Hartono, Stella

Abstract Atherosclerotic renal artery sclerosis (RAS) is a significant public health concern, affecting 7% of the population over 65 years old and conveying an increased risk for cardiovascular as well as renal disease. Recent reports have failed to show a benefit of renal artery stenting to treat RAS. We tested the hypothesis that mesenchymal stem cells (MSC) slow the progression of renal injury by reducing inflammation. MSCs were isolated and cultured from murine adipocyte tissue and injected via the carotid artery into the animals 4 weeks after inducing RAS, a time at which the stenotic kidney has undergone severe atrophy. Conjugated microsphere labeling and flow cytometry confirmed that the MSCs migrate to the stenotic kidney. Half of the treatment group demonstrated a 90% reduction in atrophy and significantly lower level of regulatory T cell (Treg) infiltration while the other half demonstrated no change when compared to vehicle treated RAS mice. Array analysis showed a significant down-regulation of NOS2 (M1 macrophages), RORc (Th 17 cells), and CCR4 (associated with M1 macrophages) within those mice with low renal atrophy. The results suggest that under certain conditions, MSCs have a possible effect on the inflammatory environment present in RAS mice. Future studies will determine whether intervention prior to development of severe atrophy will increase therapeutic efficacy of MSC.

Abstract Renovascular hypertension (RVH) affects up to 7% of individuals over 65 years of age. We have previously demonstrated that induction of oxidative stress is an early event in the development of RVH that leads to development of inflammation and renal atrophy. NLRP3 is one of the most well-known sensors of oxidative stress. However, the role of NLRP3 in the development of RVH and subsequent end-organ damage is not well understood. We therefore sought to test the hypothesis that inhibition of NLRP3 activation protects the heart and the kidneys of mice subjected to RVH. In accordance with our previous observations, wild type (WT) mice subjected to RVH developed 26±6% of renal atrophy within 1 week following cuff placement that progressed to almost 100% by 4 weeks. In contrast, although NLRP3-/- mice (KO) subjected to RVH developed similar degree of atrophy to their age-matched WT in the first week (29 ± 17 vs. 39 ± 20), the atrophy did not progress when evaluated at 4 weeks (29 ± 13 vs. 78 ± 10, p=0.013). Histologic evaluation of renal cortex of KO mice showed no significant interstitial infiltration with CD3+ T cells or F4/80+ macrophages. In addition, KO mice developed no cardiac hypertrophy, no cardiac fibrosis, or aortic lesion despite similar elevation in systolic blood pressure to the WT mice. We propose that NLRP3 activation plays a critical role in the development of renal atrophy in mice subjected to RVH and represent a potential therapeutic target.

Abstract Renal artery stenosis is a common finding in patients with cardiovascular disease. Recent studies have provided evidence that innate immunity plays a critical role in the development of chronic renal disease in renovascular hypertension. We have previously demonstrated that interstitial infiltration of lymphocytes is an early event in the development of renal atrophy in the 2K1C model of renovascular hypertension. We therefore sought to test the hypothesis that lymphocytes play a role in the development of progressive atrophy/fibrosis in this model. We subjected mice to renal artery stenosis through placement of a polytetrafluoroethylene cuff on the right renal artery. In accordance with our previous observations, wild type mice subjected to RAS developed renal atrophy within 2 weeks following placement of the cuff (70.5 +/- 11.9%). Chemokine expression in renal cortex of the stenotic kidney reflected predominantly a Th1 response (STAT1, STAT4, TBX21); CCL2, CCL17, CCL12, and TNF mRNA expression were also upregulated, but not IL-6 and IL-10. In striking contrast to wild type mice, Rag1 mice developed significantly less atrophy (23.1 +/- 10.4, p<0.0037 vs wild type). Unlike wild type mice, histologic evaluation of renal cortex of Rag1 mice showed no significant interstitial infiltration with CD3+ T cells or F4/80+ macrophages. We propose that T cell-mediated inflammation plays a critical role in the development of renal atrophy in 2K1C mice.

