Massimo Torreggiani

Induction of Diabetes in Aged C57B6 Mice Results in Severe Nephropathy : An Association with Oxidative Stress, Endoplasmic Reticulum Stress, and Inflammation

Kidney aging is a slowly progressive process that is postulated to be accelerated by intervening diseases, such as diabetes, due in part to the addition of excessive stress and inflammation from the intervening disease to the underlying aging process. This hypothesis was tested by inducing diabetes with streptozotocin in 18-month-old, aging mice. After 4 months of diabetes, these mice developed severe albuminuria, elevated creatinine levels, and renal lesions including extensive apoptotic cell death, glomerulosclerosis, afferent and efferent hyalinosis, and tubulointerstitial inflammation and fibrosis. These symptoms were associated with elevated oxidative stress. The presence of endoplasmic reticulum (ER) stress in 22-month-old diabetic kidneys resulted in up-regulation of C/EBP homologous protein (CHOP), which may play a role in increasing kidney lesions because CHOP-deficient proximal tubular cells were resistant to ER stress-induced cell death, and CHOP-deficient mice were protected from diabetic nephropathy. Moreover, CHOP-deficient mice did not develop albuminuria as they aged. Inflammation, another key component of progressive diabetic nephropathy, was prominent in 22-month-old diabetic kidneys. The expression of tumor-necrosis factor-alpha in 22-month-old diabetic kidneys may play a role in inflammation, ER stress, and apoptosis. Thus, diabetes may accelerate the underlying kidney aging process present in old mice.

Role of oxidants/inflammation in declining renal function in chronic kidney disease and normal aging

Oxidant stress (OS) and inflammation increase in normal aging and in chronic kidney disease (CKD), as observed in human and animal studies. In cross-sectional studies of the US population, these changes are associated with a decrease in renal function, which is exhibited by a significant proportion of the population. However, since many normal adults have intact renal function, and longitudinal studies show that some persons maintain normal renal function with age, the link between OS, inflammation, and renal decline is not clear. In aging mice, greater oxidant intake is associated with increased age-related CKD and mortality, which suggests that interventions that reduce OS and inflammation may be beneficial for older individuals. Both OS and inflammation can be readily lowered in normal subjects and patients with CKD stage 3-4 by a simple dietary modification that lowers intake and results in reduced serum and tissue levels of advanced glycation end products. Diabetic patients, including those with microalbuminuria, have a decreased ability to metabolize and excrete oxidants prior to observable changes in serum creatinine. Thus, OS and inflammation may occur in the diabetic kidney at an early time. We review the evidence that oxidants in the diet directly lead to increased serum levels of OS and inflammatory mediators in normal aging and in CKD. We also discuss a simple dietary intervention that helps reduce OS and inflammation, an important and achievable therapeutic goal for patients with CKD and aging individuals with reduced renal function.

AGER1 regulates endothelial cell NADPH oxidase-dependent oxidant stress via PKC-δ: implications for vascular disease

Advanced glycated end-product receptor 1 (AGER1) protects against vascular disease promoted by oxidants, such as advanced glycated end products (AGEs), via inhibition of reactive oxygen species (ROS). However, the specific AGEs, sources, and pathways involved remain undefined. The mechanism of cellular NADPH oxidase (NOX)-dependent ROS generation by defined AGEs, N(epsilon)-carboxymethyl-lysine- and methylglyoxal (MG)-modified BSA, was assessed in AGER1 overexpressing (AGER1(+) EC) or knockdown (sh-mRNA-AGER1(+) EC) human aortic endothelial (EC) and ECV304 cells, and aortic segments from old (18 mo) C57BL6-F(2) mice, propagated on low-AGE diet (LAGE), or LAGE supplemented with MG (LAGE+MG). Wild-type EC and sh-mRNA-AGER1(+) EC, but not AGER1(+) EC, had high NOX p47(phox) and gp91(phox) activity, superoxide anions, and NF-kappaB p65 nuclear translocation in response to MG and N(epsilon)-carboxymethyl-lysine. These events involved epidermal growth factor receptor-dependent PKC-delta redox-sensitive Tyr-311 and Tyr-332 phosphorylation and were suppressed in AGER1(+) ECs and enhanced in sh-mRNA-AGER1(+) ECs. Aortic ROS, PKC-delta Tyr-311, and Tyr-332 phosphorylation, NOX expression, and nuclear p65 in older LAGE+MG mice were significantly increased above that in age-matched LAGE mice, which had higher levels of AGER1. In conclusion, circulating AGEs induce NADPH-dependent ROS generation in vascular aging in both in vitro and in vivo models. Furthermore, AGER1 provides protection against AGE-induced ROS generation via NADPH.

