Dysregulated UPR and ER Stress Related to a Mutation in the Sdf2l1 Gene Are Involved in the Pathophysiology of Diet-Induced Diabetes in the Cohen Diabetic Rat.

The Cohen Diabetic rat is a model of type 2 diabetes mellitus that consists of the susceptible (CDs/y) and resistant (CDr/y) strains. Diabetes develops in CDs/y provided diabetogenic diet (DD) but not when fed regular diet (RD) nor in CDr/y given either diet. We recently identified in CDs/y a deletion in Sdf2l1, a gene that has been attributed a role in the unfolded protein response (UPR) and in the prevention of endoplasmic reticulum (ER) stress. We hypothesized that this deletion prevents expression of SDF2L1 and contributes to the pathophysiology of diabetes in CDs/y by impairing UPR, enhancing ER stress, and preventing CDs/y from secreting sufficient insulin upon demand. We studied SDF2L1 expression in CDs/y and CDr/y. We evaluated UPR by examining expression of key proteins involved in both strains fed either RD or DD. We assessed the ability of all groups of animals to secrete insulin during an oral glucose tolerance test (OGTT) over 4 weeks, and after overnight feeding (postprandial) over 4 months. We found that SDF2L1 was expressed in CDr/y but not in CDs/y. The pattern of expression of proteins involved in UPR, namely the PERK (EIF2α, ATF4 and CHOP) and IRE1 (XBP-1) pathways, was different in CDs/y DD from all other groups, with consistently lower levels of expression at 4 weeks after initiation of DD and coinciding with the development of diabetes. In CDs/y RD, insulin secretion was mildly impaired, whereas in CDs/y DD, the ability to secrete insulin decreased over time, leading to the development of the diabetic phenotype. We conclude that in CDs/y DD, UPR participating proteins were dysregulated and under-expressed at the time point when the diabetic phenotype became overt. In parallel, insulin secretion in CDs/y DD became markedly impaired. Our findings suggest that under conditions of metabolic load with DD and increased demand for insulin secretion, the lack of SDF2L1 expression in CDs/y is associated with UPR dysregulation and ER stress which, combined with oxidative stress previously attributed to the concurrent Ndufa4 mutation, are highly likely to contribute to the pathophysiology of diabetes in this model.

Predisposition to cortical neurodegenerative changes in brains of hypertension prone rats.

BACKGROUND: Substantial evidence suggests that hypertension is a significant risk factor for cognitive decline. However, it is unclear whether the genetic predisposition to hypertension is also associated with cellular dysfunction that promotes neurodegeneration. METHODS: Changes in blood pressure were evaluated following dietary salt-loading or administration of a regular diet in Sabra Normotensive (SBN/y) and Sabra Hypertension-prone rats (SBH/y). We performed quantitative RT-PCR and immunofluorescence staining in brain cortical tissues before salt loading and 6 and 9 months after salt loading. To examine the expression of brain cortical proteins involved in the gene regulation (Histone Deacetylase-HDAC2; Histone Acetyltransferase 1-HAT1), stress response (Activating Transcription Factor 4-ATF4; Eukaryotic Initiation Factor 2- eIF2α), autophagy (Autophagy related 4A cysteine peptidase- Atg4a; light-chain 3-LC3A/B; mammalian target of rapamycin complex 1- mTORC1) and apoptosis (caspase-3). RESULTS: Prior to salt loading, SBH/y compared to SBN/y expressed a significantly higher level of cortical HAT1 (protein), Caspase-3 (mRNA/protein), LC3A, and ATF4 (mRNA), lower levels of ATG4A (mRNA/protein), LC3A/B, HDAC2 (protein), as well as a lower density of cortical neurons. Following dietary salt loading, SBH/y but not SBN/y developed high blood pressure. In hypertensive SBH/y, there was significant upregulation of cortical HAT1 (protein), Caspase-3 (protein), and eIF2α ~ P (protein) and downregulation of HDAC2 (protein) and mTORC1 (mRNA), and cortical neuronal loss. CONCLUSIONS: The present findings suggest that genetic predisposition to hypertension is associated in the brain cortex with disruption in autophagy, gene regulation, an abnormal response to cellular stress, and a high level of cortical apoptosis, and could therefore exacerbate cellular dysfunction and thereby promote neurodegeneration.