Introduction: The estimated prevalence of diabetes mellitus (DM) is 8.4% among adults in the U.S. Cardiovascular disease is a major complication of DM. We have observed that diabetic mice, but not non-diabetic mice, undergoing 2kidney-1clip (2K1C) surgery inducing renovascular hypertension had high mortality attributed to ruptured aortic aneurysm. However, the effect of diabetes on formation of aortic aneurysm remains unclear. Method: In our previous experiment, 163 mice were divided into 4 groups: leptin KO 2K1C (db 2K1C, n=52), leptin KO Sham (db Sham, n=20), wild type 2K1C (dbc 2K1C, n=62), and wild type Sham (dbc Sham, n=29), respectively. We found that 42% (22/52) of db 2K1C died, from ruptured aortic aneurysms examined by gross examination, within 120 days, while others survived 100%. In this study, available H&E aortic sections of survived mice (n=74): db 2K1C (n=30), db Sham (n=15), dbc 2K1C (n=20), and dbc Sham (n=9) were examined 2K1C surgery in a blinded fashion. Aortic lesions defined as necrosis, thrombosis and dropped-out smooth muscle cells, which are signs of early formation of aortic aneurysm, were identified. Inflammation was graded as mild, moderate and severe inflammation. Result: The db 2K1C mice had a significantly greater aortic lesions (16/30, 53%) compared to the db Sham (13/15, 20%); and dbc 2K1C mice (1/20, 5%), p=0.002 and 0.0001, respectively. The number of aortic lesions in the dbc 2K1C group was not statistically different from that of the dbc Sham group (p=0.5). Similarly, the number of aortic lesions in the db Sham group was not statistically different from that of the dbc Sham group (0/9, 0%), p=0.125. Moderate-to-severe inflammation in the tunica media was more prevalence in db mice than the dbc mice. Conclusion: We report for the first time an increased incidence of aortic lesions in db 2K1C compared to dbc and sham groups. This finding suggests that diabetes contributes to aortic aneurysm formation in mice with renal artery stenosis, potentially due to induction of inflammation. Further studies to confirm a mechanism are warranted.

Background: Combined renovascular hypertension (RVH) and type II diabetes (DM II) are a major health problem. Both share common risk factors and contribute to cardiovascular morbidity and mortality. We recently observed a high mortality rate in DM II mice (dbdb) with RVH. Purpose: We sought to determine the interactions between renovascular hypertension and DM II on the cardiovascular phenotype in dbdb mice with RVH. Methods and Materials: RVH was established by placing a polytetrafluoroethylene cuff on the right renal artery of C57BL/KS (wild type, WT) or dbdb mice. We divided the mice into four groups: WT sham, WT type RAS, dbdb sham, dbdb RAS. We examined the blood pressure, plasma angiotensin I, renin, heart weight and myocyte diameter. Histologic parameters including inflammation, necrosis and fibrosis were assessed semiquantitively. Results: Blood pressure, Renin, Plasma angiotensin I: RAS produced a similar elevation in systolic blood pressure (SBP), renin mRNA expression, and plasma angiotensin I content in WT and dbdb mice with RAS. Heart weight: Despite similar elevation in SBP, db/db RAS mice showed a greater increase in heart weight than WT RAS mice (163.7 +/- 6.4 mmHg vs 145.7 +/- 2.3; n=23).. Similar trends were observed on normalization of the heart weight to tibial length. Cardiac myocyte diameter: Cardiac myocyte diameter in WT RAS (n=26) was significantly higher than in WT sham (n=15; 16.4 ± 0.3 vs. 15.0 ± 0.3 μm, p=0.0031). Myocyte diameter was higher in dbdb RAS mice (n=19, 14.76 ± 0.35) than dbdb sham (15.94 ± 0.29 μm, p= 0.0126). However, myocyte diameter was similar in dbdb RAS and WT RAS mice (16.4 ± 0.3 vs. 15.9 ± 0.3 μm, p=0.3). Histology: Sham groups had no inflammation (INF) or necrosis (NEC). Interstitial fibrous tissue was increased in dbdb RAS mice (28% by area) vs WT RAS (19% by area). Dbdb RAS mice had more severe INF (severe in 18%, mild in 82%) than WT RAS (severe in 0%, jild in 12%). Severe NEC was found in 32.1% of dbdb compared to 11.5% of WT RAS. Conclusion: The histologic changes and hypertrophy of the heart superimpose in dbdb RAS compared to WT RAS. This indicates that the risk of cardiovascular morbidity and mortality increases synergisticly in the presence of DM II and RVH.

Despite proven efficacy, ARB therapy reduces glomerular filtration in patients with unilateral renal artery stenosis (RAS). The objective of this study was to determine whether ARB exacerbates renal injury in 2K1C hypertension. RAS was established in mice by placement of a cuff on the right renal artery, which reduces blood flow by 70%. Both Hydralazine (H) and Valsartan (ARB) reduced blood pressure to a similar extent. RAS‐mediated cardiac hypertrophy (heart weight 134% sham (S) p<0.001) was prevented by ARB (102% S, p=ns) but not H (116% S, p=0.03); cardiac fibrosis was prevented in both ARB and H. Although ARB did not exacerbate renal atrophy (91.4 ± 2.1% atrophy vehicle (V) vs 75.3 ± 9.5% ARB, p=ns), H reduced renal atrophy (91.4 ± 2.1% V vs 28.4 ± 13.5% H, p<0.01). The contralateral kidney underwent compensatory hyperplasia (123% S; p<0.001), which was blunted with ARB or H treatment. No significant histopathologic alterations were observed in the contralateral kidneys of any group. RAS induced proteinuria in V mice. Urine protein excretion in ARB RAS mice was reduced to levels observed in S mice; H did not significantly alter urine protein excretion. Severe renal atrophy was observed in the cuffed kidneys of V mice, which was not exacerbated by ARB. However, ARB reduced proteinuria to baseline levels, indicating that ARB may protect overall renal function in unilateral RAS. These studies were supported by NIH grant P01 HL85307.

