**4. New technological platform for diagnostic and predictive pharmacology of aging stages and diabetes mellitus**

Timeless protein in humans (hTIM) is responsible for the production of electrical oscillations emanating from the suprachiasmatic nucleus of the hypothalamus (SCN) and determining for all circadian rhythms in the human body. This protein interacts with the key products of the activity of the oscillation genes *CLOCK, BMAL, PER1, PER2* and *PER3.* Sancar et al. investigated the role of hTIM in the work of cell cycle in humans [52]. It plays integral role in phases of G2/M and the intra-S cell cycle. In the G2/M phase of the cell cycle, hTIM binds the ATRIP subunit to the ATR protein kinase responsible for DNA damage. Binding of hTIM and ATR subsequently leads to phosphorylation of Chk1, resulting in cell cycle arrest or apoptosis. The *Timeless* gene influences to the development of human diseases. DNA damage associated with telomeres is increased in cells with reduced replication of the *Timeless* gene, along with disruption of telomere replication. Swi1 is a protein associated with the Timeless protein, which is responsible for DNA replication in the telomere region [53]. Single nucleotide polymorphism in the *Timeless* gene, which leads to the replacement of glutamine by arginine in the amino acid sequence of the protein, has not demonstrated an association with changes in morning or evening diurnal rhythms in humans [54]. The Timeless protein can be responsible for circadian rhythms in pancreatic β-cells [55]. It is believed that the Timeless protein can be identified as a

The telomeric zinc finger-associated protein (TZAP) associated with long telomeres that have low concentration of protective complex competing with TRF1 and TRF2 factors linking telomeric repeats. In telomeres, TZAP causes a purification process that leads to rapid removal of telomere repeats. The regulation of the length of telomeres in human cells has been proposed: reduced concentration of protective complex in long telomeres leads to binding of TZAP protein and initiation of telomeres purification and sets an upper limit of telomere length [57]. Telomere shortening was previously associated with the development of DM in several pilot studies and in two large studies. Zee et al. showed that telomere length was less in the study group of patients with type 2 DM than in the control group

Salpea et al. performed a study in which it was found that telomere length was less in type 2 DM and this fact corresponded to a high level of oxidative stress in these patients. Short telomeres are an independent predictor of the progression of diabetic nephropathy (DN) in patients with type 1 DM in the early onset of the disease [59, 60]. Astrup et al. showed that short telomere length is the predictor of all causes of death in type 1 DM [61]. Short telomeres were detected in arterial wall cells in patients with type 1 and type 2 DM [62]. Patients with IGT demonstrated shorter telomere length compared to healthy controls, and patients with DM and severe atherosclerosis showed the presence of shorter telomeres compared with patients with DM without atherosclerosis. The presence of obesity and insulin resistance was associated with the length of telomere leukocytes in the adult population [63]. The study found direct causal relationship between telomerase activity and insulin secretion, as well as glucose tolerance: the TERC-/- mutation showed ITG, which was caused by impaired glucosestimulated insulin secretion from pancreatic islet cells due to a decrease in pancreatic cell size

Klass showed that the life expectancy of *C. elegans* could vary with the presence of the mutation of the *Age-1* gene, and this effect is associated with caloric restriction [65]. Later,

kinase suppressor with Ras-1-like activity [56].

148 Diabetes and Its Complications

(adjusted odds ratio = 1.748) [58].

and replication damage of producing insulin β-cells [64].

Research in the field of aging and diabetology is related to recent discoveries in genomics and proteomics, new analytical equipment allows identifying biomarkers of aging and DM, the development of new drugs occurs through high-throughput screening of target molecules in human body [68].

At present, we have created unified technology platform for diagnostics and predictive pharmacology of aging and DM, taking into account interdisciplinary approach, including complex solution of problems of genomics, proteomics and metabolomics in the range of universal molecular pathways [69].

The technological platform for diagnostics and predictive pharmacology of DM includes three components taking into account a unique instrument base:


of control and prevention of aging and complications of DM in the clinic; clinical trials of drugs; economic evaluation of the platform.