Mesenchymal Stem Cell-Derived Extracellular Vesicles as Proposed Therapy in a Rat Model of Cerebral Small Vessel Disease.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been employed in the past decade as therapeutic agents in various diseases, including central nervous system (CNS) disorders. We currently aimed to use MSC-EVs as potential treatment for cerebral small vessel disease (CSVD), a complex disorder with a variety of manifestations. MSC-EVs were intranasally administrated to salt-sensitive hypertension prone SBH/y rats that were DOCA-salt loaded (SBH/y-DS), which we have previously shown is a model of CSVD. MSC-EVs accumulated within brain lesion sites of SBH/y-DS. An in vitro model of an inflammatory environment in the brain demonstrated anti-inflammatory properties of MSC-EVs. Following in vivo MSC-EV treatment, gene set enrichment analysis (GSEA) of SBH/y-DS cortices revealed downregulation of immune system response-related gene sets. In addition, MSC-EVs downregulated gene sets related to apoptosis, wound healing and coagulation, and upregulated gene sets associated with synaptic signaling and cognition. While no specific gene was markedly altered upon treatment, the synergistic effect of all gene alternations was sufficient to increase animal survival and improve the neurological state of affected SBH/y-DS rats. Our data suggest MSC-EVs act as microenvironment modulators, through various molecular pathways. We conclude that MSC-EVs may serve as beneficial therapeutic measure for multifactorial disorders, such as CSVD.

A Novel Rodent Model of Hypertensive Cerebral Small Vessel Disease with White Matter Hyperintensities and Peripheral Oxidative Stress.

Cerebral small vessel disease (CSVD) is the second most common cause of stroke and a major contributor to dementia. Manifestations of CSVD include cerebral microbleeds, intracerebral hemorrhages (ICH), lacunar infarcts, white matter hyperintensities (WMH) and enlarged perivascular spaces. Chronic hypertensive models have been found to reproduce most key features of the disease. Nevertheless, no animal models have been identified to reflect all different aspects of the human disease. Here, we described a novel model for CSVD using salt-sensitive 'Sabra' hypertension-prone rats (SBH/y), which display chronic hypertension and enhanced peripheral oxidative stress. SBH/y rats were either administered deoxycorticosteroid acetate (DOCA) (referred to as SBH/y-DOCA rats) or sham-operated and provided with 1% NaCl in drinking water. Rats underwent neurological assessment and behavioral testing, followed by ex vivo MRI and biochemical and histological analyses. SBH/y-DOCA rats show a neurological decline and cognitive impairment and present multiple cerebrovascular pathologies associated with CSVD, such as ICH, lacunes, enlarged perivascular spaces, blood vessel stenosis, BBB permeability and inflammation. Remarkably, SBH/y-DOCA rats show severe white matter pathology as well as WMH, which are rarely reported in commonly used models. Our model may serve as a novel platform for further understanding the mechanisms underlying CSVD and for testing novel therapeutics.

Diabetes induces remodeling of the left atrial appendage independently of atrial fibrillation in a rodent model of type-2 diabetes.

