Jun Yao Liu

Endoplasmic reticulum stress response and inflammatory cytokines in type 2 diabetic nephropathy: role of indoleamine 2,3-dioxygenase and programmed death-1.

We tested the hypotheses that a) type 2 diabetes increases endoplasmic reticulum (ER) stress response, production of pro-inflammatory cytokines and kidney cell death and b) downregulations of renal indoleamine 2,3-dioxygenase (IDO) and programmed death-1 (PD-1) contribute to exacerbated inflammation and tissue injury. The growth arrest and DNA damage-inducible protein 153 (GADD153; a marker of ER stress response), inflammatory cytokines and cell death were determined in the context of assessment of IDO and PD-1 in an animal model of type 2 diabetic nephropathy (i.e., db/db mouse). Peripheral blood of 4-month-old db/db mice manifested significantly greater percents of interleukin (IL)-17 and IL-23 positive cells in association with greater percents of cells that were positive for PD-1 or GADD153. Compared to kidneys of db/m controls, renal cells prepared from kidneys of db/db mice displayed a) increased percent of cells that were positive for IL-17, IL-23, PD-1 and GADD153, b) decreased JC-1 aggregates but increased JC-1 monomers suggestive of disruption of mitochondrial membrane potential and c) increased apoptotic and necrotic cell death. Immunohistochemical analyses also revealed increased staining of renal tissue of db/db mice for IL-17, IL23, GADD153, Annexin V, caspase 3, PD-1 and IDO compared to db/m kidneys; these changes were generally more prominent in the glomeruli. In conclusion, type 2 diabetes upregulates systemic and local ER stress response and pro-inflammatory mechanisms thereby contributing to renal injury. However, the accompanying upregulations of PD-1 and IDO likely reflect activation of compensatory mechanisms to curtail inflammation and cell injury.

Mitochondrial complex I and NAD(P)H oxidase are major sources of exacerbated oxidative stress in pressure-overloaded ischemic-reperfused hearts.

We tested the hypothesis that pressure overload exacerbates oxidative stress associated with augmented mitochondrial permeability transition (MPT) pore opening and cell death in ischemic-reperfused hearts. Pressure overload decreased the level of reduced glutathione but increased nitrotyrosine and 8-hydroxydeoxyguanosine levels in ischemic-reperfused hearts. The activity of catalase, but not superoxide dismutase (SOD), was lower in ischemic-reperfused hearts perfused at higher pressure. Mitochondria from ischemic-reperfused hearts subjected to higher perfusion pressure displayed significantly greater [3H]-2-deoxyglucose-6-P entrapment suggestive of greater MPT pore opening and consistent with greater necrosis and apoptosis. Tempol (SOD mimetic) reduced infarct size in both groups but it remained greater in the higher pressure group. By contrast, uric acid (peroxynitrite scavenger) markedly reduced infarct size at higher pressure, effectively eliminating the differential between the two groups. Inhibition of xanthine oxidase, with allopurinol, reduced infarct size but did not eliminate the differential between the two groups. However, amobarbital (inhibitor of mitochondrial complex I) or apocynin [inhibitor of NAD(P)H oxidase] reduced infarct size at both pressures and also abrogated the differential between the two groups. Consistent with the effect of apocynin, pressure-overloaded hearts displayed significantly higher NAD(P)H oxidase activity. Furthermore, pressure-overloaded hearts displayed increased nitric oxide synthase activity which, along with increased propensity to superoxide generation, may underlie uric acid-induced cardioprotection. In conclusion, increased oxidative and nitrosative stress, coupled with lack of augmented SOD and catalase activities, contributes importantly to the exacerbating impact of pressure overload on MPT pore opening and cell death in ischemic-reperfused hearts.