Biochemistry of DM was obtained, which stimulates the development of the phenotype of cardiomyopathy and which allowed us to analyze structural and functional changes in cardiomyocytes. The cardiomyopathic phenotype was reproduced definitively in specific cells of patients and determined by the initial clinical status. *In vitro* model was included in the stages of screening platform that identifies drugs that prevent the development of the phenotype of

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Thus, the current stage in the development of biomedical research in the field of aging and DM is associated with the introduction of new technology platform for HTS of molecules in

The newest biomedical tools of the twenty-first century are biological microchips (biochips, DNA microarrays). Developed biological microchips make it possible to realize in an accessible form very complex integrative approaches of genomics, proteomics and selomics. An important medical application of biochips is the early diagnosis of DM and the development of new therapy, as well as the correlation of diagnostic markers of DM with diagnostic markers of stages of aging of the human body. Researchers conduct on-chip simultaneous analysis of tens of thousands of genes and compare the expression of these genes in healthy and diseased cells. Biochips are also an indispensable tool for biomedical research, which can in one experiment recognize the influence of various factors (drugs, proteins, nutrition) on the work of tens of thousands of genes. The effectiveness of biochips is due to the possibility of parallel carrying out a huge number of specific reactions and interactions of molecules of biopolymers, such as DNA, proteins and polysaccharides, with each other and low molecular ligands. The task is to quickly and effectively determine the concentration of the desired com-

The use of protein chips for search of markers of aging and DM is promising direction. The following two tasks are of particular interest: simultaneous qualitative and quantitative determination of large number of proteins in cells of various tissues or in various functional states; study of interactions of cellular proteins with each other and other cellular ligands (DNA, low

Bioelectronic devices are able to raise the quality of medical analyzes to new level, they will

Highly sensitive oligonucleotide microarrays were used to evaluate mRNA levels and identify transcriptional profiles of fibroblast cultures obtained from donors of different ages. The mRNA levels were measured in actively dividing fibroblasts isolated from young, middle-aged and elderly patients, as well as patients with progeria. The study identified genes, the expression of which is associated with phenotypes of different age groups and diseases. The aging process is based on a mechanism that includes an increasing number of errors in the mitosis of

The progress and development of biosensors used in the clinic was related to the development of glucose biosensor in the 1960s and obtained on the basis of early integration of the redox enzyme glucose oxidase with an oxygen electrode [72]. New materials, sensory

contribute to a one-stage definition of stages of aging and early diagnosis of DM.

human cells and the pharmacology of the aging stages and all types of DM.

pound, for example, glucose for people with DM.

dividing cells at the post-productive stage of human life [71].

cardiomyopathy [70].

molecular compounds).

Methods of genomic and proteomic analysis of human biosamples at various stages of aging and aging-related DM are used most widely in biomedical research (**Figure 2**).

Modern systems for high-throughput screening (HTS) of molecules in cellular structures (for Image-Based High Content Screening, High Content Analysis and High Content Imaging) allow to investigate the proliferation and cytotoxicity, cell viability, cell cycles, the expression of nuclear, cytoplasmic proteins and plasma membrane proteins, mitochondrial mass, phospholipidosis, signaling pathways, the increase and the decrease of nuclear sizes, apoptosis and fragmentation of nuclei. These systems allow performing complex analysis of cellular structures in real time, obtaining universal biological information about the development of aging processes and related diseases at the molecular level in the cell. Model of diabetic cardiomyopathy has been developed with the help of Operetta High Content Screening (Perkin Elmer, UK)—the system of high-performance screening of cell structures. The ways of pharmacological influence on the key targets of the development of this complication of DM have been developed. The model of this state in vitro was developed taking into account reproduced environmental conditions and genetic factors from human pluripotent stem cells of cardiomyocytes.

**Figure 2.** Molecular pattern of patient's biosample, obtained by methods of proteomic analysis (two-dimensional polyacrylamide gel electrophoresis, MALDI-TOF-MS, HPLC / MS / MS).

