Ecto-CD38-NADase inhibition modulates cardiac metabolism and protects mice against doxorubicin-induced cardiotoxicity.

AIMS: Doxorubicin (DXR) is a chemotherapeutic agent that causes dose-dependent cardiotoxicity. Recently, it has been proposed that the NADase CD38 may play a role in doxorubicin-induced cardiotoxicity (DIC). CD38 is the main NAD+-catabolizing enzyme in mammalian tissues. Interestingly, in the heart, CD38 is mostly expressed as an ecto-enzyme that can be targeted by specific inhibitory antibodies. The goal of the present study is to characterize the role of CD38 ecto-enzymatic activity in cardiac metabolism and the development of DIC. METHODS AND RESULTS: Using both a transgenic animal model and a non-cytotoxic enzymatic anti-CD38 antibody, we investigated the role of CD38 and its ecto-NADase activity in DIC in pre-clinical models. First, we observed that DIC was prevented in the CD38 catalytically inactive (CD38-CI) transgenic mice. Both left ventricular systolic function and exercise capacity were decreased in wild-type but not in CD38-CI mice treated with DXR. Second, blocking CD38-NADase activity with the specific antibody 68 (Ab68) likewise protected mice against DIC and decreased DXR-related mortality by 50%. A reduction of DXR-induced mitochondrial dysfunction, energy deficiency, and inflammation gene expression were identified as the main mechanisms mediating the protective effects. CONCLUSION: NAD+-preserving strategies by inactivation of CD38 via a genetic or a pharmacological-based approach improve cardiac energetics and reduce cardiac inflammation and dysfunction otherwise seen in an acute DXR cardiotoxicity model.

p21 induces a senescence program and skeletal muscle dysfunction.

Recent work has established associations between elevated p21, the accumulation of senescent cells, and skeletal muscle dysfunction in mice and humans. Using a mouse model of p21 overexpression (p21OE), we examined if p21 mechanistically contributes to cellular senescence and pathological features in skeletal muscle. We show that p21 induces several core properties of cellular senescence in skeletal muscle, including an altered transcriptome, DNA damage, mitochondrial dysfunction, and the senescence-associated secretory phenotype (SASP). Furthermore, p21OE mice exhibit manifestations of skeletal muscle pathology, such as atrophy, fibrosis, and impaired physical function when compared to age-matched controls. These findings suggest p21 alone is sufficient to drive a cellular senescence program and reveal a novel source of skeletal muscle loss and dysfunction.

Low NAD+ Levels Are Associated With a Decline of Spermatogenesis in Transgenic ANDY and Aging Mice.

Advanced paternal age has increasingly been recognized as a risk factor for male fertility and progeny health. While underlying causes are not well understood, aging is associated with a continuous decline of blood and tissue NAD+ levels, as well as a decline of testicular functions. The important basic question to what extent ageing-related NAD+ decline is functionally linked to decreased male fertility has been difficult to address due to the pleiotropic effects of aging, and the lack of a suitable animal model in which NAD+ levels can be lowered experimentally in chronologically young adult males. We therefore developed a transgenic mouse model of acquired niacin dependency (ANDY), in which NAD+ levels can be experimentally lowered using a niacin-deficient, chemically defined diet. Using ANDY mice, this report demonstrates for the first time that decreasing body-wide NAD+ levels in young adult mice, including in the testes, to levels that match or exceed the natural NAD+ decline observed in old mice, results in the disruption of spermatogenesis with small testis sizes and reduced sperm counts. ANDY mice are dependent on dietary vitamin B3 (niacin) for NAD+ synthesis, similar to humans. NAD+-deficiency the animals develop on a niacin-free diet is reversed by niacin supplementation. Providing niacin to NAD+-depleted ANDY mice fully rescued spermatogenesis and restored normal testis weight in the animals. The results suggest that NAD+ is important for proper spermatogenesis and that its declining levels during aging are functionally linked to declining spermatogenesis and male fertility. Functions of NAD+ in retinoic acid synthesis, which is an essential testicular signaling pathway regulating spermatogonial proliferation and differentiation, may offer a plausible mechanism for the hypospermatogenesis observed in NAD+-deficient mice.

