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• PATHOPHYSIOLOGY OF DIABETIC NEPHROPATHY.
• Dr.Abhijit Kishore Korane
• APOLLO HOSPITALS,
• CHENNAI.

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• Definition,
• A metabolic disorder of multiple aetiology
  characterized by chronic hyperglycaemia with
  disturbances of carbohydrate, fat and protein
  metabolism resulting from defects in insulin
  secretion, insulin action or both
• Associated with a risk of developing late
  diabetic complications including
• Microvascular (retinopathy,nephropathy,neuropathy)
  
• Macrovascular (atherosclerosis)

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In 1500 BCDiabetes First Described In Writing

• Hindu healers wrote that flies and ants were
  attracted to urine of people with a mysterious
  disease that caused intense thirst, enormous
  urine output, and wasting away of the body

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250 BCThe Word Diabetes First Used

• Apollonius of Memphis coined the name "diabetes
  meaning "to go through" or siphon. He understood
  that the disease drained more fluid than a person
  could consume.
• Gradually the Latin word for honey, "mellitus,"
  was added to diabetes because it made the urine
  sweet.

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DIABETES
Top Three Countries in the world
57 million
19 million
1995
2025
King et al, Diabetes Care, 1998
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Increasing mortality from diabetes mellitus
J. Olefsky, JAMA 2001285628-632
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• PATHOPHYSIOLOGY OF DIABETIC NEPHROPATHY IS
  MULTIFACTORIAL

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• Genetic determinants of diabetic nephropathy

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• Familial clustering,
• Type I diabetic patients with and without
  diabetic nephropathy, and found 83 of diabetic
  siblings of probands with nephropathy had
  evidence of nephropathy, and only 17 in diabetic
  siblings of probands without nephropathy.
• (Seaquist, E. R., Goetz, F. C., Rich, S. and
  Barbosa, J. (1989) Familial clustering of
  diabetic kidney disease. Evidence for genetic
  susceptibility to diabetic nephropathy. N. Engl.
  J. Med. 320, 11611165)

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• Familial clustering of renal disease in Type II
  diabetes,nephropathy is observed in 46 of
  diabetic offspring if both parents have
  proteinuria, 23 if one parent has proteinuria,
  and 14 if neither have proteinuria.

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• The role of hyperglycemia in the pathogenesis of
  diabetic nephropathy

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HYPERGLYCEMIA
Overloading of mitochondrial ETC
Super oxide radicles
DNA damage
DNA repair enzyme PARP
Polyol pathway
Hexoaminase pathway
Activates Glyceraldehyde 3 phosphate dehydrogenase
PKC pathway
AGE endproducts
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• ADVANCED GLYCATION END PRODUCTS
• AGEs accumulate as a result of natural aging in
• human beings.
• Role of advanced glycation is-
• - Identify senescent proteins for
  degradation.

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• In diabetes, AGE formation is enhanced by
  persistent hyperglycemia and oxidative stress.
• It leads to modification of,
• 1) long-lived proteins such as skin collagen,
• 2) short-lived proteins also become targets
  for advanced glycation.

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• Levels of circulating AGE levels in diabetic
• patients may be a reflection of both
• Endogenously formed and exogenously ingested
  AGEs(AGE content is high in cooked and processed
  foods, especially those rich in proteins, fat,
  and sugar.)

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• GENESIS AND STRUCTURE OF AGES

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Binding of glucose non-enzymatically to the amine
group of proteins, lipids and nucleic acids
Involves the condensation of the free aldehdye
group of a sugar with an e-amino group of lysine
of the protein.
Chemically reversible glycosylation product is
formed, known as a Schiff base
Undergoes rearrangementto form an Amadori
product, which is also known as an early
glycation product.
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Amador Rearrangement
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Amino compound
Open chain D-glucose
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Open chain D-glucose
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Open chain D-glucose
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-
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• This species is unstable and will lose water to
  produce the open chain form of the glycosylamine
  with a C-N double bond

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Loss of water
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Water of dehydration
Open chain glycosylamine
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• Rearrangement of this compound will yield the
  Amador compound. This sequence of reactions is
  known as the Amador rearrangement.