BACKGROUND: Diabetic patients have an increased predisposition to thromboembolic events, in most cases originating from thrombi in the left atrial appendage (LAA). Remodeling of the LAA, which predisposes to thrombi formation, has been previously described in diabetic patients with atrial fibrillation, but whether remodeling of the LAA occurs in diabetics also in the absence of atrial fibrillation is unknown. To investigate the contribution of diabetes, as opposed to atrial fibrillation, to remodeling of the LAA, we went from humans to the animal model. METHODS: We studied by echocardiography the structure and function of the heart over multiple time points during the evolution of diabetes in the Cohen diabetic sensitive rat (CDs/y) provided diabetogenic diet over a period of 4 months; CDs/y provided regular diet and the Cohen diabetic resistant (CDr/y), which do not develop diabetes, served as controls. All animals were in sinus rhythm throughout the study period. RESULTS: Compared to controls, CDs/y developed during the evolution of diabetes a greater heart mass, larger left atrial diameter, wider LAA orifice, increased LAA depth, greater end-diastolic and end-systolic diameter, and lower E/A ratio-all indicative of remodeling of the LAA and left atrium (LA), as well as the development of left ventricular diastolic dysfunction. To investigate the pathophysiology involved, we studied the histology of the hearts at the end of the study. We found in diabetic CDs/y, but not in any of the other groups, abundance of glycogen granules in the atrial appendages , atria  and ventricles, which may be of significance as glycogen granules have previously been associated with cell and organ dysfunction in the diabetic heart. CONCLUSIONS: We conclude that our rodent model of diabetes, which was in sinus rhythm, reproduced structural and functional alterations previously observed in hearts of human diabetics with atrial fibrillation. Remodeling of the LAA and of the LA in our model was unrelated to atrial fibrillation and associated with accumulation of glycogen granules. We suggest that myocardial accumulation of glycogen granules is related to the development of diabetes and may play a pathophysiological role in remodeling of the LAA and LA, which predisposes to atrial fibrillation, thromboembolic events and left ventricular diastolic dysfunction in the diabetic heart.

Genomic Research in Rat Models of Kidney Disease.

Current understanding of the mechanisms underlying renal disease in humans is incomplete. Consequently, our ability to prevent the occurrence of renal disease or treat established kidney disease is limited. Investigating kidney disease directly in humans poses objective difficulties, which has led investigators to seek experimental animal models that simulate renal disease in humans. Animal models have thus become a tool of major importance in the study of renal physiology and have been crucial in shedding light on the complex mechanisms involved in kidney function and in our current understanding of the pathophysiology of renal disease. Among animal models, the rat has been the preferred and most commonly used species for the investigation of renal disease. This chapter reviews what has been achieved over the years, using the rat as a tool for the investigation of renal disease in humans, focusing on the contribution of rat genetics and genomics to the elucidation of the mechanisms underlying the pathophysiology of the major types of renal disease, including primary and secondary renal diseases.

A novel mutation in the NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 (Ndufa4) gene links mitochondrial dysfunction to the development of diabetes in a rodent model.

The mechanisms underlying diabetes remain unresolved. The Cohen diabetic rat represents a model of diet-induced diabetes, in which the disease is induced after exposure to a diabetogenic diet (DD) in the diabetes-sensitive (CDs/y) but not in the -resistant (CDr/y) strain. Diet imposes a metabolic strain that leads to diabetes in the appropriate genetic background. We previously identified, through whole-genome linkage analysis, a diabetes-related quantitative trait locus on rat chromosome 4 (RNO4), which incorporates NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 (Ndufa4), a nuclear gene that affects mitochondrial function. Here, we sequenced the gene and found a major deletion in CDs/y that leads to lack of expression of the NDUFA4 protein that has been reported to be involved in the activities of mitochondrial complexes I and IV. In the absence of NDUFA4 in the diabetic CDs/y on DD, complex I activity is reduced in comparison to that in nondiabetic CDs/y on regular diet and CDr/y on either diet; complex IV activity is reduced in both strains provided DD, and thus as a result of diet and unrelated to the gene mutation. ATP fails to increase in diabetic CDs/y in response to DD, in comparison to nondiabetic CDr/y on DD. Plasma malondialdehyde levels are elevated in CDs/y on DD, whereas SOD1 and SOD2 levels fail to increase, indicating increased oxidative stress and inability of the pancreas to generate an appropriate antioxidative stress response. These findings suggest that the Ndufa4 mutation in CDs/y on DD is directly associated with mitochondrial dysfunction, which we attribute to the lack of expression of NDUFA4 and to diet, and which prevents the anticipated increase in ATP production. The resulting enhanced oxidative stress impairs the ability of the pancreas to secrete insulin, leading to the development of diabetes. This is the first demonstration of an inherited mutation in a nuclear gene that adversely affects mitochondrial function and promotes diet-induced diabetes.