Biochemistry of DM was obtained, which stimulates the development of the phenotype of cardiomyopathy and which allowed us to analyze structural and functional changes in cardiomyocytes. The cardiomyopathic phenotype was reproduced definitively in specific cells of patients and determined by the initial clinical status. *In vitro* model was included in the stages of screening platform that identifies drugs that prevent the development of the phenotype of cardiomyopathy [70].

of control and prevention of aging and complications of DM in the clinic; clinical trials of

Methods of genomic and proteomic analysis of human biosamples at various stages of aging

Modern systems for high-throughput screening (HTS) of molecules in cellular structures (for Image-Based High Content Screening, High Content Analysis and High Content Imaging) allow to investigate the proliferation and cytotoxicity, cell viability, cell cycles, the expression of nuclear, cytoplasmic proteins and plasma membrane proteins, mitochondrial mass, phospholipidosis, signaling pathways, the increase and the decrease of nuclear sizes, apoptosis and fragmentation of nuclei. These systems allow performing complex analysis of cellular structures in real time, obtaining universal biological information about the development of aging processes and related diseases at the molecular level in the cell. Model of diabetic cardiomyopathy has been developed with the help of Operetta High Content Screening (Perkin Elmer, UK)—the system of high-performance screening of cell structures. The ways of pharmacological influence on the key targets of the development of this complication of DM have been developed. The model of this state in vitro was developed taking into account reproduced environmental conditions and genetic factors from human pluripotent stem cells of

**Figure 2.** Molecular pattern of patient's biosample, obtained by methods of proteomic analysis (two-dimensional

polyacrylamide gel electrophoresis, MALDI-TOF-MS, HPLC / MS / MS).

and aging-related DM are used most widely in biomedical research (**Figure 2**).

drugs; economic evaluation of the platform.

cardiomyocytes.

150 Diabetes and Its Complications

Thus, the current stage in the development of biomedical research in the field of aging and DM is associated with the introduction of new technology platform for HTS of molecules in human cells and the pharmacology of the aging stages and all types of DM.

The newest biomedical tools of the twenty-first century are biological microchips (biochips, DNA microarrays). Developed biological microchips make it possible to realize in an accessible form very complex integrative approaches of genomics, proteomics and selomics. An important medical application of biochips is the early diagnosis of DM and the development of new therapy, as well as the correlation of diagnostic markers of DM with diagnostic markers of stages of aging of the human body. Researchers conduct on-chip simultaneous analysis of tens of thousands of genes and compare the expression of these genes in healthy and diseased cells. Biochips are also an indispensable tool for biomedical research, which can in one experiment recognize the influence of various factors (drugs, proteins, nutrition) on the work of tens of thousands of genes. The effectiveness of biochips is due to the possibility of parallel carrying out a huge number of specific reactions and interactions of molecules of biopolymers, such as DNA, proteins and polysaccharides, with each other and low molecular ligands. The task is to quickly and effectively determine the concentration of the desired compound, for example, glucose for people with DM.

The use of protein chips for search of markers of aging and DM is promising direction. The following two tasks are of particular interest: simultaneous qualitative and quantitative determination of large number of proteins in cells of various tissues or in various functional states; study of interactions of cellular proteins with each other and other cellular ligands (DNA, low molecular compounds).

Bioelectronic devices are able to raise the quality of medical analyzes to new level, they will contribute to a one-stage definition of stages of aging and early diagnosis of DM.

Highly sensitive oligonucleotide microarrays were used to evaluate mRNA levels and identify transcriptional profiles of fibroblast cultures obtained from donors of different ages. The mRNA levels were measured in actively dividing fibroblasts isolated from young, middle-aged and elderly patients, as well as patients with progeria. The study identified genes, the expression of which is associated with phenotypes of different age groups and diseases. The aging process is based on a mechanism that includes an increasing number of errors in the mitosis of dividing cells at the post-productive stage of human life [71].