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Open chain glycosylamine
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Open chain glycosylamine
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Open chain Amadori compound
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• Attack by O-6 on the carbonyl group will close
  the ring producing a 1-deoxy-1-amino-D-fructopyran
  ose compound (the Amador product)

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Open chain Amadori compound
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The Amadori compound (a 1-deoxy-1-amino-D-fructopy
ranose)
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Amadori products
Dissociate
Slow chemical rearrangements.
Production of irreversible AGE
Maillard reaction.
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• The Mail lard reaction

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The Maillard reaction

• The Maillard reaction (browning reaction) is the
  responsible for turning meat brown, converting
  bread to toast and turning beer brown.
• The Maillard reaction is named for Louis-Camille
  Maillard, a French chemist who studied the
  science of browning during the early 1900s.

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• AGE-peptides are filtered by the glomerulus and
  catabolized in part by the endolysosomal system
  of the proximal convoluted tubule.
• Reabsorption could represent an
  AGE-receptor-mediated mechanism triggering
  several cell responses including cytokine
  secretion and oxidation reactions.

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• A most striking effect of Amadori-glycated serum
  proteins is the induction of glomerular
  hyperfiltration, an early functional abnormality
  implicated in the development of diabetic
  nephropathy.

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How does AGE lead to diabetic nephropathy?
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• How does AGE lead to diabetic nephropathy?
• 1) Modification of intracellular proteins.
• 2) Formation of covalent cross-links.
• 3) Interaction with AGE receptors

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• 1) Modification of intracellular proteins
• The most fundamental means of AGE induced damage
  to the kidney is via the non-enzymatic glycation
  of intracellular proteins, with consequent
  effects on cell function.

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Nonenzymatic glycosylation of intracellular
proteins in endothelial cells
Incressed expression of inflammatory mediators
bFGF
Atherosclerotic process and vascular damage
Endothelial cell growth
Impaired endothelial structure and function.
Diabetic nephropathy.
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• 2) Formation of covalent cross-links

AGE precursors can diffuse out of the cell
Form irreversible covalent cross-links between
extracellular matrix proteins e.g. collagen and
laminin
Leads to- 1) Promotes fibrosis, 2) Increased
stiffness of the protein matrix which impedes
proteolytic degradation affecting
tissue remodelling. 3) Disruption of cell-matrix
interaction, leading to increased protein
permeability in the glomerular basement
membrane
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• Cross-links form between
• glycated ECM proteins
• leading to structural
• alterations like,
• -changes in surface charge,
• -membrane permeability,
• -resistance to proteolytic digestion,
• -thermal stability.
• - sclerosis of the renal glomeruli,

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• 3) Interaction with AGE receptors,

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• AGE receptors are expressed on various cell types
  such as,
• - monocytes, macrophages.
• - endothelial cells, mesangial cells,
• - podocytes, tubular epithelial cells,
• - astrocytes, microglia,
• - smooth muscle cells

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• In the human kidney, RAGE protein is found in
• - Tubular epithelial cells,
• - Mesangial cells,
• - Podocytes,
• - Vascular and neural compartments.
• In diabetes, RAGE expression is increased at
• sites of macrovascular and microvascular injury.

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• AGEs also mediate their effects via receptors
• - Receptor for AGE (RAGE),
• - Macrophage scavenger receptor types I and
  II (types A and B1/CD36),
• -Oligosaccharyl transferase- 48 (AGE-R1),
• -80K-H phosphoprotein (AGE-R2),
• - galectin-3 (AGE-R3),
• -CD-36,
• -ezrin, radixin, and moesin proteins
  megalin also bind AGEs.

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• RAGE is a multiligand member of the
• Immunoglobulin superfamily with 394 amino
• acids, a single hydrophobic transmembrane
• domain (19 amino acids), and a highly charged
• COOH-terminal cytosolic tail (43 amino acids)
• that mediate intracellular signaling pathways.
• Extracellularly, RAGE has a terminal V-type
• ligand binding domain and 2 C-type domains
• (V-C-C)

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• RAGE binding by AGEs activates diverse signal
• transduction cascades including
• -p21ras, p38, p44/p42,
• -protein kinase/c-Jun N-terminal kinase
  -Mitogenactivated protein (MAP) kinases, -The
  Janus kinase/signal transducers and
  activators of transcription(STAT)
• -Protein kinase C (PKC) pathway.

