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.

Critical Stepwise Decline of Antibodies against SARS-CoV-2 among Chronic Hemodialysis Patients 180 Days Post Comirnaty Vaccine.

BACKGROUND: The reduced immune response of maintenance hemodialysis patients to coronavirus disease 2019 (COVID-19) vaccines is a major concern. OBJECTIVES: To analyze the late (6 months after full vaccination) antibody response and compare it to early post-vaccination titer. METHODS: We conducted a multicenter prospective study of 13 hemodialysis units in Israel. RESULTS: We demonstrated that the low titers observed among ESRD patients 2-3 months after vaccination with the Comirnaty vaccine (median 63.8 AU/ml) declined to critically lower values 6 months after full vaccination. (Mediananti S antibodies, 31 AU/ml). Seropositivity significantly declined among hemodialysis patients from 89% to 74% (P < 0.0001), although it did not significantly change among controls. CONCLUSIONS: We recommend all patients on hemodialysis receive a booster COVID-19 vaccine 6 months after the second dose.

Diminished and waning immunity to COVID-19 vaccination among hemodialysis patients in Israel: the case for a third vaccine dose.

BACKGROUND: Humoral responses to coronavirus disease 2019 (COVID-19) vaccines in hemodialysis (HD) patients can direct vaccination policy. METHODS: We compared 409 COVID-19-naïve HD patients from 13 HD units in Israel to 148 non-dialysis-dependent COVID-19-naïve controls. Twenty-four previously infected (antinucleocapsid positive) HD patients were analysed separately. Blood samples were obtained ≥14 days post-vaccination (BNT162b2, Pfizer/BioNTech) to assess seroconversion rates and titers of anti-spike (anti-S) and neutralizing antibodies. RESULTS: The median time from vaccination to blood sample collection was 82 days [interquartile range (IAR) 64-87] and 89 days (IQR 68-96) for HD patients and controls, respectively. Seroconversion rates were lower in HD patients compared with controls for both anti-S and neutralizing antibodies (89% and 77% versus 99.3%, respectively; P < 0.0001). Antibody titers were also significantly lower in HD patients compared with controls {median 69.6 [IQR 33.2-120] versus 196.5 [IQR 118.5-246], P < 0.0001; geometric mean titer [GMT] 23.3 [95% confidence interval (CI) 18.7-29.1] versus 222.7 [95% CI 174-284], P < 0.0001, for anti-S and neutralizing antibodies, respectively}. Multivariate analysis demonstrated dialysis dependence to be strongly associated with lower antibody responses and antibody titers waning with time. Age, low serum albumin and low lymphocyte count were also associated with lower seroconversion rates and antibody titers. HD patients previously infected with sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had no difference in their seroconversion rates or antibody titers compared with COVID-19-naïve patients. CONCLUSION: This study demonstrates diminished and waning humoral responses following COVID-19 vaccination in a large and diverse cohort of HD patients, including those previously infected with SARS-CoV-2. Considering these results and reduced vaccine effectiveness against variants of concern, in addition to continued social distancing precautions, a third booster dose should be considered in this population.

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.

Minimal Change Disease Following the Pfizer-BioNTech COVID-19 Vaccine.

We report on the development of minimal change disease (MCD) with nephrotic syndrome and acute kidney injury (AKI), shortly after first injection of the BNT162b2 COVID-19 vaccine (Pfizer-BioNTech). A 50-year-old previously healthy man was admitted to our hospital following the appearance of peripheral edema. Ten days earlier, he had received the first injection of the vaccine. Four days after injection, he developed lower leg edema, which rapidly progressed to anasarca. On admission, serum creatinine was 2.31 mg/dL and 24-hour urinary protein excretion was 6.9 grams. As kidney function continued to decline over the next days, empirical treatment was initiated with prednisone 80 mg/d. A kidney biopsy was performed and the findings were consistent with MCD. Ten days later, kidney function began to improve, gradually returning to normal. The clinical triad of MCD, nephrotic syndrome, and AKI has been previously described under a variety of circumstances, but not following the Pfizer-BioNTech COVID-19 vaccine. The association between the vaccination and MCD is at this time temporal and by exclusion, and by no means firmly established. We await further reports of similar cases to evaluate the true incidence of this possible vaccine side effect.

[HYPERTENSION AS A CAUSE OF RENAL DISEASE - FACT OR FICTION?]