The progress and development of biosensors used in the clinic was related to the development of glucose biosensor in the 1960s and obtained on the basis of early integration of the redox enzyme glucose oxidase with an oxygen electrode [72]. New materials, sensory configurations and technical innovations have been proposed for the determination of glucose [73–75]. Currently, glycemic control is based on the study of blood glucose, which still requires frequent blood sampling with a certain degree of inconvenience. Despite the intensive application of the existing method of blood glucose testing, precise monitoring of glucose fluctuations during the day cannot yet be performed. Therefore, today, subcutaneous biosensors are used, which measure glucose periodically during the day [76, 77]. However, the creation of an accurate implantable biosensor for glucose, acceptable for patients with DM, is an open question. Today, the most promising results were obtained on biosensors, which are based on amperometric detection of hydrogen peroxide formed by enzymes immobilized on electrodes. Updike et al. created a biosensor for glucose, implanted subcutaneously, with maximum duration of up to 5 months [78].

interactions—Ingenuity Pathway Analysis (IPA) and STRING—is published. Data about key proteins, participating in molecular pathological pathways (Wnt, APP, insulin signaling, mitochondrial apoptosis, tau-phosphorylation), were obtained on the basis of medical lit-

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Information about protein interactions in complexes is found in databases such as MINT, BioGRID, IntAct or HRPD. It is possible to provide high percentage of predicted protein-protein interactions and interactions based on literature data (PubMed database). Widely used web resource for the analysis of inter-protein interactions STRING is not only a database but also linked to several other resources with large volume of literature sources [81]. The Cytoscape graphical tool allows us to create network interactions of high degree of complexity. Recently, web platform has been launched that integrates data on molecular pathways for the development of pathological processes and the analysis of intermolecular interactions, including six different databases (KeGG, Bio-Carta, Gene Ontology, Reactome, Wiki, NCI pathways) and interacts functionally with database on molecular activity of proteins (Interpro) and database of complex information about pro-

Identification of the protein in the study of the biosample should be accompanied by a detailed analysis of its primary, secondary and tertiary structure, as well as its post-translational modifications and intermolecular interactions (BLAST search engine). The amino acid sequence of the protein can be analyzed in software products such as Pfam, Interpro, SMART or DAVID [82], whereas sequence analysis of post-translational protein modifications can be performed

Modern methods of proteomic and bioinformatic analysis allow us to investigate key genomic-proteomic interactions that underlie DN in patients with type 2 DM. The study of the formation and development of DN can become the model for studying molecular path-

We carried out prospective comparative cohort study with parallel design for the search of molecular prognostic markers of DN of different stages using methods of proteomics and bioinformatic analysis on the basis of the Department of Nephrology of the Dagestan State Medical University (Makhachkala, Dagestan, Russia) and the Department of Nephrology of the Rostov State Medical University (Rostov-on-Don, Russia), MC "Novomeditsina" (Rostovon-Don, Russia) [85]. It included 205 patients with T2DM and DN (stages 1–4). Patients corresponded to the criteria for the DN classification proposed by the Committee on Diabetic Nephropathy [86]. The duration of DN was 10.5 years. Molecular phenotyping of biosamples (urine) was processed with methods of proteomics: the prefractionation, the separation of proteins with standard sets (MB-HIC C8 Kit, MB-IMAC Cu, MB-Wax Kit, «Bruker», USA) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS, Ultraflex II, «Bruker», USA). The partially identified sequences were then submitted to "BLAST protein-protein" and screened against the *Homo sapiens* Swissprot database to check if this identification matched the MASCOT-identification (Matrix Science). The data of the molecular interactions and functional features of proteins were received with STRING

erature and data of proteomic analysis of the HEK293 cell line.

using algorithms such as MotifX or PhosphoMotif [83].

ways of aging of kidney tissue [84].

teins (Corum).

10.0 database.