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ACTIVATION OF RAGE
Activation of transcription factors, nuclear
factor kappa B (NF-B).
Release of proinflammatory cytokines, growth
factors
1)Transforming growthfactor-1 (TGF-1), 2)
Vascular endothelial growth factor, 3)
Connective tissue growth factor, 4) Interleukin-1
and -6, insulin-like growth factor-1, platelet-der
ived growth factor, tumor necrosis factor (TNF)-,
and vascular cell adhesion molecule (VCAM)-1
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• OTHER AGE RECEPTORS,
• AGE-R1 may have a protective effect against
  AGE-induced injury.
• In diabetic kidney disease, AGE-R1 expression is
  suppressed in human beings.
• In mesangial cells, up-regulation of AGE-R1
  enhances AGE removal and down-regulates RAGE and
  downstream signaling pathways such as NF-B
  activity and MAP kinase phosphorylation, whereas
  downregulation of AGE-R1 increases AGE-induced
  MAP kinase activation.

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• AGE-R2 or P90 is involved in the intracellular
  signaling of various receptors, including the
  fibroblast growth factor receptor.

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• AGE-R3 or galectin-3 is a 32-kd protein that
  binds to carbohydrates, laminin, and
  immunoglobulin E and is associated with several
  cellular functions including activation,
  inflammation, tumor growth activity, and
  apoptosis.

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• Oxidative Stress and Antioxidant Defence

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• Oxidative stress is defined as the excess
  formation or insufficient removal by antioxidant
  defenses of highly reactive molecules including
  ROS and reactive nitrogen species (RNS).

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• ROS include free radicals such as
• superoxide (O2-), hydroxyl (HO-), peroxyl (O2-),
  hydroperoxyl (HO2-), and nonradical species such
  as hydrogen peroxide (H2O2) and hydrochlorous
  acid(HOCl).
• RNS include free radicals such as nitric oxide
  (NO-) and nitrogen dioxide(NO2-), and nonradicals
  such as peroxynitrite (ONOO), alkyl
  peroxynitrates (RONOO), and nitrous oxide (HNO2).

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• The major free radical implicated in diabetic
  complications is O2-, which can be produced by
  various sources including the mitochondrial
  electron transport chain (ETC) during normal
  oxidative phosphorylation, by,
• - NADPH reduced oxidase,
• - xanthine oxidase, cyclooxygenase,
• - lipoxygenase, cytochrome P-450, and - nitric
  oxide synthase.

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• Effects of the various ROS include,
• Oxidation of macromolecules like lipids, DNA,
  proteins, and carbohydrates.
• - Oxidants increase signaling molecules such as
  p38 or c-Jun N-terminal kinase MAP kinases.

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Oxidative Stress in Diabetes
Nonenzymatic sources
Enzymatic sources
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• Nonenzymatic sources
• Nonenzymatic sources of oxidative stress
• induced by diabetes includes,
• - glucose auto-oxidation,
• - advanced glycation,
• - polyol pathway,
• - mitochondrial ETC.

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• Primary initiating event in the development of
  diabetic complications is O2- formation by
  mitochondria,

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• Hyperglycemia induces changes in the
  mitochondrial voltage gradient by increasing
• electron donors of the ETC or via uncoupling
• protein-1.
• - Hyperglycemia may inhibit adenosine
  triphosphate (ATP) synthase, slowing electron
  transfer and ATP synthesis, leading to an excess
  of electrons that would combine with molecular O2
  to form O2-

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• Enzymatic sources,
• NAD(P)H oxidase is a major source of cellular O2-
  and is an important source of vascular O2- in
  both nondiabetic and diabetic patients.
• Changes in the antioxidants enzymes GPx,
  catalase, CuZnSOD, and MnSOD also may contribute
  to oxidative stress in diabetes.

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• INTERPLAY BETWEEN AGES AND ROS.
• Oxidative stress may facilitate both the
  formation of intracellular AGEs and cross linking
  in diabetes
• (Fu MX, Knecht KJ, Thorpe SR, et al. Role of
  oxygen in cross-linking and chemical modification
  of collagen by glucose. Diabetes. 199241 Suppl
  242-8.)