Three interacting genomic loci incorporating two novel mutations underlie the evolution of diet-induced diabetes.

We investigated the pathophysiology of diet-induced diabetes in the Cohen diabetic rat (CDs/y) from its induction to its chronic phase, using a multi-layered integrated genomic approach. We identified by linkage analysis two diabetes-related quantitative trait loci on RNO4 and RNO13. We determined their functional contribution to diabetes by chromosomal substitution, using congenic and consomic strains. To identify within these loci genes of relevance to diabetes, we sequenced the genome of CDs/y and compared it to 25 other rat strains. Within the RNO4 locus, we detected a novel high impact deletion in the Ndufa4 gene that was unique to CDs/y. Within the RNO13 locus, we found multiple SNPs and INDELs that were unique to CDs/y but were unable to prioritize any of the genes. Genome wide screening identified a novel third locus not detected by linkage analysis that consisted of a novel high impact deletion on RNO11 that was unique to CDs/y and that involved the Sdf2l1 gene. Using co-segregation analysis, we investigated in silico the relative contribution to the diabetic phenotype and the interaction between the three genomic loci on RNO4, RNO11 and RNO13. We found that the RNO4 locus plays a major role during the induction of diabetes, whereas the genomic loci on RNO13 and RNO11, while interacting with the RNO4 locus, contribute more significantly to the diabetic phenotype during the chronic phase of the disease. The mechanisms whereby the mutations on RNO4 and 11 and the RNO13 locus contribute to the development of diabetes are under continuing investigation.

[THE ISRAELI RAT GENOME CENTER].

INTRODUCTION: The Israeli Rat Genome Center, which is located at the Barzilai Medical Center Campus of the Faculty of Health Sciences of the Ben-Gurion University of the Negev in Ashkelon, was established to provide a repository of unique genetic strains of rats that were created in Israel and that simulate complex diseases. The Center incorporates models of: salt-sensitive hypertension (SBN/y and SBH/y rats), type 2 diabetes (CDr and CDs rats), combined hypertension and diabetes (CRDH) and additional genetic strains (transgenics, consomics, congenics). All these strains are available to researchers who are interested in the study of complex diseases, on the basis of collaboration. The Laboratory for Molecular Medicine within the Center also performs independent research on the genetic basis of complex diseases, including hypertension, diabetes, kidney disease and target organ involvement in cardiovascular and metabolic diseases. The Center collaborates with researchers from research centers in other medical centers and universities in Israel and worldwide.

Macrophages dictate the progression and manifestation of hypertensive heart disease.

BACKGROUND: Inflammation has been implicated in the initiation, progression and manifestation of hypertensive heart disease. We sought to determine the role of monocytes/macrophages in hypertension and pressure overload induced left ventricular (LV) remodeling. METHODS AND RESULTS: We used two models of LV hypertrophy (LVH). First, to induce hypertension and LVH, we fed Sabra salt-sensitive rats with a high-salt diet. The number of macrophages increased in the hypertensive hearts, peaking at 10 weeks after a high-salt diet. Surprisingly, macrophage depletion, by IV clodronate (CL) liposomes, inhibited the development of hypertension. Moreover, macrophage depletion reduced LVH by 17% (p<0.05), and reduced cardiac fibrosis by 75%, compared with controls (p=0.001). Second, to determine the role of macrophages in the development and progression of LVH, independent of high-salt diet, we depleted macrophages in mice subjected to transverse aortic constriction and pressure overload. Significantly, macrophage depletion, for 3 weeks, attenuated LVH: a 12% decrease in diastolic and 20% in systolic wall thickness (p<0.05), and a 13% in LV mass (p=0.04), compared with controls. Additionally, macrophage depletion reduced cardiac fibrosis by 80% (p=0.006). Finally, macrophage depletion down-regulated the expression of genes associated with cardiac remodeling and fibrosis: transforming growth factor beta-1 (by 80%) collagen type III alpha-1 (by 71%) and atrial natriuretic factor (by 86%). CONCLUSIONS: Macrophages mediate the development of hypertension, LVH, adverse cardiac remodeling, and fibrosis. Macrophages, therefore, should be considered as a therapeutic target to reduce the adverse consequences of hypertensive heart disease.