Interstitial levels of lactate should reflect its systemic level when hypoxia appears in the tissues. There is an opportunity to control lactate *in vivo* in the interstitium; however, it is difficult to introduce monitoring methods into the clinic because of their unreliability due to the influence on the level of lactate of a lot of endogenous factors. Several types of biosensors for the determination of lactate are presented in the literature. A microfluidic biochip was developed, which is integrated with a highly sensitive fiber-optic biosensor of glucose. Experimental results showed that the biochip determines an ultra-low glucose concentration (1 nM) [79]. Due to the fact that DM as an aging-related disease progresses and due to complex and stepwise processes of malfunctioning of the pancreatic β-cells at the molecular level that can be registered in the blood, early detection of DM requires the use of supersensitive systems for detecting molecular changes. To this end, a protein microchip was developed, including the use of polyfluorophor technology. The innovative system is characterized by high sensitivity: the possibility of the determining of biomarkers at the level of femtograms in 10 μl of the biosample is 92% within 10 minutes [80].

It is believed that the stages of aging and related diseases, including DM, are characterized by their bar code—a change in the level of transcription of a set of genes specific for this disease. It is assumed that in the future, according to the barcode, changes in the expression in particular set of genes can be diagnosed with specific diseases and the stages of their development and, consequently, develop targeted treatment regimen.

At present, bioinformatics is ready to provide data about tens of thousands of new drug targets, predicting the function of genes and deciphering the sequence of proteins. Promising bioinformatic developments are presented in such sections of medicine as gerontology and diabetology. The main directions of bioinformatics are distinguished, depending on the objects of study: sequence bioinformatics, structural bioinformatics and computer genomics. The main task of bioinformatics in the development of new drugs is to provide technologies that allow the formation of target targets for directed action of drug having specific structure for this target.

Today, an extended analysis is available, including the molecular pathways presented in most databases; so, large array of information about altered proteins can be obtained, including their expression and/or post-translational modifications in molecular pathways. Comparative analysis of two web products for the analysis of pathways and intermolecular interactions—Ingenuity Pathway Analysis (IPA) and STRING—is published. Data about key proteins, participating in molecular pathological pathways (Wnt, APP, insulin signaling, mitochondrial apoptosis, tau-phosphorylation), were obtained on the basis of medical literature and data of proteomic analysis of the HEK293 cell line.

configurations and technical innovations have been proposed for the determination of glucose [73–75]. Currently, glycemic control is based on the study of blood glucose, which still requires frequent blood sampling with a certain degree of inconvenience. Despite the intensive application of the existing method of blood glucose testing, precise monitoring of glucose fluctuations during the day cannot yet be performed. Therefore, today, subcutaneous biosensors are used, which measure glucose periodically during the day [76, 77]. However, the creation of an accurate implantable biosensor for glucose, acceptable for patients with DM, is an open question. Today, the most promising results were obtained on biosensors, which are based on amperometric detection of hydrogen peroxide formed by enzymes immobilized on electrodes. Updike et al. created a biosensor for glucose, implanted subcutaneously, with

Interstitial levels of lactate should reflect its systemic level when hypoxia appears in the tissues. There is an opportunity to control lactate *in vivo* in the interstitium; however, it is difficult to introduce monitoring methods into the clinic because of their unreliability due to the influence on the level of lactate of a lot of endogenous factors. Several types of biosensors for the determination of lactate are presented in the literature. A microfluidic biochip was developed, which is integrated with a highly sensitive fiber-optic biosensor of glucose. Experimental results showed that the biochip determines an ultra-low glucose concentration (1 nM) [79]. Due to the fact that DM as an aging-related disease progresses and due to complex and stepwise processes of malfunctioning of the pancreatic β-cells at the molecular level that can be registered in the blood, early detection of DM requires the use of supersensitive systems for detecting molecular changes. To this end, a protein microchip was developed, including the use of polyfluorophor technology. The innovative system is characterized by high sensitivity: the possibility of the determining of biomarkers at the level of femtograms in

It is believed that the stages of aging and related diseases, including DM, are characterized by their bar code—a change in the level of transcription of a set of genes specific for this disease. It is assumed that in the future, according to the barcode, changes in the expression in particular set of genes can be diagnosed with specific diseases and the stages of their development

At present, bioinformatics is ready to provide data about tens of thousands of new drug targets, predicting the function of genes and deciphering the sequence of proteins. Promising bioinformatic developments are presented in such sections of medicine as gerontology and diabetology. The main directions of bioinformatics are distinguished, depending on the objects of study: sequence bioinformatics, structural bioinformatics and computer genomics. The main task of bioinformatics in the development of new drugs is to provide technologies that allow the formation of target targets for directed action of drug having specific structure

Today, an extended analysis is available, including the molecular pathways presented in most databases; so, large array of information about altered proteins can be obtained, including their expression and/or post-translational modifications in molecular pathways. Comparative analysis of two web products for the analysis of pathways and intermolecular

maximum duration of up to 5 months [78].