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• Converse occurs with AGE formation triggering ROS
  production.
• AGEs induce decreases in the activities of
  antioxidant enzymes such as SOD and catalase,
  decreases glutathione stores, or can directly
  stimulate ROS production.

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• POTENTIAL INTERVENTIONS FOR DIABETIC COMPLICATIONS

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• AGE formation inhibitors,
• These agents act in a variety of ways
• - trapping of reactive carbonyl and dicarbonyl
• compounds,
• chelation of transition metal ions,
• direct inhibition of the conversion of Amadori
  intermediates to AGEs

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• AGE formation inhibitors have been described
• - Aminoguanidine,
• - ALT-946,
• - Pyridoxamine,
• - OPB-9195.
• (Coughlan MT. Can advanced glycation end product
  inhibitors
• modulate more than one pathway to enhance
  renoprotection in
• diabetes? Ann N Y Acad Sci. 20051043750-8.)

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• Aminoguanidine,
• a nucleophilic hydrazine compound that inhibits
  formation of AGEs. via binding to early glycation
  products, dicarbonyl intermediates, and aldehyde
  products.
• Aminoguanidine has been shown to slow the
  development of diabetic complications including
  nephropathy.
• (Forbes JM et al. Renoprotective effects of a
  novel inhibitor of advanced glycation.
  Diabetologia. 200144108-14.)

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• Aminoguanidine interferes with several important
  regulatory systems(NO synthatase activity)and
  toxic side effects were observed with use of this
  agent in clinical trials. Thus, it has been
  discontinued for further clinical development.
• (Freedman BI, Wuerth JP, Cartwright K, et al.
  Design and baselin
• characteristics for the aminoguanidine Clinical
  Trial in Overt Type 2 Diabetic
• Nephropathy (ACTION II). Control Clin Trials.
  199920493-510.)

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• Angiotensin- converting enzyme inhibitors, have
  been identified as potent inhibitors of AGE
  formation and it is postulated that at least some
  of the nonhemodynamic renoprotection conferred by
  angiotensin-converting enzyme inhibitors may
  involve effects on AGE accumulation.
• (Forbes JM, Thorpe SR, Thallas-Bonke V, et al.
  Modulation of soluble receptor for advanced
  glycation endproducts by angiotensin-converting
  enzyme-1 inhibition in diabetic nephropathy. J Am
  Soc Nephrol.2005162363-72.)

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• AGE cross-link breakers
• AGE cross-link breakers are compounds that
• reduce AGE accumulation by cleavage of
• Preformed AGE-mediated cross-links.
• AGE cross-link breakers include,
• - N-phenacylthiazolium bromide (PTB),
• - Alagebrium chloride,
• - 4,5-Dimethyl-3-(2-oxo2-phenylethyl)
• thiazolium chloride (ALT-711).

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• ALT-711 has been reported to attenuate renal
  injury in experimental Diabetes and is deemed
  safe in human clinical trials in other
  nondiabetic diseases.
• (Forbes JM, Thallas V, Thomas MC, et al. The
  Breakdown of preexisting advanced
• glycation end products is associated with
  reduced renal fibrosis in experimental
• diabetes. FASEB J. 2003171762-4.)

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• Inhibitors of AGE binding
• Inhibitors of AGE receptor ligand binding include
• soluble RAGE and RAGE-specific neutralizing
• antibodies, which have been used in both in vivo
• and in vitro studies to block the biological
• Effects of RAGE.

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• RAGE-specific neutralizing antibodies
  administered to diabetic mice prevent
  diabetes-induced renal changes including
  mesangial expansion and albuminuria.
• (Flyvbjerg A, Denner L, Schrijvers BF, et al.
  Long-term renal effects of a neutralizing RAGE
  antibody in obese type 2 diabetic mice. Diabetes.
  200453166-72.)

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• TARGETING ROS,
• A large number of experimental studies have been
  performed using a range of antioxidants to assess
  their potential actions as renoprotective agents.
• This has included the use of vitamins C and E and
  alpha-lipoic acid.