Dihydronicotinamide Riboside Is a Potent NAD+ Precursor Promoting a Pro-Inflammatory Phenotype in Macrophages.

Nicotinamide adenine dinucleotide (NAD) metabolism plays an important role in the regulation of immune function. However, a complete picture of how NAD, its metabolites, precursors, and metabolizing enzymes work together in regulating immune function and inflammatory diseases is still not fully understood. Surprisingly, few studies have compared the effect of different forms of vitamin B3 on cellular functions. Therefore, we investigated the role of NAD boosting in the regulation of macrophage activation and function using different NAD precursors supplementation. We compared nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and nicotinamide (NAM) supplementation, with the recently described potent NAD precursor NRH. Our results show that only NRH supplementation strongly increased NAD+ levels in both bone marrow-derived and THP-1 macrophages. Importantly, NRH supplementation activated a pro-inflammatory phenotype in resting macrophages, inducing gene expression of several cytokines, chemokines, and enzymes. NRH also potentiated the effect of lipopolysaccharide (LPS) on macrophage activation and cytokine gene expression, suggesting that potent NAD+ precursors can promote inflammation in macrophages. The effect of NRH in NAD+ boosting and gene expression was blocked by inhibitors of adenosine kinase, equilibrative nucleoside transporters (ENT), and IκB kinase (IKK). Interestingly, the IKK inhibitor, BMS-345541, blocked the mRNA expression of several enzymes and transporters involved in the NAD boosting effect of NRH, indicating that IKK is also a regulator of NAD metabolism. In conclusion, NAD precursors such as NRH may be important tools to understand the role of NAD and NADH metabolism in the inflammatory process of other immune cells, and to reprogram immune cells to a pro-inflammatory phenotype, such as the M2 to M1 switch in macrophage reprogramming, in the cancer microenvironment.

CD38 inhibitor 78c increases mice lifespan and healthspan in a model of chronological aging.

Nicotinamide adenine dinucleotide (NAD) levels decline during aging, contributing to physical and metabolic dysfunction. The NADase CD38 plays a key role in age-related NAD decline. Whether the inhibition of CD38 increases lifespan is not known. Here, we show that the CD38 inhibitor 78c increases lifespan and healthspan of naturally aged mice. In addition to a 10% increase in median survival, 78c improved exercise performance, endurance, and metabolic function in mice. The effects of 78c were different between sexes. Our study is the first to investigate the effect of CD38 inhibition in naturally aged animals.

The CD38 glycohydrolase and the NAD sink: implications for pathological conditions.

Nicotinamide adenine dinucleotide (NAD) acts as a cofactor in several oxidation-reduction (redox) reactions and is a substrate for a number of nonredox enzymes. NAD is fundamental to a variety of cellular processes including energy metabolism, cell signaling, and epigenetics. NAD homeostasis appears to be of paramount importance to health span and longevity, and its dysregulation is associated with multiple diseases. NAD metabolism is dynamic and maintained by synthesis and degradation. The enzyme CD38, one of the main NAD-consuming enzymes, is a key component of NAD homeostasis. The majority of CD38 is localized in the plasma membrane with its catalytic domain facing the extracellular environment, likely for the purpose of controlling systemic levels of NAD. Several cell types express CD38, but its expression predominates on endothelial cells and immune cells capable of infiltrating organs and tissues. Here we review potential roles of CD38 in health and disease and postulate ways in which CD38 dysregulation causes changes in NAD homeostasis and contributes to the pathophysiology of multiple conditions. Indeed, in animal models the development of infectious diseases, autoimmune disorders, fibrosis, metabolic diseases, and age-associated diseases including cancer, heart disease, and neurodegeneration are associated with altered CD38 enzymatic activity. Many of these conditions are modified in CD38-deficient mice or by blocking CD38 NADase activity. In diseases in which CD38 appears to play a role, CD38-dependent NAD decline is often a common denominator of pathophysiology. Thus, understanding dysregulation of NAD homeostasis by CD38 may open new avenues for the treatment of human diseases.

