# Presentation: 155418

## Slide 1

## INTRODUCTION

** ****Renal failure is a common and serious complication of longstanding diabetes mellitus.**
** ****Diabetes is now the most common cause of end-stage renal failure requiring dialysis.**
** ****Diabetes accounts for almost 40% of all new dialysis patients.**

## INTRODUCTION (continuation) 

**The incidence of renal failure caused by diabetes is rising dramatically in the United States especially in minorities (e.g. African Americans, Hispanics and Native Americans).  **
** ****This tragedy is compounded by the grim reality that the survival of patients with renal failure caused by diabetes is much worse than that of patients with renal failure resulting from other causes.**

## DIABETIC NEPHROPATHY

**Diabetic nephropathy refers to a characteristic set of structural and functional kidney abnormalities that occur in patients with diabetes which leads to End Stage Renal Disease (ESRD). **

## Structural abnormalities include :

_**Structural abnormalities include**_** :**
**hypertrophy of the kidney **
**Increase in the thickness of glomerular basement membranes**
**Accumulation of extracellular matrix components in the glomerulus**
**Tubular atrophy and interstitial fibrosis.**

##  Thus, excessive cellular growth in the glomeruli is a major contributor to pathological changes associated with diabetic nephropathy.

**  ****Furthermore, high glucose-induced growth of glomerular mesangial cells is a characteristic feature of diabetes-induced renal complications. **

## Slide 7

##  Glomerular mesangial cells are responsive to traditional growth factors.  

** ****However, in diabetes this occurs in the context of an environment enriched in both circulating vasoactive mediators and high glucose.**

##  The vasoactive peptide Angiotensin II (ANG II) has been implicated in the pathogenesis of diabetic renal disease.

** ****Recent findings suggest that both high glucose and ANG II activate intracellular signaling processes leading to growth which include the JAK/STAT Pathway**

##  For the last few years we have been actively investigating the importance of the JAK/STAT cell-signaling cascade in the progression of  Diabetic Nephropathy.

## Members of the JAK/STAT family

**    **_**JAKs**_
**JAK1**
**JAK2**
**JAK3**
**Tyk2**

**    **_**STATs**_
**STAT1**
**STAT3**
**STAT5 A/B**
**STAT6**
**STAT2**
**STAT4**

##  There are four members of the JAK family in mammals, Jak1, Jak2, Jak3 and Tyk2. 

** ****They are over 1000 amino acids in length, ranging in molecular weight from 120 to 130 kDa. **
** ****Jak1, Jak2 and Tyk2 are expressed ubiquitously, whereas the expression of Jak3 is restricted to cells of the myeloid and lymphoid lineages**** **

## Slide 13

## 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. **

##  Several studies have  suggested that JAK’s associate with growth factor (e.g. Insulin, EGF and PDGF) and with G-protein coupled (e.g. Angiotensin II) receptors.

** ****These associations and JAK’s activation enables these receptors to activate the STAT’s.**

## Slide 16

## THE JAK/STAT PATHWAY IS NECESSARY FOR THE ANGIOTENSIN II-MEDIATED KIDNEY MESANGIAL CELL GROWTH

## Hypothesis

**Activation of the JAK/STAT signaling cascade by Angiotensin II is altered by hyperglycemia in glomerular mesangial cells.**

## Methods

**GMC were isolated by collagenase digestion of the rat glomeruli.  **
**Cells from passages 1-6 were grown in DMEM:**
**Normal Glucose (5.5 mM) (NG)**
**High Glucose (25.0 mM) (HG)**
**Normal Glucose + High Mannitol (19.5 mM)**
**Stimulated with 100 nM Ang II for various times**

## Tyr-P-JAK2 

**0       1      5     10    30    60   min**

**JAK2**

**0      1      5      10    30    60 **

**NG**

**HG**

**NG**

**HG**

**HG effects on Ang II-induced JAK2 activation in mesangial cells**

**Ang II incubation time (minutes)**

**Notes:**
Figure 1

## HG effects on Ang II-induced STAT activation in mesangial cells

**HG effects on Ang II-induced STAT activation in mesangial cells**

**Ang II incubation time (minutes)**

##     0     1      3     6    12   24     

**sense**

**antisense**

**JAK2**

**Time, hours**

## Slide 23

## Slide 24

## Diabetes-induced changes

- Altered regulation of the vasoactive peptide angiotensin II receptor and its early growth signaling pathways ( e.g. JAK/STAT).
- Increased growth  (accumulation of extracellular matrix proteins (e.g., collagen) and proliferation of kidney mesangial cells.