Unmasking of proteinuria in the course of genetic dissection of nonproteinuric diabetic nephropathy.

We previously described the development of nonproteinuric diabetic nephropathy (NPDN) in the Cohen diabetic rat (CDs), a model that simulates Type 2 diabetes in humans. Using linkage analysis in an F2 cross, we currently set out to investigate the mechanisms underlying NPDN. We crossbred between CDs and SBN/y, a nondiabetic rat strain, generated F1 and F2 progenies, fed them diabetogenic diet that elicits diabetes and NPDN in CDs but not in SBN/y, and determined metabolic and renal phenotypes. Over 5 mo, ∼75% of F2 developed a diabetic phenotype. In parallel, a nephropathy developed in F2, with glomerular filtration rate (GFR) declining in ∼25% and, unexpectedly, significant proteinuria appearing in ∼75%. We scanned the F2 genome with microsatellite markers and used linkage analysis to identify quantitative trait loci (QTLs). We detected diabetes-related QTLs on RNO4 and 13. We also detected two QTLs for the decline in GFR on RNO4 and 13 and another QTL for proteinuria on RNO13. The metabolic and renal-related QTLs overlapped. These results suggest that the mechanisms underlying the nephropathy in F2 are related to genes that map to RNO4 and 13, as well as a common genetic background for the development of diabetes and the renal disease. Our findings further indicate that proteinuria is inhibited in parental diabetic CDs, thus accounting for the nonproteinuric phenotype, but "unmasked" in diabetic F2 whose genome has been modified. Identifying the nature of the factor inhibiting proteinuria in diabetic CDs but not in F2 may provide a clue to treatment and prevention of proteinuria in diabetes.

Geno-transcriptomic dissection of proteinuria in the uninephrectomized rat uncovers a molecular complexity with sexual dimorphism.

Investigation of proteinuria, whose pathophysiology remains incompletely understood, is confounded by differences in the phenotype between males and females. We initiated a sex-specific geno-transcriptomic dissection of proteinuria in uninephrectomized male and female Sabra rats that spontaneously develop focal and segmental glomerulosclerosis, testing the hypothesis that different mechanisms might underlie the pathophysiology of proteinuria between the sexes. In the genomic arm, we scanned the genome of 136 male and 111 female uninephrectomized F2 populations derived from crosses between SBH/y and SBN/y. In males, we identified proteinuria-related quantitative trait loci (QTLs) on RNO2 and 20 and protective QTLs on RNO6 and 9. In females, we detected proteinuria-related QTLs on RNO11, 13, and 20. The only QTL overlap between the sexes was on RNO20. Using consomic strains, we confirmed the functional significance of this QTL in both sexes. In the transcriptomic arm, we searched on a genomewide scale for genes that were differentially expressed in kidneys of SBH/y and SBN/y with and without uninephrectomy. These studies identified within each sex differentially expressed genes of relevance to proteinuria. Integrating genomics with transcriptomics, we identified differentially expressed genes that mapped within the boundaries of the proteinuria-related QTLs, singling out 24 transcripts in males and 30 in females, only 4 of which (Tubb5, Ubd, Psmb8, and C2) were common to both sexes. Data mining revealed that these transcripts are involved in multiple molecular mechanisms, including immunity, inflammation, apoptosis, matrix deposition, and protease activity, with no single molecular pathway predominating in either sex. These results suggest that the pathophysiology of proteinuria is highly complex and that some of the underlying mechanisms are shared between the sexes, while others are sex specific and may account for the difference in the proteinuric phenotype between males and females.