FBF1 deficiency promotes beiging and healthy expansion of white adipose tissue.

Preadipocytes dynamically produce sensory cilia. However, the role of primary cilia in preadipocyte differentiation and adipose homeostasis remains poorly understood. We previously identified transition fiber component FBF1 as an essential player in controlling selective cilia import. Here, we establish Fbf1tm1a/tm1a mice and discover that Fbf1tm1a/tm1a mice develop severe obesity, but surprisingly, are not predisposed to adverse metabolic complications. Obese Fbf1tm1a/tm1a mice possess unexpectedly healthy white fat tissue characterized by spontaneous upregulated beiging, hyperplasia but not hypertrophy, and low inflammation along the lifetime. Mechanistically, FBF1 governs preadipocyte differentiation by constraining the beiging program through an AKAP9-dependent, cilia-regulated PKA signaling, while recruiting the BBS chaperonin to transition fibers to suppress the hedgehog signaling-dependent adipogenic program. Remarkably, obese Fbf1tm1a/tm1a mice further fed a high-fat diet are protected from diabetes and premature death. We reveal a central role for primary cilia in the fate determination of preadipocytes and the generation of metabolically healthy fat tissue.

A methionine-Mettl3-N6-methyladenosine axis promotes polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic disorder marked by numerous progressively enlarging kidney cysts. Mettl3, a methyltransferase that catalyzes the abundant N6-methyladenosine (m6A) RNA modification, is implicated in development, but its role in most diseases is unknown. Here, we show that Mettl3 and m6A levels are increased in mouse and human ADPKD samples and that kidney-specific transgenic Mettl3 expression produces tubular cysts. Conversely, Mettl3 deletion in three orthologous ADPKD mouse models slows cyst growth. Interestingly, methionine and S-adenosylmethionine (SAM) levels are also elevated in ADPKD models. Moreover, methionine and SAM induce Mettl3 expression and aggravate ex vivo cyst growth, whereas dietary methionine restriction attenuates mouse ADPKD. Finally, Mettl3 activates the cyst-promoting c-Myc and cAMP pathways through enhanced c-Myc and Avpr2 mRNA m6A modification and translation. Thus, Mettl3 promotes ADPKD and links methionine utilization to epitranscriptomic activation of proliferation and cyst growth.

CD38 ecto-enzyme in immune cells is induced during aging and regulates NAD+ and NMN levels.

Decreased NAD+ levels have been shown to contribute to metabolic dysfunction during aging. NAD+ decline can be partially prevented by knockout of the enzyme CD38. However, it is not known how CD38 is regulated during aging, and how its ecto-enzymatic activity impacts NAD+ homeostasis. Here we show that an increase in CD38 in white adipose tissue (WAT) and the liver during aging is mediated by accumulation of CD38+ immune cells. Inflammation increases CD38 and decreases NAD+. In addition, senescent cells and their secreted signals promote accumulation of CD38+ cells in WAT, and ablation of senescent cells or their secretory phenotype decreases CD38, partially reversing NAD+ decline. Finally, blocking the ecto-enzymatic activity of CD38 can increase NAD+ through a nicotinamide mononucleotide (NMN)-dependent process. Our findings demonstrate that senescence-induced inflammation promotes accumulation of CD38 in immune cells that, through its ecto-enzymatic activity, decreases levels of NMN and NAD+.