Effects of chromium picolinate on glycemic control and kidney of the obese Zucker rat

BackgroundChromium picolinate (Cr(pic)3) is advocated as adjuvant therapy for impaired glycemic control, despite concerns for DNA damage. Potential toxicity of Cr(pic)3 should be greater for the kidney that accumulates chromium. Therefore, we tested the hypothesis that Cr(pic)3 treatment of obese Zucker rats (OZR) exacerbates renal abnormalities associated with dysglycemia.MethodsMale OZR were treated with diets lacking or containing 5 and 10 mg/kg of chromium, as Cr(pic)3, for 20 weeks; lean Zucker rats (LZR) served as controls. Glycemic and renal effects of Cr(pic)3 were determined in the context of indices of oxidative stress and inflammation.ResultsThe OZR displayed increased fasting plasma glucose and insulin in association with enlarged pancreatic islets exhibiting collagen and periodic acid Schiff-positive deposits compared to LZR; Cr(pic)3 treatment did not affect these parameters. The OZR, irrespective of Cr(pic)3, excreted more albumin than LZR. Also, other indices of renal function or histopathology were not affected by Cr(pic)3 treatment. Urinary excretion of 8-hydroxydeoxyguanosine (8-OHdG), an index of oxidative DNA damage, was greater in the OZR than LZR; dietary Cr(pic)3 treatment attenuated 8-OHdG excretion. However, immunostaining of kidney for 8-OHdG revealed similar staining pattern and intensity, despite significant renal accumulation of chromium in Cr(pic)3-treated groups. Finally, increased renal nitrotyrosine and cyclooxygenase-2 levels and urinary excretion of monocyte chemoattractant protein-1 of OZR were partially reversed by Cr(pic)3 treatment.ConclusionDietary Cr(pic)3 treatment of OZR does not beneficially influence glycemic status or increase the risk for oxidative DNA damage; rather, the treatment attenuates indices of oxidative stress and inflammation.

Renal and glycemic effects of high-dose chromium picolinate in db/db mice: Assessment of DNA damage

This study examined renal and glycemic effects of chromium picolinate [Cr(pic)3] supplementation in the context of its purported potential for DNA damage. In preventional protocol, male obese diabetic db/db mice were fed diets either lacking or containing 5, 10 or 100 mg/kg chromium as Cr(pic)3 from 6 to 24 weeks of age; male lean nondiabetic db/m mice served as controls. Untreated db/db mice displayed increased plasma glucose and insulin, hemoglobin A1c, renal tissue advanced glycation end products, albuminuria, glomerular mesangial expansion, urinary 8-hydroxydeoxyguanosine (an index of oxidative DNA damage) and renal tissue immunostaining for γH2AX (a marker of double-strand DNA breaks) compared to db/m controls. Creatinine clearance was lower in untreated db/db mice than their db/m controls, while blood pressure was similar. High Cr(pic)3 intake (i.e., 100-mg/kg diet) mildly improved glycemic status and albuminuria without affecting blood pressure or creatinine clearance. Treatment with Cr(pic)3 did not increase DNA damage despite marked renal accumulation of chromium. In interventional protocol, effects of diets containing 0, 100 and 250 mg/kg supplemental chromium, from 12 to 24 weeks of age, were examined in db/db mice. The results generally revealed similar effects to those of the 100-mg/kg diet of the preventional protocol. In conclusion, the severely hyperglycemic db/db mouse displays renal structural and functional abnormalities in association with DNA damage. High-dose Cr(pic)3 treatment mildly improves glycemic control, and it causes moderate reduction in albuminuria, without affecting the histopathological appearance of the kidney and increasing the risk for DNA damage.

Reciprocal relation between GADD153 and Del-1 in regulation of salivary gland inflammation in Sjögren syndrome.

Endoplasmic reticulum (ER) stress response is a pivotal regulator of inflammation and cell death. An integral component of ER stress-induced apoptosis is expression of growth arrest- and DNA damage-inducible protein 153 (GADD153). Further, ER stress response is implicated in leukocyte adhesion and recent studies have discovered endogenous inhibitors of leukocyte adhesion including the developmental endothelial locus-1 (Del-1). Accordingly, we tested the hypothesis that Sjögrens syndrome (SS) is associated with increased salivary gland expression of GADD153 and increased leukocyte infiltration in association with decreased Del-1 thereby contributing to inflammation and cell death. We utilized the non-obese diabetic (NOD) mice, a model of SS-like disease, in association with immunostaining and flow cytometry-based studies. Salivary glands of 14-week-old NOD mice displayed a) increased GADD153 expression, b) marked reduction in Del-1, c) inflammatory cell infiltrates including CD3+ T and CD19+ B lymphocytes as well as M1 and M2 macrophages and d) increased pro-inflammatory interleukin (IL)-17 but reduced anti-inflammatory cytokine, IL-10. These changes were accompanied with disruption of mitochondrial membrane potential and significant increase in apoptosis and necrosis of salivary gland cells of NOD than control mice. Our collective observations suggested that GADD153 directly and/or indirectly through downregulation of Del-1 contributes importantly to salivary gland inflammation and cell death. To establish the relevance of GADD153 and Del-1 for the human condition, lower lip biopsy samples of non-SS subjects and those with a diagnosis of SS were subjected to immunohistochemistry. The results show intense GADD153 immunostaining but marked reduction in Del-1 expression in biopsy samples of SS compared to non-SS subjects. Collectively, the results indicate that GADD153 regulates inflammation and cell death in salivary gland in SS. Further, Del-1 expression likely provides a mechanistic link between increased GADD153 and leukocyte infiltration and accompanying inflammation of salivary gland tissue in this condition.

