1. HDAC inhibitors have been shown to be renoprotective in diabetes by preventing the accumulation of extracellular matrix proteins in the glomerular mesangium and tubulointerstitium. 2. Histone hyperacetylation by the nonselective HDAC inhibitor trichostatin A prevents TGF-β1-induced downregulation of E-cadherin and upregulation of collagen I in human renal proximal tubular epithelial cells. 3. TGF-β1 expression is increased in experimental diabetic animal models and in human diabetic nephropathy, and plays a key role in the development of renal changes through inducing epithelial-mesenchymal transition and extracellular matrix accumulation.

1. Presented by
Nitin Kshirsagar
M.S. (Pharm.) second semester
Under the guidance of
Dr. G.B. Jena
Assistant professor
Department of Pharmacology and Toxicology
National Institute of Pharmaceutical Education and Research (NIPER)
Mohali

2. Flow of presentation
Introduction
Literature review
Hypothesis
Study objectives
Study design
Material and Methods
Expected outcomes
1

3. Introduction
A microvascular diabetic complication characterized by renal pathological alterations
that leads to renal dysfunction
Occurs in 25-40% patients of diabetes
MacIsaac, et al. (2014).Am. J. Kidney Dis 63: S39-S62.
Epigenetics involves changes in gene expression and function without a
corresponding alteration in DNA sequence
Histone deacetylases brings about deacetylation of histones thereby HDACi are
one of the epigenetic modifiers, currently being tested as therapeutic interventions for
cancer in clinical trials
Recent data from Hyonam Kidney Laboratory and others have demonstrated
antifibrotic and renoprotective effect of HDAC inhibitors in diabetic kidneys
Epigenetic basis of diabetic nephropathy
Diabetic nephropathy
2

4. Literature review
 Most common complication among diabetic patients also called DKD
 The global increase in the prevalence of diabetes leads to acceleration of micro-
and macrovascular complications
 The important causative factor in the development of complications in patients
with diabetes is hyperglycemia
Afkarian, M (2014) Pediatr Nephrol: 1-10.
The points of concern:
I. Progression of diabetic kidney disease to end-stage renal disease (ESRD)
leading to increased CVS morbidity and mortality
II. Prevalence in middle and low income countries
 With central role of hyperglycemia, the diabetic complications like nephropathy
leads to dysfunctioning of renal system
Diabetes and nephropathy
3

5. Glycation of proteins
Formation of AGE
Nonenzyma
tic pathways
Sorbitol pathway
Hexosamine pathway
Activation of PKC
Metabolic
pathways
General pathophysiology
4

6. Pathophysiology of renal damage
• Renal hemodynamic alterations
• Glomerular structural changes
• Tubular structural changes
Early Renal
hemodynamic
alterations
Single nephron hyperfiltration
Increased Glomerular blood flow
Increased intra-glomerular pressure
• Increased glomerular basement permeability
• Decreased glomerular filtration
Late renal
hemodynamic
alterations
5

7. Bleasel, AF, Yong, LCJ (1982) Experientia 38: 129-130.
Cont.
Tubular structural changes
Glomerular structural changes
Armanni-Ebstein lesions- accumulation of fluid and glycogen granules
in the cytoplasm
Tubular basement membrane thickening
Tubulointerstitial fibrosis - late structural change
Glomerular hypertrophy
Mesangial matrix expansion
Nodular diabetic glomeruloesclerosis (Kimmelstiel-Wilson nodules)
6

8. Epigenetic regulation by histone deacetylases
Histones
 Octamers
 Wraps about 146 base pairs of DNA
Exist as : H1,H5, H2A, H2B, H3 and
H4 , H1 and H5 are known as the
linker histones, H1,H5 has the
highest lysine/arginine ratio
Modification brings about
epigenetic control
Acetylation of lysine residue of histone decreases its positive charge ultimately
decreasing tight interaction with DNA. These processes are regulated by two groups of
enzymes i.e. HATs and HDACs.
HDACHAT
7

9.  Classified according to functional and phylogenetic criteria, there are 18
HDACs, Classified into four classes
HDAC and HDACi
Khan, O, La Thangue, NB (2012) Immunol Cell Biol 90: 85-94
Zn++ dependent
• Class I, II, IV
• Class II translocates
NAD+ dependent
• Class III
• SIRT 1-7
 Based on chemical nature, HDACis are classified as follows:
 Although roughly classified, mostly pan-selective
8

10. HDAC inhibitors in DN
Recently a number of potential mechanisms have been proposed for the action of
HDACis
HDACs overexpression in DN leads to,
HB Lee et al., Kidney International (2007) 72, S61–S66
Proinflammatory and profibrotic
proteins,
eg. vascular endothelial growth factor, -smooth
muscle actin (a-SMA), extracellular matrix
proteins,TGF-β, CTGF,
Antifibrotic cytokine,
eg. E-cadherin, bone morphogenetic protein-7
The mechanisms for HDAC activity are pleiotropic and likely to be cell context-
dependent, HDACI are believed to act by selective inhibition of HDAC thus regulating
protein expression
9