Advanced Glycation End Product Receptor-1 Transgenic Mice Are Resistant to Inflammation, Oxidative Stress, and Post-Injury Intimal Hyperplasia

The high levels of oxidative stress (OS) and inflammation associated with cardiovascular disease are linked to pro-oxidants such as advanced glycation end products (AGEs). AGEs interact with multiple receptors, including receptor 1 (AGER1), which promotes AGE removal and blocks OS and inflammation, and RAGE, which enhances inflammation. In this study, we evaluated metabolic and vascular changes in AGER1 transgenic mice (AGER1-tg) subjected to an atherogenic diet and arterial wire-injury. Both baseline and postatherogenic diet serum and tissue AGEs as well as plasma 8-isoprostane levels were lower in AGER1-tg mice than in wild-type mice. The levels of injected (125)I-AGE in tissues were decreased as well in AGER1-tg mice. After ingesting a high-fat diet, AGER1-tg mice had a normal glucose tolerance and only 7% were hyperglycemic, whereas 53% of wild-type mice had stable hyperglycemia. After wire-injury, intimal lesions in AGER1-tg mice were small, whereas wild-type mice had diffuse intimal hyperplasia, a high intima/media ratio, and inflammatory cell infiltrates. In addition, AGER1 staining, prominent in AGER1-tg mice, was attenuated in 30 to 40% of wild-type cells, although all cells were strongly positive for AGEs. Thus, AGER1 overexpression in mice reduces basal levels of AGEs and OS, enhances resistance to diet-induced hyperglycemia and OS, and protects against injury-induced arterial intimal hyperplasia and inflammation, providing protection against OS and inflammation induced by AGEs and high-fat diets in vivo.

Identifying advanced glycation end products as a major source of oxidants in aging: implications for the management and/or prevention of reduced renal function in elderly persons.

Aging is characterized by increasing inflammation and oxidant stress (OS). Reduced renal function was present in more than 20% of normal-aged individuals sampled in the National Health and Nutrition Examination Survey (NHANES) cross-sectional study of the US population. Longitudinal studies in the United States and Italy showed that renal function does not decline in some individuals, suggesting that a search for causes of the loss of renal function in some persons might be indicated and interventions to reduce this outcome should be sought. Because advanced glycation end products (AGEs) induce both inflammation and OS, accumulate with age, and primarily are excreted by the kidney, one outcome of reduced renal function in aging could be decreased AGE disposal. The build-up of AGEs with reduced renal function could contribute to inflammation, increased oxidant stress, and accumulation of AGEs in aging. In fact, results from a longitudinal study of normal aging adults in Italy showed that the most significant correlation with mortality was the level of renal function. A clear link between inflammation, OS, AGEs, and chronic disease was shown in studies of mice that showed that reduction of AGE levels by drugs or decreased intake of AGEs reduces chronic kidney disease (CKD) and cardiovascular disease of aging. The data support a role for AGEs in the development of renal lesions in aging mice and reveal that AGEs in the diet are very important contributors to renal and cardiovascular lesions. AGEs signal through two receptors, one of which is anti-inflammatory (AGER1) and the other is proinflammatory (RAGE). Overexpression of AGER1 protects against OS and acute vascular injury. The reduction of AGEs in the diet is as efficient in preventing aging-related cardiovascular and renal lesions in mice as that seen with calorie restriction. Studies in normal adults of all ages and those with CKD suggest that the findings in mice may be directly applicable to both aging and CKD. Namely, the dietary content of AGEs determines the serum levels of AGEs and inflammatory mediators and urine AGE levels in both normal subjects and CKD patients. Importantly, reduction of AGEs controls these changes in both normal subjects and CKD patients, and the phenotypic changes in AGER1 are reduced in CKD patients by decreasing the amount of AGEs consumed with the diet. These data suggest that the changes in renal function in normal aging may be subject to control and this subject deserves renewed attention.

Risk Factors for Chronic Renal Dysfunction in Lung Transplant Recipients

Several factors predispose to renal dysfunction (RD), a common complication of solid organ transplants. We evaluated the impact of clinical and laboratory parameters on the decline of renal function in lung and heart-lung transplant recipients. We enrolled 45 patients who survived more than 6 months after transplantation, had normal renal function and urinalysis before the surgery. The prognostic value of variables for the occurrence of RD was calculated by univariate analysis. Thirty patients developed RD, defined as doubling of serum creatinine or creatinine steadily >1.5 mg/dL after a median time of 12 months. Serum creatinine above 0.9 mg/dL during the month preceding lung transplant, systolic blood pressure above 130 mmHg, and pretransplant idiopathic pulmonary hypertension were significantly associated with the development of RD. Our findings indicate that increased systolic blood pressure, reduced glomerular filtration rate, and idiopathic pulmonary hypertension are risk factors for chronic RD in lung transplant recipients.