Sex-specific genetic dissection of diabetes in a rodent model identifies Ica1 and Ndufa4 as major candidate genes.

The aim of the study was to initiate a sex-specific investigation of the molecular basis of diabetes, using a genomic approach in the Cohen Diabetic rat model of diet-induced Type 2 diabetes. We used an F2 population resulting from a cross between Cohen Diabetic susceptible (CDs) and resistant (CDr) and consisting of 132 males and 159 females to detect relevant QTLs by linkage and cosegregation analyses. To confirm the functional relevance of the QTL, we applied the "chromosome substitution" strategy. We identified candidate genes within the quantitative trait locus (QTL) and studied their differential expression. We sequenced the differentially expressed candidate genes to account for differences in their expression. We confirmed in this new cross in males a previously detected major QTL on rat chromosome 4 (RNO4); we identified in females this major QTL as well. We found three additional diabetes-related QTLs on RNO11, 13, and 20 in females only. We pursued the investigation of the QTL on RNO4 and generated a CDs.4(CDr) consomic strain, which provided us with functional confirmation for the contribution of the QTL to the diabetic phenotype in both sexes. We successfully narrowed the QTL span to 2.6 cM and identified within it six candidate genes, but only two of which, Ica1 (islet cell autoantigen 1) and Ndufa4 (NADH dehydrogenase ubiquinone) were differentially expressed between CDs and CDr. We sequenced the exons and promoter regions of Ica1 and Ndufa4 but did not identify sequence variations between the strains. The detection of the QTL on RNO4 in both sexes suggests involvement of Ica1, Ndufa4, the Golgi apparatus, the mitochondria and genetic susceptibility to dietary-environmental factors in the pathophysiology of diabetes in our model. The additional sex-specific QTLs are likely to account for differences in the diabetic phenotype between the sexes.

Tumor necrosis factor-alpha: a possible priming agent for the polymorphonuclear leukocyte-reduced nicotinamide-adenine dinucleotide phosphate oxidase in hypertension.

In the Sabra rat, oxidative stress (OS) and inflammation precede the development of hypertension. Inhibition of the phagocytic NADPH oxidase attenuates the rise in blood pressure. The present study was set to identify possible priming agents for this enzyme and to test the hypothesis that the phagocytic NADPH oxidase contributes to OS and inflammation. Sabra salt-sensitive and Sabra salt-resistant rats were salt loaded or provided regular chow for 60 days with or without apocynin to inhibit NADPH oxidase. Levels of interleukin 6, tumor necrosis factor-alpha, and interferon-gamma served as indices of inflammation. Extracellular and intracellular levels of the polymorphonuclear leukocyte tumor necrosis factor-alpha receptors (p55 and p75) were assessed by flow cytometry in young and adult rats. NADPH oxidase activity and expression of p47phox were measured in polymorphonuclear leukocytes and aortic rings. Malondialdehyde and carbonylated fibrinogen served as indices of OS. Inflammatory and OS indices excluding interferon-gamma were higher in the hypertensive state and reduced by apocynin. Levels of malondialdehyde and tumor necrosis factor-alpha were elevated already in the prehypertensive state. No differences were found in the levels of p75. The extracellular expression of p55 was higher in adult Sabra salt-resistant compared with Sabra salt-sensitive rats (7.46+/-2.2% versus 2.1+/-0.5%; P<0.05), whereas levels of the intracellular p55 were higher in adult Sabra salt-sensitive rats (3.2+/-2% versus 1.1+/-0.5%; P<0.05). In young normotensive rats, the extracellular levels of p55 were higher in Sabra salt-sensitive compared with Sabra salt-resistant rats (10.6+/-5.2% versus 2.9+/-1.5%; P<0.01). Tumor necrosis factor-alpha plays a role in activation of the polymorphonuclear leukocyte NADPH oxidase, thereby contributing to systemic OS, inflammation, and the development of hypertension in this model.