11. Cont…
↑VEGF
↑ Collagen
↑ MCP
↑ ROS
Fibronectin
Increased
Proinflammatory
cytokines
HDAC 
Mesangial
expansion
Renal fibrosis
Tubular
inflammation
Glomerular
hypertrophy
Renal complications
10

12. Sodium phenylbutyrate as a HDAC inhibitor
Sodium salt of aromatic fatty acid
White to beige in color, salty taste
Molecular weight: 186
Solubility : ≥ 5mg/ml
pKa : 4.76
Stable at the room temperature
Peak plasma levels of
phenylbutyrate occur within 1
hour after a single dose.
SBP gets converted in vivo into
phenylacetate (PAA) by β-oxidation
Classified as class I and II selective HDAC
inhibitor
As HDAC
inhibitor
11

13. cont..
Phenylbutyrate can suppress the expression of the glucose-regulated protein 78 (an ER stress
indicators) further suppresses the expression of inflammatory cytokines
It inhibited the increase in urinary protein excretion and urinary monocyte chemoattractant
protein–1 (MCP-1) 1
It is reported to suppress NADPH oxidase activity, NF-κB activity, and expression of
nitrotyrosine, Nrf2 (NF-E2-related factor) in renal tissue2
In radiation therapy-induced skin damage PBA found to be protective ,by inhibiting ER stress
and unfolded protein response (UPR) signaling3
1Wei Qi et al., metabolism 60 (2011) 594–603,
2Zhi-Feng Luo et al., Toxicol Appl Pharmacol 246 (2010) 49–57, 2009,
3Wang, Jian-Qing, et al., Toxicol Appl Pharmacol 266.2 (2013): 307-316
4Srinivasan, K, and Sharma, S, Behav Brain Res 225.1 (2011): 110-116
In type 2 diabetes sodium phenylbutyrate ameliorated focal cerebral ischemic/reperfusion
injury by reducing endoplasmic reticulum stress and DNA fragmentation 4
12

14. Hypothesis
13

15. ↑ Transcription of profibrotic proteins
(TGF-β,CTGF,VEGF)
↑ Mesangial collagen Renal fibrosis
Diabetic nephropathy
AGE
ROS
NF-
Diacylglycerol
PKC-
↑ HDAC activity
Hyperglycemia
Sodium phenylbutyrate
14

16. Objectives
I. To evaluate the efficacy of sodium phenylbutyrate in
dose dependent manner in type-1 diabetic nephropathy
II. To evaluate the efficacy of sodium phenylbutyrate in
time dependent manner in type-1 diabetic nephropathy
15

17. Study design
 Minimum 3 weeks period is required for development of DN after induction
of type-1 diabetes by STZ¹
Tesch, Greg H., and Terri J.Allen Nephrology 12.3, (2007) 261-266
 To evaluate the efficacy of Sodium phenyl butyrate in streptozotocin
induced DN (4 weeks model)
 Group 1: Normal control (normal saline, oral.)
 Group 2: Diabetic control (STZ, 55mg/kg, single i.p inj.)
 Group 3: Sodium phenylbutyrate control (200mg/kg Sodium
phenylbutyrate, i.p.)
 Group 4: Diabetic + Sodium phenylbutyrate (100mg/kg Sodium
phenylbutyrate, i.p.)
 Group 5: Diabetic + Sodium phenylbutyrate (200mg/kg Sodium
phenylbutyrate i.p)
16

18.  II. To evaluate the efficacy of Sodium phenylbutyrate in streptozotocin
induced DN (8 weeks model).
 Group 1: Normal control (normal saline, oral.)
 Group 2: Diabetic control (STZ, 55mg/kg, single i.p inj.)
 Group 3: Sodium phenylbutyrate control (200mg/kg Sodium
phenylbutyrate, i.p.)
 Group 4: Diabetic + Sodium phenylbutyrate (100mg/kg Sodium
phenylbutyrate, i.p.)
 Group 5: Diabetic + Sodium phenylbutyrate (200mg/kg Sodium
phenylbutyrate i.p)
17

19. Material and methods
 Animals
 Juvenile, male​ rats
 Induction and validation of diabetic model
 Using single dose of STZ (55 mg/kg)​
 Animals are starved overnight before STZ treatment
 Confirmation
By estimation of glucose by GOD-POD method, after 48 hrs of STZ injection,
(glucose level more than 250 mg/dl)
17

20. Methods of evaluation
• Albumin ,Creatinine,BUNBiochemical markers:
• Malondialdehyde (MDA) level, GSH assayDetermination of
oxidative stress:
• COMET assay (single cell gel electrophoresis)DNA damage:
• Differential staining, immunofluorescenceHistopathology:
• Western blottingMolecular targets
18