152 Diabetes and Its Complications

10 μl of the biosample is 92% within 10 minutes [80].

and, consequently, develop targeted treatment regimen.

for this target.

Information about protein interactions in complexes is found in databases such as MINT, BioGRID, IntAct or HRPD. It is possible to provide high percentage of predicted protein-protein interactions and interactions based on literature data (PubMed database). Widely used web resource for the analysis of inter-protein interactions STRING is not only a database but also linked to several other resources with large volume of literature sources [81]. The Cytoscape graphical tool allows us to create network interactions of high degree of complexity. Recently, web platform has been launched that integrates data on molecular pathways for the development of pathological processes and the analysis of intermolecular interactions, including six different databases (KeGG, Bio-Carta, Gene Ontology, Reactome, Wiki, NCI pathways) and interacts functionally with database on molecular activity of proteins (Interpro) and database of complex information about proteins (Corum).

Identification of the protein in the study of the biosample should be accompanied by a detailed analysis of its primary, secondary and tertiary structure, as well as its post-translational modifications and intermolecular interactions (BLAST search engine). The amino acid sequence of the protein can be analyzed in software products such as Pfam, Interpro, SMART or DAVID [82], whereas sequence analysis of post-translational protein modifications can be performed using algorithms such as MotifX or PhosphoMotif [83].

Modern methods of proteomic and bioinformatic analysis allow us to investigate key genomic-proteomic interactions that underlie DN in patients with type 2 DM. The study of the formation and development of DN can become the model for studying molecular pathways of aging of kidney tissue [84].

We carried out prospective comparative cohort study with parallel design for the search of molecular prognostic markers of DN of different stages using methods of proteomics and bioinformatic analysis on the basis of the Department of Nephrology of the Dagestan State Medical University (Makhachkala, Dagestan, Russia) and the Department of Nephrology of the Rostov State Medical University (Rostov-on-Don, Russia), MC "Novomeditsina" (Rostovon-Don, Russia) [85]. It included 205 patients with T2DM and DN (stages 1–4). Patients corresponded to the criteria for the DN classification proposed by the Committee on Diabetic Nephropathy [86]. The duration of DN was 10.5 years. Molecular phenotyping of biosamples (urine) was processed with methods of proteomics: the prefractionation, the separation of proteins with standard sets (MB-HIC C8 Kit, MB-IMAC Cu, MB-Wax Kit, «Bruker», USA) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS, Ultraflex II, «Bruker», USA). The partially identified sequences were then submitted to "BLAST protein-protein" and screened against the *Homo sapiens* Swissprot database to check if this identification matched the MASCOT-identification (Matrix Science). The data of the molecular interactions and functional features of proteins were received with STRING 10.0 database.


**Protein name** β2-microglobulin

Podocin

MCP-1 **Table 1.**

Qualitative profile of urine proteins in T2DM patients with DN.

11 *P*CG-1 = 0.011

39 *P*CG-2 = 0.000

48 *P*CG-3 = 0.000

46 *P*CG-4 = 0.000

1

2583

23 *P*CG-1 = 0.000

45 *P*CG-2 = 0.000

63 *P*CG-3 = 0.000

49 *P*CG-4 = 0.000

1

42,201

Podocyte-specific protein, interact

with the PI3K/AKT-signaling pathway

for maintenance of functional integrity

Chemotactic factor for monocytes;