Metalloproteinase PAPP-A regulation of IGF-1 contributes to polycystic kidney disease pathogenesis.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end-stage renal disease (ESRD). The treatment options for ADPKD are limited. We observed an upregulation in several IGF-1 pathway genes in the kidney of Pkd1RC/RC mice, a model of ADPKD. Pregnancy-associated plasma protein A (PAPP-A), a metalloproteinase that cleaves inhibitory IGF binding proteins (IGFBPs), increasing the local bioactivity of IGF-1, was highly induced in the kidney of ADPKD mice. PAPP-A levels were high in cystic fluid and kidneys of humans with ADPKD. Our studies further showed that PAPP-A transcription in ADPKD was mainly regulated through the cAMP/CREB/CBP/p300 pathway. Pappa deficiency effectively inhibited the development of cysts in the Pkd1RC/RC mice. The role of PAPP-A in cystic disease appears to be regulation of the IGF-1 pathway and cellular proliferation in the kidney. Finally, preclinical studies demonstrated that treatment with a monoclonal antibody that blocks the proteolytic activity of PAPP-A against IGFBP4 ameliorated ADPKD cystic disease in vivo in Pkd1RC/RC mice and ex vivo in embryonic kidneys. These data indicated that the PAPP-A/IGF-1 pathway plays an important role in the growth and expansion of cysts in ADPKD. Our findings introduce a therapeutic strategy for ADPKD that involves the inhibition of PAPP-A.

The NADase CD38 is induced by factors secreted from senescent cells providing a potential link between senescence and age-related cellular NAD+ decline.

Tissue nicotinamide adenine dinucleotide (NAD+) decline has been implicated in aging. We have recently identified CD38 as a central regulator involved in tissue NAD+ decline during the aging process. CD38 is an ecto-enzyme highly expressed in endothelial and inflammatory cells. To date, the mechanisms that regulate CD38 expression in aging tissues characterized by the presence of senescent cells is not completely understood. Cellular senescence has been described as a hallmark of the aging process and these cells are known to secrete several factors including cytokines and chemokines through their senescent associated secretory phenotype (SASP). Here we investigated if the cellular senescence phenotype is involved in the regulation of CD38 expression and its NADase activity. We observed that senescent cells do not have high expression of CD38. However, the SASP factors secreted by senescent cells induced CD38 mRNA and protein expression and increased CD38-NADase activity in non-senescent cells such as endothelial cells or bone marrow derived macrophages. Our data suggest a link between cellular senescence and NAD+ decline in which SASP-mediated upregulation of CD38 can disrupt cellular NAD+ homeostasis.

A Potent and Specific CD38 Inhibitor Ameliorates Age-Related Metabolic Dysfunction by Reversing Tissue NAD+ Decline.

Aging is characterized by the development of metabolic dysfunction and frailty. Recent studies show that a reduction in nicotinamide adenine dinucleotide (NAD+) is a key factor for the development of age-associated metabolic decline. We recently demonstrated that the NADase CD38 has a central role in age-related NAD+ decline. Here we show that a highly potent and specific thiazoloquin(az)olin(on)e CD38 inhibitor, 78c, reverses age-related NAD+ decline and improves several physiological and metabolic parameters of aging, including glucose tolerance, muscle function, exercise capacity, and cardiac function in mouse models of natural and accelerated aging. The physiological effects of 78c depend on tissue NAD+ levels and were reversed by inhibition of NAD+ synthesis. 78c increased NAD+ levels, resulting in activation of pro-longevity and health span-related factors, including sirtuins, AMPK, and PARPs. Furthermore, in animals treated with 78c we observed inhibition of pathways that negatively affect health span, such as mTOR-S6K and ERK, and attenuation of telomere-associated DNA damage, a marker of cellular aging. Together, our results detail a novel pharmacological strategy for prevention and/or reversal of age-related NAD+ decline and subsequent metabolic dysfunction.

CD38 Dictates Age-Related NAD Decline and Mitochondrial Dysfunction through an SIRT3-Dependent Mechanism.

Nicotinamide adenine dinucleotide (NAD) levels decrease during aging and are involved in age-related metabolic decline. To date, the mechanism responsible for the age-related reduction in NAD has not been elucidated. Here we demonstrate that expression and activity of the NADase CD38 increase with aging and that CD38 is required for the age-related NAD decline and mitochondrial dysfunction via a pathway mediated at least in part by regulation of SIRT3 activity. We also identified CD38 as the main enzyme involved in the degradation of the NAD precursor nicotinamide mononucleotide (NMN) in vivo, indicating that CD38 has a key role in the modulation of NAD-replacement therapy for aging and metabolic diseases.