21. Statistical analysis
Results will be expressed as mean ±SEM for each
group
Statistical differences between the groups will be
determined by one-way ANOVA
Followed by multiple comparisons with Tukey’s test
using Sigma Stat statistical software
The level of statistical significance will be set at P <
0.05
19

22. Expected outcome of the study
Different level of damage may be characterized in diabetic rats
Protection at each level can be elucidated
Molecular targets may be evaluated
20

23. THANK YOU
21

24. 24
DATA SUGGESTING THAT HDAC INHIBITORS ARE
RENOPROTECTIVE IN DIABETES
Progressive accumulation of ECM in glomerular mesangium and
tubulointerstitium is the hallmark of diabetic nephro-pathy.17 Epithelial–
mesenchymal transition of renal tubular epithelial cells, characterized by
loss of epithelial cell characteristics and gain of myofibroblast
characteristics, is an important mechanism involved in tubulointerstitial
fibrosis.18 TGF-b1 is the major mediator inducing epithe-lial–mesenchymal
transition19,20 and ECM accumulation,21,22 and has been recognized as
the final common pathway to
Histone hyperacetylation by a nonselective HDAC in-hibitor trichostatin A (TSA)
was recently shown to prevent TGF-b1-induced downregulation of E-cadherin and
upregulation of collagen I in human renal proximal tubular epithelial cells in
association with induction of Id2 and bone morphogenetic protein-7 mRNAs,
inhibitors of TGF-b1 signals.
It is observed that TSA effectively inhibited TGF-b1-induced fibronectin, collagen I,
and a-SMA upregulation and E-cadherin downregulation in normal rat kidney
epithelial cells (NRK-52E) (Ha H, Noh H, Yu MR et al.
HB Lee et al., Kidney International (2007) 72, S61–S66

25. 25
Recently, the increased glomerular TGF-b1 expression in
experimental animals [37] and in human diabetic nephropathy
[37,38] has been reported. The involvement of TGF-b1 in the
development of early renal changes could be shown by using
neutralizing anti-TGF-b1 antibodies [39]. Important to note,
Clinica Chimica Acta Volume 297 issue 1-2 2000 [doi
10.1016%2Fs0009-8981%2800%2900240-0] Rainer Lehmann;
Erwin D Schleicher -- Molecular mechanism of diabetic
nephropathy
Some studies suggest that they promote the
accumulation of ROS in tumor cells but induce
the ROS scavenger
thioredoxin in non-transformed normal cells

26. Hyperglycemia
↑ Renin ↑ Ang-II ↑ Aldosterone ↑ NADPH ↑ PKC
↑VEGF
Mesangial
expansion
↑ Collagen
Renal fibrosis
↑ MCP
Tubular
inflammation
↑ ROS
Oxidative stress
↑ TGF-β
Glomerular
hypertrophy
Fibronectin
Diabetic
nephropathy
26

27. 27
Flyvbjerg A. Putative pathophysiological role of growth factors and
cytokines in experimental diabetic kidney disease. Diabetologia 2000;
43: 1205–23. TGF-BETA INHIBITION

28. 28

29. 29
Approved
Vorinostat
Romidepsin
Clinical trials
Started phase III clinical trials
Panobinostat
Valproic acid -muscular atrophy.
Started pivotal phase II clinical trials
Belinostat
Mocetinostat
Abexinostat
Entinostat in phase II for Hodgkin lymphoma, lung cancer and breast cancer

30. 30
Histone acetylation with alterations in gene expression
that effect cell cycle arrest and limit cell growth, including
up-regulation of genes such as p21, p27, and other genetic
markers of differentiation; and down-regulation of genes
involved in growth such as cyclin D (22–24);
2. Acetylation of nonhistone proteins such as p53, HIF-
1alpha, pRb, STAT-3, Rel A/p65, or estrogen receptor that
mayimpair their function and thereby influence cell
growth or survival (25–27);
3. Acetylation of Hsp90, with its attendant loss of ability to
chaperone client proteins resulting in their ubiquitinylation
and proteasomal degradation (28, 29);
4. A prometaphase cell cycle arrest that results from reduced
premitotic phosphorylation of pericentromeric histone
H3 and disruption of kinetochore assembly(30);
5. An antiangiogenic effect potentiallymediated byimpairing
HIF-1a stability(31);
6. Direct activation of apoptotic pathways through reduction
of antiapoptotic proteins such as Bcl-2 and increased
expression of proapoptotic proteins such as BAX and BAK
(32, 33);
7. Enhanced production of reactive oxygen species (ROS)
(refs. 34, 35);
8. Disruption of aggresome formation through acetylation of
tubulin (36);
9. Enhanced antitumor immunitythrough enhancement of
TRAIL or up-regulation of antigen expression that could
facilitate cancer cell recognition (37–40);
10. Disruption of DNA repair through acetylation or downregulation
of proteins such as Ku70, Ku86, BRCA1, and
RAD51 (41–43).