regulates the memory T lymphocytes,

NK cells; increases with TNFα and

IL-6

in damaged kidneys

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6 *P*CG-1 > 0.05

37 *P*CG-2 = 0.000

62 *P*CG-3 = 0.000

49 *P*CG-4 = 0.000

1

11,774

The indicator of incipient DN;

detecting injured epithelial cells in the

proximal tubules

**Group 1 (n = 42)**

**Group 2 (n = 48)**

**Group 3 (n = 65)**

**Group 4 (n = 50)**

**Control group** 

**MW (Dа)**

**Functional process (sources: InterPro,** 

**Entrez, SWISS-PROT, NRDB, PDB,** 

**KEGG**

**(n = 30)**


**Protein name**

TGF-β1 E-cadherin

Cystatin C Collagen IV

MMP 9 Fibronectin

NGAL Ceruloplasmin

12 *P*CG-1 = 0.006

37 *P*CG-2 = 0.000

42 *P*CG-3 = 0.000

35 *P*CG-4 = 0.000

1

122,205

Marker of damaged glomerulus

10 *P*CG-1 = 0.043

42 *P*CG-2 = 0.000

62 *P*CG-3 = 0.000

48 *P*CG-4 = 0.000

1

22,588

Kidney development; it loses through

the damaged glomerulus, injured

tubular cells produce NGAL as a

compensatory mechanism against

intracellular oxidative stress and

complement- induced apoptosis.

4 *P*CG-1 > 0.05

35 *P*CG-2 = 0.000

52 *P*CG-3 = 0.000

43 *P*CG-4 = 0.000

1

262,625

Adhesive glycoprotein, locally

stimulated mesangial and epithelial

cell production

8 *PC*G-1 > 0.05

32 *P*CG-2 = 0.000

60 *P*CG-3 = 0.000

49 *P*CG-4 = 0.000

1

78,458

Potent modulator of ECM turnover

and also of shedding of syndecans

4 *P*CG-1 > 0.05

32 *P*CG-2 = 0.000

48 *P*CG-3 = 0.000

42 *P*CG-4 = 0.000

1

164,038

Constituent of mesangial matrix,

marker of the phase of compromised

renal filtration function

15 *P*CG-1 = 0.001

40 *P*CG-2 = 0.000

60 *P*CG-3 = 0.000

45 *P*CG-4 = 0.000

1

15,799

Cysteine proteinase inhibitor, tubular

damage marker

22 *P*CG-1 = 0.000

38 *P*CG-2 = 0.000

62 *P*CG-3 = 0.000

49 *P*CG-4 = 0.000

1

97,456

The regulation of tubular EMT; the

maintenance of epithelial integrity,

cell phenotype; the progression of

renal fibrosis

18 *P*CG-1 = 0.001

27 *P*CG-2 = 0.000

52 *P*CG-3 = 0.000

48 *P*CG-4 = 0.000

2

44,341

Pro-fibrotic and anti-inflammatory

154 Diabetes and Its Complications

activities, the regulation of tubular

EMT

**Group 1 (n = 42)**

**Group 2 (n = 48)**

**Group 3 (n = 65)**

**Group 4 (n = 50)**

**Control group** 

**MW (Dа)**

**Functional process (sources: InterPro,** 

**Entrez, SWISS-PROT, NRDB, PDB,** 

**KEGG**

**(n = 30)**

> **Table 1.** Qualitative profile of urine proteins in T2DM patients with DN.

All changes in patients with DN are associated with a higher expression of urine proteins in the progression of epithelial-to-mesenchymal transition (EMT) and changes in the extracellular matrix (ECM) in kidneys in T2DM patients with DN (**Table 1**). Proteomic analysis helps in the detection of differences in the component composition of the urine proteins in patients with DN of varying stages compared with the control group. Molecules interact among themselves and with other molecules as participants in universal pathways in T2DM patients with DN, which are the key elements for EMT formation and changes in ECM: Smad, p38 MAPK, TLRs, Wnt, mTOR, Notch, small GTPase and Hedgehog and PI3K/AKT-signaling pathways.