Blockade of CCR2 reduces macrophage influx and development of chronic renal damage in murine renovascular hypertension.

Renovascular hypertension (RVH) is a common cause of both cardiovascular and renal morbidity and mortality. In renal artery stenosis (RAS), atrophy in the stenotic kidney is associated with an influx of macrophages and other mononuclear cells. We tested the hypothesis that chemokine receptor 2 (CCR2) inhibition would reduce chronic renal injury by reducing macrophage influx in the stenotic kidney of mice with RAS. We employed a well-established murine model of RVH to define the relationship between macrophage infiltration and development of renal atrophy in the stenotic kidney. To determine the role of chemokine ligand 2 (CCL2)/CCR2 signaling in the development of renal atrophy, mice were treated with the CCR2 inhibitor RS-102895 at the time of RAS surgery and followed for 4 wk. Renal tubular epithelial cells expressed CCL2 by 3 days following surgery, a time at which no significant light microscopic alterations, including interstitial inflammation, were identified. Macrophage influx increased with time following surgery. At 4 wk, the development of severe renal atrophy was accompanied by an influx of inducible nitric oxide synthase (iNOS)+ and CD206+ macrophages that coexpressed F4/80, with a modest increase in macrophages coexpressing arginase 1 and F4/80. The CCR2 inhibitor RS-102895 attenuated renal atrophy and significantly reduced the number of dual-stained F4/80+ iNOS+ and F4/80+ CD206+ but not F4/80+ arginase 1+ macrophages. CCR2 inhibition reduces iNOS+ and CD206+ macrophage accumulation that coexpress F4/80 and renal atrophy in experimental renal artery stenosis. CCR2 blockade may provide a novel therapeutic approach to humans with RVH.

Inhibition of p38 MAPK attenuates renal atrophy and fibrosis in a murine renal artery stenosis model.

Renal artery stenosis (RAS) is an important cause of chronic renal dysfunction. Recent studies have underscored a critical role for CCL2 (MCP-1)-mediated inflammation in the progression of chronic renal damage in RAS and other chronic renal diseases. In vitro studies have implicated p38 MAPK as a critical intermediate for the production of CCL2. However, a potential role of p38 signaling in the development and progression of chronic renal disease in RAS has not been previously defined. We sought to test the hypothesis that inhibition of p38 MAPK ameliorates chronic renal injury in mice with RAS. We established a murine RAS model by placing a cuff on the right renal artery and treated mice with the p38 inhibitor SB203580 or vehicle for 2 wk. In mice treated with vehicle, the cuffed kidney developed interstitial fibrosis, tubular atrophy, and interstitial inflammation. In mice treated with SB203580, the RAS-induced renal atrophy was reduced (70% vs. 39%, P < 0.05). SB203580 also reduced interstitial inflammation and extracellular matrix deposition but had no effect on the development of hypertension. SB203580 partially blocked the induction of CCL2, CCL7 (MCP-3), CC chemokine receptor 2 (CCR2), and collagen 4 mRNA expression in the cuffed kidneys. In vitro, blockade of p38 hindered both TNF-α and TGF-ß-induced CCL2 upregulation. Based on these observations, we conclude that p38 MAPK plays a critical role in the induction of CCL2/CCL7/CCR2 system and the development of interstitial inflammation in RAS.

Genetic deficiency of Smad3 protects the kidneys from atrophy and interstitial fibrosis in 2K1C hypertension.

