Frailty is a syndrome in which the accumulation of small, individually insignificant deficits leads to heightened vulnerability to adverse events and predisposes to potential catastrophic decompensation.

Objective of this study is to clarify the underlying genetic and immunological mechanisms responsible of frailty condition focused on: i. nephrosclerosis ageing kidney phenotype related to salt effects on immunosystem, ii. immunological aspect of sarcopenia, iii. psychological disorder related to immunosystem activation, iv. detection of new biomarkers of frailty.

The cross-sectional, observational cohort study include 1500 subjects (age >65 years) subdivided in 750 cases and 750 controls according to frailty.

Subjects who met the following criteria are included: 1) older than 65; 2) able to walk 500 m without assistance; 3) a Mini-Mental State Examination (MMSE) score <18; and 4) no severe health problems (eg, uncontrolled hypertension, recent upper or lower extremity fractures, myocardial infarction within the past 1 year).

According to definition for the 'phenotype' of frailty (cases) is as follows: presence of three or more of the following features:

1. Decreased grip strength
2. Self-reported exhaustion
3. Unintentional weight loss of more than 4.5 kg over the past year
4. Slow walking speed
5. Low physical activity

The investigators select as control subjects with presence of less than 1 criteria . All the control subjects are evaluated by the same protocol than the cases. Subjects will be collected in collaboration with senior housing (RSA) of Milano city, country clubs, social club for elderly, and elderly university. The study will be approved by the Ethics Committee of ASL of Milano city and at individual centers by local ethical committees. All the study participants signed informed consent. At the baseline evaluation, a large amount of data is collected. First, the participants completed a battery of self-reported questionnaires concerning the demographic characteristics, psychosocial adjustment, quality of life, and health condition, in the presence of a trained psychologist in order to clarify any doubts. Second, an expert in physical education and adapted physical activity for older adults administered physical tests. Finally, people with expertise in the eld of ergonomics take anthropometric measurements. Data collection is always carried out in the same order and individually for each participant.

In all the subjects following clinical and laboratoristic data are collected:

1. Clinical evaluation:

1. Collection of anamnestic data.

2. Blood pressure measurement. Blood pressure is measured by a trained nurse using an authomatic device (OMRON) and an appropriately sized cuff. Readings are obtained after 5 min of seated rest. The mean of 3 blood pressure measurements obtained at 1-min intervals during the medical evaluation is used to define SBP and DBP.

3. Evaluation of kidney function :

   a. CKD is defined as a glomerular filtration rate (GFR) lower than 60 mL/min/1.73 m2 or markers of kidney damage, such as albuminuria,for greater than 3 months. A creatinine-based formula is used to estimate GFR, applying Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations.

   b. Urine analysis and albumin /creatinine and sodium /creatinine ratio urinary excretion.

2. Physical frailty measure. To identify physically frail older adults, an adapted version of the frailty phenotype of Fried et al is used. Subjects with three or more criteria are classified as frail, those with one or two as prefrail, and those meeting none as robust.

1. Body composition: Height and weight are detected. Bioimpedance analysis (BIA) is a system of evaluation of body composition, able to calculate adjunctive information: total body water (TBW), extra and intracellular water (ECW and ICW), body cell mass (BCM), muscle mass (MM), fat mass (FM), fat free mass (FFM), basal metabolic rate.
2. SPPB: The short physical performance battery (SPPB) is a group of measures that combines the results of the gait speed, chair stand and balance tests. It has been used as a predictive tool for possible disability and can aid in the monitoring of function in older people. The scores range from 0 (worst performance) to 12 (best performance).
3. PASE: Physical Activity Scale for the Elderly (PASE) is a brief (5 minutes) and easily scored survey designed specifically to assess physical activity in epidemiological studies of persons age 65 years and older. The PASE score combines information on leisure, household and occupational activity. The PASE assesses the types of activities typically chosen by older adults (walking, recreational activities, exercise, housework, yard work, and caring for others. It uses frequency, duration, and intensity level of activity over the previous week to assign a score, ranging from 0 to 793, with higher scores indicating greater physical activity.

Sarcopenia will be defined as fat-free mass index (FFMI) below the 25th percentile combined with one of the following functional parameter: either an SPPB score ≤8 or a comfortable gait speed <1.0 m/s.

3. Psychological test will be used to assess self conditions

1. Mini Mental State Examination (MMSE): The Mini-Mental Stat Examination (MMSE) is a 10-minute measure of cognitive impairment in undeveloped, uneducated, diseased, or very old populations.
2. Geriatric Depression Scale-short form (GDS-15): The Geriatric Depression Scale (GDS) is a 15- item self-report validated measure for rating depression in elderly adults. Additionally, the GDS is sensitive to depression among elderly adults suffering from mild to moderate dementia and elderly adults with physical illnesses by excluding questions pertaining to somatic complaints and cognitive dimension of depression. In this scale, a score of 6 or higher may be indicative of depression and must be evaluated jointly with patient's clinical data.
3. SF-36 Medical Outcomes Study (MOS) 36-item Short Form Health Survey: This instrument should capture both mental and physical aspects of health. The 36 6-likert item assess health across eight domains, namely bodily pain, general health perceptions, mental health, physical functioning, role limitations due to emotional health problems, role limitations due to physical health problems, social functioning, and vitality. All items use categorical response options (range: 2-6 options). Two component summary scores for physical and mental health (MPS and MCS, respectively) can also be calculated.

4. Laboratory Methods

1. Genotyping. OpenArray technology (ThermoFisher) for genetic characterization of Single Nucleotide Polymorphisms (SNPs), based on TaqMan chemistry. DNA will be used for genotyping of genetic variants in loci for Klotho (KL) gene, Adducins (ADD1, ADD2 and ADD3) and EO synthesis (LSS, HSD3B1), metabolism (CYP11A1, ABCB1), and activity (SLC8A1, SIK1) genes, and for immunological pathway (TLR4, HMGB1, IL6, IL1).

2. Biochemical measurements.

   1. Endogenous Ouabain measurement
   2. Plasma Klotho measurement.

3. Immunological determinations. Simultaneous assessment of serum concentrations of epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), FMS-like tyrosine kinase 3 ligand (Flt-3L), granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte colonystimulating factor (GM-CSF), interferon-α2 (IFN-α2), INF-γ, IL-10, IL-15, IL-17, IL-1β, IL-2, IL-6, IL-8, INF-γ inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1β (MIP-1 β), platelet-derived growth factor-AA (PDGF-AA), soluble CD40 ligand (sCD40L), transforming growth factor alpha (TGF-α), TNF-α and vascular endothelial growth factor (VEGF).

Inclusion Criteria:

• Age ≥ 65 years
• Male and female patients
• Patients able to walk > 500 m without assistance
• MMSE < 18
• No severe health problems (Uncontrolled hypertension, recent upper or lower extremity fractures, myocardial infarction within the past 1 year).

Exclusion Criteria:

• Severe cardiological or pulmonary disease (NYHA IV, Gold IV)
• Intracranial interventions
• Dying patients (palliative situation)
• Not enough language skills
• Non-consenting patients
• Participation in another prospective intervention study for study inclusion or throughout the study period.

Community sample of the elderly population

Clinical data and sample alredy collectd are available,