analysis revealed that Neuropeptide Y ( Npy ) was the most highly down-regulated transcript in insulin-resistant vs. insulin-sensitive podocytes. Analysis of patient cohorts also revealed a significant reduction in glomerular NPY expression in both early and late-stage diabetic nephropathy (DN), as well as in several other human albuminuric conditions. This contrasts with the increased plasma and urinary levels of NPY that are observed in diabetes and CKD ( 19 ⇓ ⇓ – 22 ). This prompted us to further investigate the potential role of NPY (and NPY signaling) in the podocyte and glomerulus. Results Npy Expression Is Reduced in Diabetic, Insulin-Resistant Podocytes and Glomeruli. To explore factors associated with podocyte injury, we initially performed nonbiased RNA sequencing of highly insulin-sensitive mouse podocytes [with stable insulin receptor expression ( 14 )] and insulin-resistant mouse podocytes [with insulin resistance induced via exposure to a combination of inflammatory cytokines, high insulin, and high glucose, as previously reported ( 14 )]. Of over 23,000 transcripts, Npy was the most highly down-regulated in the insulin-resistant cells (46.53-fold reduction, P < 5.22 × 10 −181 ), in comparison with highly insulin-sensitive podocytes ( Fig. 1 A and SI Appendix , Fig. S1 ). A significant reduction in Npy was also observed in RNA sequencing of control mouse podocytes (insulin-sensitive, without stable insulin receptor expression), when compared to the insulin-resistant cells (5.56-fold reduction, P < 3.06 × 10 −33 , SI Appendix , Fig. S1 ). We also performed focused insulin-signaling qPCR arrays on podocytes exposed to the inflammatory cytokines TNF-α and IL-6, which become insulin-resistant with respect to glucose uptake ( SI Appendix , Fig. S2 ). Results from these arrays also revealed that Npy was again the most significantly down-regulated transcript of 84 genes related to insulin signaling ( Fig. 1 B and SI Appendix , Fig. S1 ). This reduction in Npy expression was further confirmed in our insulin-resistant podocyte model using end-point PCR ( Fig. 1 C ) and qPCR ( Fig. 1 D ). Analyzing data from the large kidney transcriptomics database Nephroseq, we also found a significant reduction in Npy expression in the glomeruli of diabetic mice ( Fig. 1 E ).