Although the two-kidney, one-clip (2K1C) model is widely used as a model of human renovascular hypertension, mechanisms leading to the development of fibrosis and atrophy in the cuffed kidney and compensatory hyperplasia in the contralateral kidney have not been defined. Based on the well-established role of the transforming growth factor (TGF)-ß signaling pathway in renal fibrosis, we tested the hypothesis that abrogation of TGF-ß/Smad3 signaling would prevent fibrosis in the cuffed kidney. Renal artery stenosis (RAS) was established in mice with a targeted disruption of exon 2 of the Smad3 gene (Smad3 KO) and wild-type (WT) controls by placement of a polytetrafluoroethylene cuff on the right renal artery. Serial pulse-wave Doppler ultrasound assessments verified that blood flow through the cuffed renal artery was decreased to a similar extent in Smad3 KO and WT mice. Two weeks after surgery, systolic blood pressure and plasma renin activity were significantly elevated in both the Smad3 KO and WT mice. The cuffed kidney of WT mice developed renal atrophy (50% reduction in weight after 6 wk, P < 0.0001), which was associated with the development of interstitial fibrosis, tubular atrophy, and interstitial inflammation. Remarkably, despite a similar reduction of renal blood flow, the cuffed kidney of the Smad3 KO mice showed minimal atrophy (9% reduction in weight, P = not significant), with no significant histopathological alterations (interstitial fibrosis, tubular atrophy, and interstitial inflammation). We conclude that abrogation of TGF-ß/Smad3 signaling confers protection against the development of fibrosis and atrophy in RAS.

Genetic deficiency of Smad3 protects against murine ischemic acute kidney injury.

TGF-ß1 contributes to chronic kidney disease, at least in part, via Smad3. TGF-ß1 is induced in the kidney following acute ischemia, and there is increasing evidence that TGF-ß1 may protect against acute kidney injury. As there is a paucity of information regarding the functional significance of Smad3 in acute kidney injury, the present study explored this issue in a murine model of ischemic acute kidney injury in Smad3(+/+) and Smad3(-/-) mice. We demonstrate that, at 24 h after ischemia, Smad3 is significantly induced in Smad3(+/+) mice, whereas Smad3(-/-) mice fail to express this protein in the kidney in either the sham or postischemic groups. Compared with Smad3(+/+) mice, and 24 h following ischemia, Smad3(-/-) mice exhibited greater preservation of renal function as measured by blood urea nitrogen (BUN) and serum creatinine; less histological injury assessed by both semiquantitative and qualitative analyses; markedly suppressed renal expression of IL-6 and endothelin-1 mRNA (but comparable expression of MCP-1, TNF-α, and heme oxygenase-1 mRNA); and no increase in plasma IL-6 levels, the latter increasing approximately sixfold in postischemic Smad3(+/+) mice. We conclude that genetic deficiency of Smad3 confers structural and functional protection against acute ischemic injury to the kidney. We speculate that these effects may be mediated through suppression of IL-6 production. Finally, we suggest that upregulation of Smad3 after an ischemic insult may contribute to the increased risk for chronic kidney disease that occurs after acute renal ischemia.

n-3 Fatty acids block TNF-α-stimulated MCP-1 expression in rat mesangial cells.

Monocyte chemoattractant protein 1 (MCP-1) is a CC cytokine that fundamentally contributes to the pathogenesis of inflammatory renal disease. MCP-1 is highly expressed in cytokine-stimulated mesangial cells in vitro and following glomerular injury in vivo. Interventions to limit MCP-1 expression are commonly effective in assorted experimental models. Fish oil, an abundant source of n-3 fatty acids, has anti-inflammatory properties, the basis of which remains incompletely defined. We examined potential mechanisms whereby fish oil reduces MCP-1 expression and thereby suppresses inflammatory responses to tissue injury. Cultured mesangial cells were treated with TNF-α in the presence of the n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA); equimolar concentrations of the n-6 fatty acids LA and OA served as controls. MCP-1 mRNA expression was assessed by Northern blotting, and transcriptional activity of the MCP-1 promoter was assessed by transient transfection. The involvement of the ERK and NF-κB pathways was evaluated through transfection analysis and the use of the MEK inhibitor U0126. DHA and EPA decreased TNF-α-stimulated MCP-1 mRNA expression by decreasing transcription of the MCP-1 gene. DHA and EPA decreased p-ERK expression and nuclear translocation of NF-κB, both of which are necessary for TNF-α-stimulated MCP-1 expression. Both NF-κB and AP-1 sites were involved in transcriptional regulation of the MCP-1 gene by DHA and EPA. We conclude that DHA and EPA inhibit TNF-α-stimulated transcription of the MCP-1 gene through interaction of signaling pathways involving ERK and NF-κB. We speculate that such effects may contribute to the salutary effect of fish oil in renal and vascular disease.