Sirolimus Prevents Short-Term Renal Changes Induced by Ischemia-Reperfusion Injury in Rats

Background: Ischemia-reperfusion (I/R) is present at various degrees in kidney transplants. I/R plays a major role in early function and long-term survival of renal allograft. The purpose of our study was to determine if immunosuppressants modulate I/R in a model that separates I/R from all immune responses. Methods: Sprague-Dawley rats with monolateral renal I/R received daily cyclosporine (A), tacrolimus (B), sirolimus (C) or saline (D). Sham-operated rats received saline (E). After 30 days, glomerular filtration rate for each kidney was measured by inulin clearance. Kidney injury was examined, and TGF-β, fibronectin and metalloproteases were evaluated by real-time PCR, Western blot and zymography. Results: Sirolimus, but not cyclosporine and tacrolimus, prevented a glomerular filtration rate decrease in I/R kidneys (403 ± 303 vs. 1,006 ± 484 µl/min, p < 0.05; 126 ± 170 vs. 567 ± 374 µl/min, p < 0.05; 633 ± 293 vs. 786 ± 255; A, B and C group, respectively, I/R vs. contralateral kidneys). Sirolimus reduced ED-1+ cell infiltrate, interstitial fibrosis and intimal thickening of small vessels observed in I/R kidneys of controls and calcineurin inhibitor-treated rats. Tacrolimus and cyclosporine increased fibronectin and TGF-β expression and matrix deposition. Only sirolimus increased metalloprotease activity. Conclusions: Sirolimus but not calcineurin inhibitors prevented I/R-induced kidney injury.

Loss of Renal Function in the Elderly Italians: A Physiologic or Pathologic Process?

BACKGROUND Nowadays it seems that chronic kidney disease (CKD) is outbreaking, mostly in the elderly participants. The aim of this study was to assess the progression of CKD in different ages. METHODS We conducted a monocentric, retrospective, observational study enrolling 116 patients afferent to our outpatient clinic. INCLUSION CRITERIA age >18 years, follow-up ≥5 years, estimated glomerular filtration rate (eGFR) <60mL/min/1.73 m(2), and/or diagnosed renal disease and/or presence of renal damage. Patients were divided into four groups according to their age: 25-55 years (n = 27), 56-65 (25), 66-75 (42), and 76-87 (22). eGFR was calculated using the modification of diet in renal disease and the CKD-epidemiology collaboration formulas. RESULTS Younger patients had a significantly longer follow-up and less comorbidities, evaluated by the cumulative illness rating scale score, compared with the other groups. There was no difference between creatinine at baseline and at the end-of-follow-up period among the groups. Even though renal function significantly decreased in all groups, we noticed a slower progression as the age increased, and the difference between basal and end-of-follow-up eGFR was minimal in the group of patients aged 76-87 years. Analyzing the eGFR of every ambulatory control plotted against the year of follow-up, we showed a more rapid loss of filtrate in the younger group. Instead, loss of renal function decreased as the age of patients increased. CONCLUSIONS This study demonstrates that, in elderly Italian participants, progression of CKD occurs more slowly than in younger patients. This implies that we may probably face an epidemic of CKD but that most of elderly patients diagnosed with CKD may not evolve to end-stage renal disease and require renal replacement therapy.

Increased Asymmetric Dimethylarginine Serum Levels are Associated With Acute Rejection in Kidney Transplant Recipients

Asymmetric dimethylarginine (ADMA) has been identified as a marker of endothelial dysfunction and an independent risk factor for cardiovascular events in uremic subjects. This study evaluated ADMA plasma levels in kidney transplant recipients. ADMA levels were serially measured during the first year posttransplantation in 41 recipients treated with cyclosporine regimen (CY), sirolimus (SIR), or low-dose cyclosporine plus everolimus (E). Homocysteine, C reactive protein (CRP), nitric oxide (NO), and standard routine laboratory analyses were determined serially. ADMA significantly increased at 6 months posttransplantation, but was significantly lower among patients on SIR or E. NO was only slightly reduced in patients with increased ADMA levels. Interestingly, ADMA was significantly increased during the first 4 days posttransplantation in patients who experienced acute rejection during the first 6 months after transplantation. The same group of patients demonstrated higher levels of CRP and systolic blood pressure before transplantation. Our results demonstrated that ADMA was increased in patients on CY at 6 months. When increased soon after transplantation ADMA may be associated with episodes of acute rejection in kidney transplant recipients. The presence of elevated systolic blood pressure, as well as CRP and ADMA levels, suggested a role for endothelial dysfunction in the development of acute rejection episodes among deceased donor kidney transplant recipients.

Microalbuminuria and sRAGE in High-Risk Hypertensive Patients Treated with Nifedipine/Telmisartan Combination Treatment: A Substudy of TALENT

Some antihypertensive drugs have also renoprotective and anti-inflammatory properties that go beyond their effect on blood pressure. It has been suggested that microalbuminuria and glomerular filtration rate (GFR) are associated with circulating levels of the soluble form of the receptor, sRAGE (soluble receptor for advanced glycation ends-products). In the present analysis, we used data from the TALENT study to evaluate soluble receptor for advanced glycation end-products (sRAGE) plasma levels in patients with hypertension and high-cardiovascular risk-treated nifedipine and telmisartan in combination. Treatment with nifedipine-telmisartan significantly decreased mean systolic and diastolic ambulatory blood pressure and resulted in a significant increase in sRAGE plasma concentrations after 24 weeks of therapy. We concluded that in hypertensive patients with early-stage renal disease, sRAGE concentrations are not influenced by either microalbuminuria or GFR. Long-term treatment with a combination of nifedipine-telmisartan may have a beneficial effect increasing sRAGE plasma levels, thus exerting an atheroprotective and anti-inflammatory activity.