BACKGROUND: The possible link between hypertension and chronic kidney disease has been preoccupying the medical world for over a century. The cause-effect relationship between malignant hypertension and renal disease is clear and agreed upon, but when hypertension is at milder levels, this relationship becomes blurred. Does indeed non-malignant hypertension also cause kidney disease that can lead to end-stage kidney failure? In this review, we provide evidence based primarily on epidemiologic data, from which it can be concluded that the higher the blood pressure is, starting from normal blood pressure and above, the higher is the risk of developing kidney disease and loss of renal function. Does treatment of hypertension prevent kidney injury secondary to hypertension? The evidence points to the issue that hypertension slows down the rate of decline in glomerular filtration rate, but only in the presence of significant urinary protein excretion, whereas in the absence of proteinuria, lowering blood pressure has no effect on the development of hypertensive renal disease.

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.

Anti-TGF-ß1 Antibody Therapy in Patients with Diabetic Nephropathy.

TGF-ß has been implicated as a major pathogenic factor in diabetic nephropathy. This randomized, double-blind, phase 2 study assessed whether modulating TGF-ß1 activity with a TGF-ß1-specific, humanized, neutralizing monoclonal antibody (TGF-ß1 mAb) is safe and more effective than placebo in slowing renal function loss in patients with diabetic nephropathy on chronic stable renin-angiotensin system inhibitor treatment. We randomized 416 patients aged ≥25 years with type 1 or type 2 diabetes, a serum creatinine (SCr) level of 1.3-3.3 mg/dl for women and 1.5-3.5 mg/dl for men (or eGFR of 20-60 ml/min per 1.73 m2), and a 24-hour urine protein-to-creatinine ratio ≥800 mg/g to TGF-ß1 mAb (2-, 10-, or 50-mg monthly subcutaneous dosing for 12 months) or placebo. We assessed a change in SCr from baseline to 12 months as the primary efficacy variable. Although the Data Monitoring Committee did not identify safety issues, we terminated the trial 4 months early for futility on the basis of their recommendation. The placebo group had a mean±SD change in SCr from baseline to end of treatment of 0.33±0.67 mg/dl. Least squares mean percentage change in SCr from baseline to end of treatment did not differ between placebo (14%; 95% confidence interval [95% CI], 9.7% to 18.2%) and TGF-ß1 mAb treatments (20% [95% CI, 15.3% to 24.3%], 19% [95% CI, 14.2% to 23.0%], and 19% [95% CI, 14.0% to 23.3%] for 2-, 10-, and 50-mg doses, respectively). Thus, TGF-ß1 mAb added to renin-angiotensin system inhibitors did not slow progression of diabetic nephropathy.

Adherence rates to ferric citrate as compared to active control in patients with end stage kidney disease on dialysis.

INTRODUCTION: Oral phosphate binders are the main stay of treatment of hyperphosphatemia. Adherence rates to ferric citrate, a recently approved phosphate binder, are unknown. METHODS: We conducted a post-hoc analysis to evaluate whether adherence rates were different for ferric citrate vs. active control in 412 subjects with end stage kidney disease (ESKD) who were randomized to ferric citrate vs. active control (sevelamer carbonate and/or calcium acetate). Adherence was defined as percent of actual number of pills taken to total number of pills prescribed. FINDINGS: There were no significant differences in baseline characteristics including gender, race/ethnicity, and age between the ferric citrate and active control groups. Baseline phosphorus, calcium, and parathyroid hormone levels were similar. Mean (SD) adherence was 81.4% (17.4) and 81.7% (15.9) in the ferric citrate and active control groups, respectively (P = 0.88). Adherence remained similar between both groups after adjusting for gender, race/ethnicity, age, cardiovascular disease (CVD), and diabetic nephropathy (mean [95% CI]: 81.4% [78.2, 84.6] and 81.5% [77.7, 85.2] for ferric citrate and active control, respectively). Gender, race/ethnicity, age, and diagnosis of diabetic nephropathy did not influence adherence to the prescribed phosphate binder. Subjects with CVD had lower adherence rates to phosphate binder; this was significant only in the active control group. DISCUSSION: Adherence rates to the phosphate binder, ferric citrate, were similar to adherence rates to active control. Similar adherence rates to ferric citrate are notable since tolerance to active control was an entry criteria and the study was open label. Gender, race/ethnicity, nor age influenced adherence.

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.

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.

[Medical research improves quality of care].

INTRODUCTION: The Barzilai Medical Center has recently been designated as an Academic Medical Center. The challenge to become an academic center arose from the realization that the level and quality of care provided to patients are necessarily improved when clinical medicine is practiced in a teaching and research environment. Indeed, after years of close working ties between the Barzilai Medical Center in Ashkelon and the Ben-Gurion University of the Negev, the center officially became an Academic Medical Center and was recognized as one of the two leading clinical campuses of the Faculty of Health Sciences. The academic status of a medical center incorporates the necessity for the medical staff to become actively involved and excel in medical service, educational activities and research. In this issue of Harefuah, we publish original research and review articles that reflect, in part, the clinical and research interests and activities of the medical staff of the Barzilai University Medical Center during the past few years.