Temporal analysis of signaling pathways activated in a murine model of two-kidney, one-clip hypertension.

Unilateral renal artery stenosis (RAS) leads to atrophy of the stenotic kidney and compensatory enlargement of the contralateral kidney. Although the two-kidney, one-clip (2K1C) model has been extensively used to model human RAS, the cellular responses in the stenotic and contralateral kidneys, particularly in the murine model, have received relatively little attention. We studied mice 2, 5, and 11 wk after unilateral RAS. These mice became hypertensive within 1 wk. The contralateral kidney increased in size within 2 wk after surgery. This enlargement was associated with a transient increase in expression of phospho-extracellular signal-regulated kinase (p-ERK), the proliferation markers proliferating cell nuclear antigen and Ki-67, the cell cycle inhibitors p21 and p27, and transforming growth factor-beta, with return to baseline levels by 11 wk. The size of the stenotic kidney was unchanged at 2 wk but progressively decreased between 5 and 11 wk. Unlike the contralateral kidney, which showed minimal histopathological alterations, the stenotic kidney developed progressive interstitial fibrosis, tubular atrophy, and interstitial inflammation. Surprisingly, the stenotic kidney showed a proliferative response, which involved largely tubular epithelial cells. The atrophic kidney had little evidence of apoptosis, despite persistent upregulation of p53; expression of cell cycle regulatory proteins in the stenotic kidney was persistently increased through 11 wk. These studies indicate that in the 2K1C model, the stenotic kidney and contralateral, enlarged kidney exhibit a distinct temporal expression of proteins involved in cell growth, cell survival, apoptosis, inflammation, and fibrosis. Notably, an unexpected proliferative response occurs in the stenotic kidney that undergoes atrophy.

Signaling pathways modulated by fish oil in salt-sensitive hypertension.

Although many studies have indicated that fish oil (FO) improves cardiovascular risk factors and reduces histopathological manifestations of injury in experimental renal injury models, potential mechanisms underlying this protective effect have not been adequately defined. The objective of this study was to identify potential signaling pathways that confer protection in the Dahl rat model of salt-sensitive hypertension. Male Dahl salt-sensitive rats (n = 10/group) were provided with formulated diets containing 8% NaCl, 20% protein, and 25% FO or 25% corn oil (CO) for 28 days. FO reduced blood pressure (-11% at 4 wk; P < 0.05), urine protein excretion (-45% at 4 wk; P < 0.05), plasma cholesterol and triglyceride levels (-54%, P < 0.001; and -58%, P < 0.05), and histopathological manifestations of renal injury, including vascular hypertrophy, segmental and global glomerular sclerosis, interstitial fibrosis, and tubular atrophy. Interstitial inflammation was significantly reduced by FO (-32%; P < 0.001), as assessed by quantitative analysis of ED1-positive cells in sections of the renal cortex. FO reduced tubulointerstitial proliferative activity, as assessed by Western blot analysis of cortical homogenates for PCNA (-51%; P < 0.01) and quantitative analysis of Mib-1-stained sections of the renal cortex (-42%; P < 0.001). Decreased proliferative activity was associated with reduced phospho-ERK expression (-37%; P < 0.005) and NF-kappaB activation (-42%; P < 0.05). FO reduced cyclooxygenase (COX)-2 expression (-63%; P < 0.01) and membrane translocation of the NADPH oxidase subunits p47(phox) and p67(phox) (-26 and -34%; P < 0.05). We propose that FO ameliorates renal injury in Dahl salt-sensitive rats through the inhibition of ERK, decreased NF-kappaB activation, inhibition of COX-2 expression, and decreased NADPH oxidase activation.