Genomic research in rat models of kidney disease.

Current understanding of the mechanisms underlying renal disease in humans is incomplete. Consequently, our ability to prevent the occurrence of renal disease or treat kidney disease once it develops is limited. There are objective difficulties in investigating kidney disease directly in humans, leading investigators to resort to experimental animal models that simulate renal disease in humans. Animal models have thus been a tool of major importance in the study of normal renal physiology and have been crucial in shedding light on the complex mechanisms involved in normal kidney function and in our current understanding of and ability to treat renal disease. Among the animal models, rat has been the preferred and most commonly used species for the investigation of renal disease. This chapter reviews what has been achieved over the years, using rat as a tool for the investigation of renal disease in humans, focusing on the contribution of rat genetics and genomics to the elucidation of the mechanisms underlying the pathophysiology of the major types of renal disease, including primary and secondary renal diseases.

ACE2 expression and activity are enhanced during pregnancy.

In the current study, we investigated the expression and activity of ACE2 during pregnancy in normotensive and hypertensive rats, focusing on the relative contribution of the uterus and the placentas, the kidney serving as a reference. We used the Sabra rat model of salt-sensitive hypertension. We confirmed a systemic vasodilatory state during the third trimester of pregnancy, as evidenced by a reduction in blood pressure, both in normotensive and hypertensive rats. At the time that blood pressure was reduced, ACE2 was expressed abundantly in the reproductive organs. The relative levels of ACE2 mRNA in the pregnant animal were placenta > kidneys > or = uterus and of ACE2 activity kidney > placenta > uterus. In the uterus and the placenta, ACE2 expression was unaffected by strain, salt-loading, or the level of blood pressure. ACE2 activity in the uterus of the nonpregnant rat was not affected by any of these variables either, but during pregnancy increased in salt-loaded animals. When estimating the total contribution of the uterus to ACE2 mRNA and activity during pregnancy, we found that the amount of ACE2 mRNA increased in both strains irrespective of diet, but that ACE2 activity increased only in salt-loaded animals. We further estimated the relative total contribution of the uterus, placentas, and kidneys to ACE2 expression and activity during pregnancy by adjusting for mass and number of organs and found that the placentas were the major contributors, followed by the kidney and the uterus. We conclude that during pregnancy, the placentas, in particular, but also the uterus, constitute important sources of ACE2, in addition to its normal production in the kidney, leading to an estimated twofold increase in total ACE2 activity. These data are consistent the hypothesis that transient ACE2 overexpression and increased activity during pregnancy may be important in modulating systemic, as well as local hemodynamics in the uteroplacental unit.

Impaired glucose-stimulated insulin secretion is coupled with exocrine pancreatic lesions in the Cohen diabetic rat.