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• Diabetic rats treated with the ROS scavenger
  nitecapone normalized,
• - urinary sodium excretion and oxidative stress
  parameters,
• - prevented hyperfiltration,
• - focal glomerulosclerosis,
• - reduced albuminuria,
• - activation of glomerular PKC activity.
• (Lal MA, Korner A, Matsuo Y, et al. Combined
  antioxidant and COMT inhibitor treatment reverses
  renal abnormalities in diabetic rats. Diabetes.
  2000491381-9.)

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• The potential beneficial effects of antioxidant
  therapy in human beings remain controversial.

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• Type 1 diabetic patients with highdose vitamin E
  supplementation have normalized baseline retinal
  blood flow and creatinine clearance, suggesting a
  role in improving retinal hemodynamics and renal
  function in diabetic patients.
• (Bursell SE, Clermont AC, Aiello LP, et al.
  High-dose vitamin E supplementation normalizes
  retinal blood flow and creatinine clearance in
  patients with type 1 diabetes. Diabetes Care.
  1999221245-51.)

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• It remains to be determined if such a strategy,
  potentially targeting mitochondrial ROS
  generation, may be useful in patients with
  diabetic nephropathy.

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• POLYOL PATHWAY

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HYPERGLYCEMIA
SORBITOL (ALDOSE REDUCTASE)
Utilises NADPH
FRUCTOSE (SDH)
PENTOSE PHOSPHATE PATHWAY
ACTIVATES PROTEIN KANASE C
INCRESSED PROSTAGLIANDIN
GLOMERULAR HYPERFILTERATION
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• Modification of protein kinase C activity

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• Incressed PKC leads to-
• 1)Decressed Na/KATPase
• or gene expressions of extracellular matrix
  components and contractile proteins.
• 2) Changes in retinal and renal blood flow,
  contractility, permeability, and cellproliferation

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• Transforming growth factor b contributes to
  progressive diabetic nephropathy

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Role of TGF-ß

• Stimulates extracellular matrix synthesis
• Inhibits extracelluular matrix degradation
• Up regulates protease inhibitors down regulates
  matrix degrading enzymes
• Stimulates synthesis of integrins (matrix
  receptors)
• Key role in glomerular and tubuloepithelial
  hypertrophy, basement membrane thickening, and
  mesangial matrix expansion.

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• Angiogenesis in Diabetic Nephropathy

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• Neovascularization has been implicated in the
  genesis of diverse diabetic complications such as
  retinopathy, impaired wound healing, neuropathy,
  diabetic nephropathy.

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THE JAK/STAT PATHWAY IS NECESSARY FOR THE
ANGIOTENSIN II-MEDIATED KIDNEY MESANGIAL CELL
GROWTH
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• JAK/STAT Activation
• Upon ligand stimulation, receptors undergo
  conformational changes.
• These changes attract JAKs which subsequently are
  activated by trans-phosphorylation.
• Phosphorylated JAKs once activated phosphorylate
  downstream signaling molecules such as STATs.

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• Several studies have suggested that JAKs
  associate with growth factor (e.g. Insulin, EGF
  and PDGF) and with G-protein coupled (e.g.
  Angiotensin II) receptors.
• These associations and JAKs activation enables
  these receptors to activate the STATs.

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Angiotensin II
S100B
High Glucose
RAGE
AT1
High Glucose
PLC-gamma1
Aldose Reductase
O2
ROS
PLD2
NADPH oxidase
Polyol Pathway
SHP-1 and SHP-2
DAG
PKC-beta
JAK2 and STATs
Cell Growth
High Glucose and S100B augmentation of the Ang
II-induced JAK/STAT pathway in kidney mesangial
cell growth.
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• Endothelial Cell Dysfunction Leading to Diabetic
  Nephropathy role of Nitric Oxide

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• Extracellular Matrix Metabolism in Diabetic
  Nephropathy

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• Diabetic nephropathy is characterized by
  excessive deposition of extracellular matrix
  proteins in the mesangium and basement membrane
  of the glomerulus and in the renal
  tubulointerstitium.
• ROS may activate intracellular signaling pathways
  leading to incressed expression of genes encoding
  extracellular matrix proteins and the protease
  systems responsible for their turnover.

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• THANK YOU