Inflammatory cytokines as predictive markers for early detection and progression of diabetic nephropathy

Diabetic nephropathy is a major complication of diabetes mellitus and the leading cause of end-stage renal disease. Both hyperglycemia and hypertension (systemic and/or intraglomerular) are established causal factors for diabetic nephropathy. Nonetheless, there is growing evidence that activated innate immunity and inflammation are also contributing factors to the pathogenesis of diabetic nephropathy. This notion is based on increasing evidence indicating that both cytokines-chemokines and pro-fibrotic growth factors are important players in the progression of diabetic nephropathy, effectively accelerating and exacerbating inflammatory and fibrotic processes leading to end-stage renal disease. In this review, we focus on several predominant cytokines-chemokines as potential predictive markers for diabetic nephropathy. These cytokines-chemokines may also be helpful as biomarkers to monitor the progression of the disease and the impact of interventional modalities aimed at halting eventual manifestation of end-stage renal disease in diabetic patients.

Aryl hydrocarbon receptor agonist, leflunomide, protects the ischemic-reperfused kidney: role of Tregs and stem cells

The aryl hydrocarbon receptor (AHR) has emerged as a major modulator of inflammatory processes. We tested the hypothesis that AHR activation protects the ischemic-reperfused kidney in association with the suppression of the inflammatory response. Accordingly, male mice were treated with the nondioxin AHR agonist, leflunomide (40 mg/kg ip); vehicle-treated animals served as controls. Thereafter, the right kidney was subjected to an ischemia (45 min)-reperfusion (4 h) insult, while the left kidney served as a sham control. Renal cells prepared from ischemic-reperfused kidneys of leflunomide-treated mice displayed preservation of mitochondrial membrane potential (Ψ(m)) and decreased apoptosis and necrosis compared with vehicle-treated ischemic-reperfused kidneys. Leflunomide treatment increased regulatory T cells (Tregs; forkhead box P3+) and IL-10-positive cells but reduced IL-17- and IL-23-expressing cells in both the peripheral blood and kidney cells, indicative of down-regulation of inflammatory responses. Leflunomide treatment also increased mobilization of stems cells subsets (i.e., mesenchymal and hematopoietic stem cells and endothelial progenitor cells) in the peripheral blood and promoted their recruitment into the ischemic-reperfused kidney. Collectively, the results indicate that AHR stimulation may represent a novel renoprotective mechanism likely involving mobilization and recruitment of Tregs and stem cells into the damaged kidney.

Renal distal tubule proliferation and increased aquaporin 2 level but decreased urine osmolality in db/db mouse: treatment with chromium picolinate.

Hallmark features of type 2 diabetes mellitus include glucosuria and polyuria. Further, renal aquaporin 2 is pivotal to regulation of fluid excretion and urine osmolality. Accordingly, we tested the hypothesis that the db/db mouse displays increased glucosuria and fluid excretion but reduced urine osmolality in association with decreased renal aquaporin 2 level. In addition, we examined the effect of chromium picolinate (Cr(pic)3) which is purported to improve glycemic control. The db/db mice excreted more urine in association with marked glucose excretion but lower urine osmolality than db/m control group. Light microscopic examination of renal tissue revealed proliferation of tubular structures in db/db compared to the db/m mice, a feature validated with Ki67 immunostaining. Further, these tubules showed generally similar immunostaining intensity and pattern for aquaporin 2 indicating that proliferated tubules are of distal origin. On the other hand, renal aquaporin 2 protein level was significantly higher in the db/db than db/m group. Treatment of db/db mice with Cr(pic)3 reduced plasma glucose and hemoglobin A1c (~15-17%, p<0.05) and Ki67 positive cells but other parameters were similar to their untreated counterparts. Collectively, these findings suggest that proliferation of renal distal tubules and increased aquaporin 2 level likely represent an adaptive mechanism to regulate fluid excretion to prevent dehydration in the setting of marked glucosuria in the db/db mouse, features not affected by Cr(pic)3 treatment. These observations are of relevance to increasing interest in developing therapeutic agents that facilitate renal glucose elimination.