Thus, the future progress in biomedical research of the aging stages of the human body and DM is associated with the development of experimental genomics, transcriptomics, proteomics and selomics and with the development of typical human development scenario, starting from the postnatal period on the basis of modern technological platforms. The development of methods for the systematic analysis of molecular interactions in cell and the subsequent study of their functional activity makes it possible to discover new pathways for the

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Medical Centre "Novomeditsina,"Rostov State Medical University, Rostov-on-Don, Russia

[1] American Diabetes Association. Standards of medical care in diabetes. Diabetes Care.

[2] International Diabetes Federation. IDF Diabetes Atlas – 7th edition. Available at: https:// www.idf.org/sites/default/files/EN\_6E\_Atlas\_Full\_0.pdf [Accesed: 1 Feb 2016]

[3] Silink M. UN Resolution 61/225: 'World Diabetes Day'; A United Nations Resolution on Diabetes—The Result of a Joint Effort. US Endocrinology. 2007;**12**(1):12-14. DOI:

[4] Dedov I, Shestakova M, Mayorov A. Standards of specialized diabetes care. Diabetes

[5] Morley J. Diabetes and aging: Epidemiologic overview. Clinics in Geriatric Medicine.

[6] Ford E, Giles W, Dietz W. Prevalence of the metabolic syndrome among US adults: Findings from the third National Health and nutrition examination survey. JAMA.

[7] Smirnov V, Freidlin I. Immunodeficiency Conditions. St. Petersburg: Foliant; 2000. p. 568p.

[8] Zatz M, Goldstein A.Thymosins, lymphokines, and the immunology of aging. Gerontology.

[9] Poveshchenko A, Orlov N, Kazakov O, Poveshchenko O, Kim I, et al. Age and gender differences in cytokine profile of lymph and blood serum. Advances in Aging Research.

2017;**40**(Supplement 1):S4-S5. DOI: https://doi.org/10.2337/dc17-S003

Mellitus. 2017;**20**(1S):1-112. DOI: 10.14341/DM20171S8

2008;**24**(3):395-405. DOI: 10.1016/j.cger.2008.03.005. Review

2002;**287**:356-359. DOI: https://doi:10.1001/jama.287.3.356

1985;**31**(4):263-277. DOI: https://doi.org/10.1159/000212709

DOI: https://doi:10.1016/j.ijantimicag.2015.03.001

2014;**3**:216-221. DOI: 10.4236/aar.2014.33030

development of human pathology associated with aging.

Address all correspondence to: isarvilina@mail.ru

10.17925/USE.2007.00.1.12

**Author details**

Irina Sarvilina

**References**

Each protein molecule in the functional group interacts with other protein molecules. For example, the molecular interactions of NGAL are presented in **Figure 3**. The concentration of NGAL increases in the urine of T2DM patients with DN. The study identified the biomarkers of tubular damage that have a key role in the development and progression of DN. The research into signaling pathways and molecules that are involved in ECM formation may help in developing strategies to prevent DN. Molecular pathways for the development of DN constitute a model for the study of molecular pathways in the development of aging of kidney tissue.

**Figure 3.** Molecular interactions of NGAL (STRING 10.0 database). LCN2, lipocalin-2; MMP-9, matrix metallopeptidase 9; LRP2, low density lipoprotein-related protein 2; ERBB2, erythroblastic leukemia viral oncogene homolog 2 (neuro/ glioblastoma derived oncogene homolog); IL3, interleukin 3 (colony-stimulating factor, multiple); HMOX1, heme oxygenase (decycling) 1; IL-17A, interleukin 17A; LEP, leptin; INS, insulin; TLR2, toll-like receptor 2 and CDH1, cadherin 1, type 1, E-cadherin (epithelial cadherin).

Thus, the future progress in biomedical research of the aging stages of the human body and DM is associated with the development of experimental genomics, transcriptomics, proteomics and selomics and with the development of typical human development scenario, starting from the postnatal period on the basis of modern technological platforms. The development of methods for the systematic analysis of molecular interactions in cell and the subsequent study of their functional activity makes it possible to discover new pathways for the development of human pathology associated with aging.