## Conclusion.  

**Angiotensin II-induced activation of the JAK/STAT pathway ****	**
**	****1.****	****Enhanced under HG conditions *****in vitro*****.**
**	****2.****	****HG-induced growth (DNA and ****	****	****	****	****collagen IV synthesis) are blocked by JAK2 ****	****	****antisense.**
**These results provide evidence that activation of the JAK/STAT pathway by HG or/and Ang II may be of importance in the increased growth in the matrix proteins, collagen IV synthesis and cell proliferation that is seen in diabetic nephropathy.**

## What are the Molecular Mechanisms by which high glucose augments the Angiotensin II-induced activation of the JAK/STAT pathway?

## Effects of High Glucose

**High Glucose**

**Aldose Reductase**
**Pathway**

**Increases**
**NADH:NAD**
**Ratio**

**DAG synthesis**

**PKC**

**ROS**

**AGE**
**(S100B)**

**RAGE**

## Effects of zopolrestat on the HG or S100B  augmentation on the Ang II-induced JAK2 tyrosine phosphorylation

**Ang II incubation time (minutes)**

**pJAK2**

**S100B**

**with zopolrestat **

**Normal Glucose**

**High Glucose **

**Effects of zopolrestat on the HG or S100B  augmentation on the Ang II-induced JAK2 tyrosine phosphorylation**

**JAK2**

**0       5      10**

**pJAK2**

**JAK2**

**with zopolrestat**

**no zopolrestat**

**no zopolrestat**

**0       5      10**

## Effects of electroporating rabbit IgG or anti-p47phox  antibodies on the HG or S100B augmentation on the Ang II-induced JAK2 tyrosine phosphorylation

