Mechanisms of Diabetic Nephropathy in Ecuador

1. Determinations of social, environmental and biological factor implicated in diabetic nephropathy in Ecuador Investigators will study subjects from the general population. Investigators will contact community and religious leaders in order to invite adult subjects from the general population to participate in this study and 10000 persons will be enrolled.

   After teaching basic concepts about prevention of diabetic nephropathy in all subjects, investigators will collect demographics data, house conditions and localization (rural, urban or around the cities), number of people that share a home, education level, employment and economical condition, personal health insurance, medical checkup history, toxic habits (alcohol, cigarette abuse of addictive substances) and physical exercises habits. Investigators will collect personal and family antecedent of diabetes mellitus, diabetic nephropathy, hypertension, kidney diseases, diabetes gestational, current treatments and other chronic diseases. Investigators will measure body weight (Kg), height (m) and investigators will calculate body mass index. Investigators will also measure blood pressure, blood glucose, glycosylated hemoglobin, microalbuminuria and proteinuria. Trained personnel will fill out the person's survey. Investigators will supervise and collect data from 2% of the sample.

2. Role of extracellular matrix proteins and growth factors signaling in diabetic nephropathy

   1. Systemic and urine studies: proteins involved in diabetic nephropathy pathogenesis as vascular endothelial factor A (VEGF) and extracellular matrix protein as tenascin C (tenascin) concentration will be measured by a commercially available kits in plasma and urine (respectively). Samples will be obtained from diabetics with (n: 20) and without diabetic nephropathy (n: 20) and from non-diabetic subjects (controls) from the general population (n: 2000). Determinations of social, environmental and biological factor will be completed as in aim 1.
   2. Kidney tissue studies: investigators will determine the role of protein involved in diabetic nephropathy, VEGF signaling and tenascin in:

I) Paraffin embedded kidney tissue obtained from the sample libraries of pathology laboratories will be studied. Slides from non-diabetics (n: 10) and diabetics with diabetic nephropathy (n: 20) and without diabetic nephropathy (n: 10) will be collected. The role of proteins involved in diabetic nephropathy as VEGF signaling and tenascin will be studied by immunohistochemistry and proximity link assay.

II) Paraffin embedded kidney slides from diabetics with diabetic nephropathy (n: 12), non-diabetic control (n: 6) with primary glomerulopathy as minimal change disease, and diabetic controls without diabetic nephropathy (n: 6), will be studied. Investigators will recruit patients with kidney biopsy indicated, and scheduled to be performed, by their medical doctors. After pathology diagnosis completion, some of the remnant paraffin embedded tissue will be used for this study. The role of proteins involved in diabetic nephropathy, VEGF signaling and tenascin will be studied by immunohistochemistry and proximity link assay. Urine and plasma will be collected prior to the kidney biopsy in order to measure protein concentration of potentially useful biomarkers of diabetic nephropathy; VEGF and tenascin levels will be quantified. Patients will be informed and signed consent will be required.

Blood glucose will be measured by the blood glucose meter and test strip. Glycosylated hemoglobin A1c and microalbuminuria (albumin/creatinine in spot urine) will be quantified using the portable machine (Siemens).

In fasting conditions investigators will consider normal blood glucose values \<99 mg%, pre-diabetes between 100 and 125 mg% and diabetes ≥126 mg%. In non-fasting conditions values of blood glucose >200mg% will be considered as diabetes. Investigators will deem normal glycosylated A1c hemoglobin values \<5.6 %, pre-diabetes between 5.7 and 6.4% and diabetes ≥6.5%.

Investigators will deem normal albuminuria values of albumin/creatinine ratio \<29mg/g, microalbuminuria values between 30 and 299 mg/g and macro-albuminuria values ≥300mg/g. Proteinuria will also be evaluated by reactive urine strips.

In the case of inconsistent results or out of range values the analysis will be repeated in the same sample or re analyzed by standard laboratory equipment and techniques.

Investigators will consider diabetic nephropathy as persistent proteinuria associated to diabetic retinopathy and decreased endogen glomerular filtration rate (in subjects without a diagnosis of other kidney or urinary tract diseases). In Type 1 diabetics, diabetic nephropathy will be diagnosed if the history of diabetes is longer than 10 years prior to the onset of proteinuria; in diabetic type 2, proteinuria associated to a decreased glomerular filtration rate at diagnosis time, investigators will deem diabetic nephropathy.

In all paraffin embedded kidney samples we will measure the protein expression and localization. Investigators will use kits, primary and secondary antibodies commercially available to perform immunohistochemistry and proximity link assay. Investigators will perform those experiments in cooperation with pathologist.

All subjects recruited from general population will be identified by a unique code. Number and capital letters code will be placed in the survey and in the sample labels as M0001 (M: city, number 0001: person number 0001). Data will be uploaded and saved in a secure data base. Trained personnel will be in charge of handling the data base. Investigators will supervise the data entry. An investigator at least once every 6 months will randomly select 2% survey and will match it against the information uploaded in the database. Furthermore, investigators will analyze and report all collected data from the surveys even if they are incomplete by adjusting the analysis parameters of the incomplete ones.

Investigators will use descriptive statistic tools to describe groups (mean, standard deviation and standard error or median and interquartile range). For group comparisons, unpaired T test, Mann Whitney or Fisher's test will be used. ANOVA, Kruskal Wallis and Chi Square test will be performed for to compare unmatched groups; to quantify association between two variables Pearson and Spearman correlation test will be used. Simple or multiple regressions will be considered to predict value from variables. P value \<0.05 will be deemed significant. A sample from the general population was calculated considering the adult population between 21-70 years old in Ecuador.