OBJECTIVE: The Cohen diabetes-sensitive rat develops postprandial hyperglycemia when fed a high-sucrose, copper-poor diet, whereas the Cohen diabetes-resistant rat maintains normoglycemia. The pathophysiological basis of diabetes was studied in the Cohen diabetic rat centering on the interplay between the exocrine and endocrine compartments of the pancreas. RESEARCH DESIGN AND METHODS: Studies used male Cohen diabetes-sensitive and Cohen diabetes-resistant rats fed 1-month high-sucrose, copper-poor diet. Serum insulin and glucose levels were measured during glucose and insulin tolerance tests. The pancreas was evaluated for weight, insulin content, macrophage, and fat infiltration. Glucose-stimulated insulin secretion (GSIS) was determined in isolated perfused pancreas and in islets. RESULTS: Hyperglycemic Cohen diabetes-sensitive rats exhibited reduced pancreatic weight with lipid deposits and interleukin-1beta-positive macrophage infiltration in the exocrine pancreas. Islet morphology was preserved, and total pancreatic insulin content did not differ from that of Cohen diabetes-resistant rats. Lipids did not accumulate in skeletal muscle, nor was insulin resistance observed in hyperglycemic Cohen diabetes-sensitive rats. Intravenous glucose-tolerance test revealed markedly elevated glucose levels associated with diminished insulin output. Insulin release was induced in vivo by the non-nutrient secretagogues arginine and tolbutamide, suggesting a selective unresponsiveness to glucose. Decreased GSIS was observed in the isolated perfused pancreas of the hyperglycemic Cohen diabetes-sensitive rat, whereas islets isolated from these rats exhibited glucose-dependent insulin secretion and proinsulin biosynthesis. CONCLUSIONS: The association of the in vivo insulin secretory defect with lipid accumulation and activated macrophage infiltration in the exocrine pancreas suggests that changes in the islet microenvironment are the culprit in the insulin secretory malfunction observed in vivo.

The polymorphonuclear leukocyte contributes to the development of hypertension in the Sabra rat.

BACKGROUND: We previously showed that priming of the polymorphonuclear leukocyte (PMNL), inflammation and oxidative stress antecede the development of hypertension in the Sabra rat model of hypertension. The actual role of PMNLs and PMNL-mediated oxidative stress and inflammation in the development of hypertension in this model has remained, however, unresolved. OBJECTIVE: The aim of our study was to test the hypothesis that PMNLs and that the PMNL-associated NADPH oxidase contribute to the development of hypertension in the Sabra rat model. METHODS: To determine the contribution of the PMNL to the development of hypertension, we depleted Sabra hypertension-prone (SBH/y) animals from PMNLs with an anti-PMNL antibody, salt-loaded them and monitored their blood pressure over a period of 30 days. To determine the contribution of the NADPH oxidase on the development of hypertension, we inhibited the activity of this enzyme with phenylarsine oxide or apocynin in SBH/y rats while salt-loading the animals and followed the course of their blood pressure over 60 days. RESULTS: PMNL depletion attenuated significantly the development of hypertension in SBH/y rats. Inhibition of NADPH oxidase with phenylarsine oxide and apocynin markedly inhibited the development of hypertension in SBH/y rats, as well as decreased the rate of superoxide release, the level of PMNL CD11b and the PMNL count. CONCLUSION: These data are consistent with a significant contribution of PMNLs to the development of hypertension, and suggest that the mechanism may be related, at least in part, to PMNL-mediated oxidative stress and inflammation.

Metabolic and genomic dissection of diabetes in the Cohen rat.

We investigated the metabolic and genetic basis of diabetes in the Cohen Diabetic rat, a model of diet-induced diabetes, as a means to identify the molecular mechanisms involved. By altering individual components in the diabetogenic diet, we established that the dietary susceptibility that leads to the development of diabetes in this model is directly related to the high casein and low copper content in chow. The development of diabetes is accompanied by depletion of the acini from the exocrine pancreas and replacement with fat cells, while the appearance of the islets of Langerhans remains intact. With reversion back from diabetogenic to regular diet, the diabetic phenotype disappears but the histological changes in the exocrine pancreas prevail. Using positional cloning, we detected a major quantitative trait locus (QTL) on rat chromosome 4 with a chromosomal span of 4.9 cM, and two additional loci on chromosomes 7 and X. A screen for genes within that QTL in the rat and in the syntenic regions in mouse and man revealed only 23 candidate genes. Notable among these genes is Ica1, which has been causally associated with diabetes and bovine casein. We conclude that the development of diabetes in our model is dependent upon high casein and low copper in diet, that it is accompanied by histomorphological changes in the exocrine but not endocrine pancreas, that it is reversible, and that it is associated with a major QTL on chromosome 4 in which we detected Ica1, a high priority candidate gene.