Upregulation of Programmed death-1 and its ligand in cardiac injury models: Interaction with GADD153

Purpose Programmed Death-1 (PD-1) and its ligand, PD-L1, are regulators of immune/ inflammatory mechanisms. We explored the potential involvement of PD-1/PD-L1 pathway in the inflammatory response and tissue damage in cardiac injury models. Experimental Design Ischemic-reperfused and cryoinjured hearts were processed for flow cytometry and immunohistochemical studies for determination of cardiac PD-1 and PD-L1 in the context of assessment of the growth arrest- and DNA damage-inducible protein 153 (GADD153) which regulates both inflammation and cell death. Further, we explored the potential ability of injured cardiac cells to influence proliferation of T lymphocytes. Results The isolated ischemic-reperfused hearts displayed marked increases in expression of PD-1 and PD-L1 in cardiomyocytes; however, immunofluorescent studies indicate that PD-1 and PD-L1 are not primarily co-expressed on the same cardiomyocytes. Upregulation of PD-1/PD-L1 was associated with a) marked increases in GADD153 and interleukin (IL)-17 but a mild increase in IL-10 and b) disruption of mitochondrial membrane potential (ψm) as well as apoptotic and necrotic cell death. Importantly, while isotype matching treatment did not affect the aforementioned changes, treatment with the PD-L1 blocking antibody reversed those effects in association with marked cardioprotection. Further, ischemic-reperfused cardiac cells reduced proliferation of T lymphocytes, an effect partially reversed by PD-L1 antibody. Subsequent studies using the cryoinjury model of myocardial infarction revealed significant increases in PD-1, PD-L1, GADD153 and IL-17 positive cells in association with significant apoptosis/necrosis. Conclusions The data suggest that upregulation of PD-1/PD-L1 pathway in cardiac injury models mediates tissue damage likely through a paracrine mechanism. Importantly, inhibition of T cell proliferation by ischemic-reperfused cardiac cells is consistent with the negative immunoregulatory role of PD-1/PD-L1 pathway, likely reflecting an endogenous cardiac mechanism to curtail the deleterious impact of infiltrating immune cells to the damaged myocardium. The balance of these countervailing effects determines the extent of cardiac injury.

Submandibular gland and caries susceptibility in the obese Zucker rat.

BACKGROUND Obesity is a prevalent disorder characterized as marked insulin resistance and low grade inflammation. We tested the hypothesis that obesity upregulates inflammatory markers in the submandibular gland in association with derangements of its architecture and pre-disposition to caries in obese Zucker rats (OZR). We also examined the potential impact of chromium picolinate (Cr(Pic)3), a nutritional supplement suggested to improve glycemic control, on the aforementioned parameters. DESIGN Male OZR were treated with diets lacking and containing 5 or 10 mg/kg chromium (as Cr(Pic)3) from 6 weeks to about 6 months of age; lean Zucker rats (LZR) served as controls. Thereafter, glycemic status, salivary tissue architecture, and the levels of several inflammatory markers were determined in association with caries susceptibility. RESULTS OZR showed reduced insulin sensitivity, increased ratio of phospho-nuclear factor-kappa B (NF-κB) to total NF-κB, and increased intercellular adhesion molecule-1 level but similar histological features compared to LZR. Importantly, compared to LZR, OZR displayed rampant caries and a tendency for reduced dentin mineral density. Treatment of OZR with Cr(Pic)3 attenuated upregulation of these proinflammatory indicators in association with reduced severity of caries without improving insulin sensitivity. CONCLUSIONS Obesity promotes proinflammatory changes within the submandibular gland, without affecting glandular architecture, in association with rampant caries; Cr(Pic)3 treatment provided some protective effects.