**anti-IgG**

**anti-p47phox**

**pJAK2**

**Normal Glucose**

**High Glucose**

**S100B**

**anti-IgG**

**anti-p47phox**

**pJAK2**

**JAK2**

**JAK2**

**  ****0      5      10**

** ****0       5      10**

**0       5      10**

**Ang II incubation time (minutes)**

## 0          5        10 

**Ang II incubation time (minutes)**

**S100B (5ug/ml)**

**S100B (5ug/ml) + rabbit IgG (100 ug/ml)**

**S100B (5ug/ml) + anti-RAGE (70 ug/ml)**

**Control**

**pJAK2**

**Effects of anti-RAGE on the S100B effects on the Ang II-induced tyrosine phosphorylation of JAK2.**

**Control**

**S100B (5ug/ml)**

**S100B (5ug/ml) + rabbit IgG (100 ug/ml)**

**pJAK2**

**pJAK2**

**pJAK2**

**JAK2**

**JAK2**

**JAK2**

**S100B (5ug/ml) + anti-RAGE (70 ug/ml)**

**JAK2**

## Effects of 1-Butanol or 2-Butanol on the S100B effects on the Ang II-induced tyrosine phosphorylation of JAK2.

**S100B (5ug/ml)**

**S100B (5ug/ml) + 0.2% 1-Butanol**

**S100B (5ug/ml) + 0.2% 2-Butanol**

**0         5         10**

**Ang II incubation time (minutes)**

**pJAK2**

**Effects of 1-Butanol or 2-Butanol on the S100B effects on the Ang II-induced tyrosine phosphorylation of JAK2.**

**Control**

**S100B (5ug/ml)**

**S100B (5ug/ml) + 0.2% 1-Butanol**

**S100B (5ug/ml) + 0.2% 2-Butanol**

**pJAK2**

**pJAK2**

**pJAK2**

**JAK2**

**JAK2**

**JAK2**

**JAK2**

## sense

**sense**

**antisense**

**0       2       6       12    24     **

**PLD2**

**Time, hours**

## Effects of PLD2 antisense on the S100B effects on the Ang II-induced tyrosine phosphorylation of JAK2.

**Control**

**S100B (5ug/ml)**

**S100B (5ug/ml) + PLD2 antisense**

**S100B (5ug/ml) + PLD2 sense**

**0         5         10**

**Ang II incubation time (minutes)**

**pJAK2**

**Control**

**S100B (5ug/ml)**

**S100B (5ug/ml) + PLD2 antisense**

**S100B (5ug/ml) + PLD2 sense**

**pJAK2**

**pJAK2**

**pJAK2**

**JAK2**

**JAK2**

**JAK2**

**JAK2**

**Effects of PLD2 antisense on the S100B effects on the Ang II-induced tyrosine phosphorylation of JAK2.**

## Effects of PLD2 antisense on the S100B and Ang II-induced cell proliferation.

**Effects of PLD2 antisense on the S100B and Ang II-induced cell proliferation.**

## Effects of PKC-betainhibitor LY379196 on the Ang II-, HG- and S100B-induced H2O2 production.

**Effects of PKC-beta********inhibitor LY379196 on the Ang II-, HG- and S100B-induced H****2****O****2**** production.**

## Effects of PKC-beta inhibitor LY379196 on the Ang II-, HG-induced JAK2 activation.

**Ang II incubation time (minutes)**

**pJAK2**

**Normal Glucose**

**High Glucose **

**JAK2**

**0       5      10**

**pJAK2**

**JAK2**

**with  LY379196**

**no inhibitor**

**no inhibitor**

**with  LY379196**

**Effects of PKC-beta inhibitor LY379196 on the Ang II-, HG-induced JAK2 activation.**

## O2

**O****2**

**ROS**

**NADPH oxidase**

**Angiotensin II**

**AT****1**

**High Glucose and S100B augmentation of the Ang II-induced JAK/STAT pathway in kidney mesangial cell growth.**

**High Glucose**

**Aldose**
**Reductase**

**Polyol Pathway**

**High Glucose**

**RAGE**

**PKC-beta**

**S100B**

**PLD2**

**JAK2 and STATs**

**Cell Growth**

**SHP-1 and SHP-2**

**PLC-gamma******

**DAG**

## Activation of the JAK/STAT pathway by Ang II is also altered by High Glucose in vivo.

## Material and Methods (in vivo)

- Male Sprague-Dawley rats (225-250g)
- STZ (60mg/kg) rats were considered hyperglycemic if blood glucose > 15.0 mM 48 hrs after injection
- Seven groups:
- STZ
- STZ + high insulin
- STZ + AG-490
- STZ + Candesartan (10 mg/kg/day)
- Candesartan (10 mg/kg/day)
- AG-490
- Control (Sham)
- Animals were treated for 7, 14 and 28 days

## Effects of Candesartan on the STZ-induced JAKs activation in the rat kidney glomeruli after 14 days of hyperglycemia

**pJAK2**

**JAK2**

**pJAK1**

**JAK1**

**TYK2**

**pTYK2**

-    **C    Can   STZ   STZ + Can**
**                             **

## Effects of Candesartan on the STZ-induced STATs phosphorylation in the rat kidney glomeruli after 14 days of hyperglycemia

**pTyr-STAT1**

**STAT1**

**STAT3**

**pTyr-STAT3**

**pTyr-STAT5**

**STAT5**

**pTyr-STAT6**

**STAT6**

-   **C     Can   STZ    STZ+Can**

## Slide 43

## Slide 44

## Slide 45

## Slide 46

## Slide 47

## Slide 48

## Slide 49

## Slide 50

## Slide 51

## Slide 52

## Slide 53

## Acknowledgments

- Dr. Douglas C. Eaton
- Dr. David Fulton
- Dr. Carlos Isales
- Dr. David Pollock
- Dr. R. Dan Rudic
- Dr. Ann Marie Schmidt
- Dr. David Stepp
- Dr. David Stern
_**Dr. Marrero’s Laboratory**_
- Dr. Gwen Alexander
- Dr. Farhad Amiri
- Dr. Amy Banes
- Dr. Sean Shaw
- Mr. Kenjiro Muta
- Ms. Amanda Shirley
- Ms. Xiaodan Wang