Abstract PurposeTo determine the utility of GLILD CT scoring system for assessment of GLILD in patients with CVID to avoid the need for lung biopsy. MethodsThe CT scoring system was developed as a consensus based on review of literature. Two radiologists, blinded to patient clinical information, retrospectively scored CT scans of selected patients with histopathologic diagnosis of GLILD. Discrepancies were settled by a third radiologist. Inter-observer reproducibility was calculated with intra-class correlation coefficients. ResultsWe identified 25 CVID patients with histologically confirmed GLILD (12 male, 13 female). The median age was 38 years (range 12 – 72 years). Inter-observer reproducibility was 0.76 (95% CI 0.53 – 0.89). Splenomegaly/splenectomy and basilar predominant perilymphatic nodules greater than or equal to 4mm have good interobserver agreement in our CT scoring system. Discrepancy between observers occurred most often in differentiating intermediate versus high confidence diagnosis of GLILD as opposed to differentiating between low versus intermediate confidence. ConclusionGLILD CT scoring system of pulmonary disease in CVID patients can stratify patients into categories that may assist in avoiding lung biopsy for diagnosis in a subset of patients.

The sequence of events that lead to inflammation and fibrosing nonalcoholic steatohepatitis (NASH) is incompletely understood. Hence, we investigated the chronology of whole body, tissue, and cellular events that occur during the evolution of diet-induced NASH. Male C57Bl/6 mice were assigned to a fast-food (FF; high calorie, high cholesterol, high fructose) or standard-chow (SC) diet over a period of 36 wk. Liver histology, body composition, mitochondrial respiration, metabolic rate, gene expression, and hepatic lipid content were analyzed. Insulin resistance [homeostasis model assessment-insulin resistance (HOMA-IR)] increased 10-fold after 4 wk. Fibrosing NASH was fully established by 16 wk. Total hepatic lipids increased by 4 wk and remained two- to threefold increased throughout. Hepatic triglycerides declined from sixfold increase at 8 wk to threefold increase by 36 wk. In contrast, hepatic cholesterol levels steadily increased from baseline at 8 wk to twofold by 36 wk. The hepatic immune cell population altered over time with macrophages persisting beyond 16 wk. Mitochondrial oxygen flux rates of FF mice diet were uniformly lower with all the tested substrates (13–276 pmol·s−1·ml−1 per unit citrate synthase) than SC mice (17–394 pmol·s−1·ml−1 per unit citrate synthase) and was accompanied by decreased mitochondrial:nuclear gene copy number ratios after 4 wk. Metabolic rate was lower in FF mice. Mitochondrial glutathione was significantly decreased at 24 wk in FF mice. Expression of dismutases and catalase was also decreased in FF mice. The evolution of NASH in the FF diet-induced model is multiphasic, particularly in terms of hepatic lipid composition. Insulin resistance precedes hepatic inflammation and fibrosis. Mitochondrial dysfunction and depletion occur after the histological features of NASH are apparent. Collectively, these observations provide a unique overview of the sequence of changes that coevolve with the histological evolution of NASH. NEW & NOTEWORTHY This study demonstrates in a first of kind longitudinal analysis, the evolution of nonalcoholic steatohepatitis (NASH) on a fast-food diet-induced model. Key findings include 1) hepatic lipid composition changes in a multiphasic fashion as NASH evolves; 2) insulin resistance precedes hepatic inflammation and fibrosis, answering a longstanding chicken-and-egg question regarding the relationship of insulin resistance to liver histology in NASH; and 3) mitochondrial dysfunction and depletion occur after the histological features of NASH are apparent.

Purpose of review Norovirus infection is an emerging chronic infection in immunocompromised hosts. The aim of this review is to discuss the pathophysiology of Norovirus infection and explore mechanistic models for chronic infection/shedder state, especially in patients with immune deficiency diseases. Recent findings Chronic Norovirus infection is increasingly associated with enteropathy associated with both primary and secondary immune deficiency diseases. There is an ongoing debate in the immune deficiency community whether it is truly a causative agent for the enteropathy or it is an innocent bystander. We describe the historic aspects of Norovirus infection, its immunology and viral structure and the basis for preventive and vaccination strategies. We also postulate in this review a disease model in immune deficiency subjects which creates a milieu for it to become a chronic and explore newer frontiers for disease modification and prevention. Summary Norovirus is the most common cause of acute gastroenteritis in general population but the factors that lead to its persistence in patients with immune deficiency need further holistic studies. This should include host assessment, microbiome signatures, and viral pathogenic factors assessment.

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.