Journal Club 03/04/2012

Lama Nazzal

NOTCH and the kidneys

Is an evolutionarily conserved cell–cell communication mechanism.

Is a regulator of cell specification, differentiation, and tissue patterning.

Lack of or reduced Notch signalling in patients causes renal developmental abnormalities.

Notch is reactivated in the kidney in different disease conditions and is though to play a role in orchestrating chronic kidney disease development.

Structure

There are four receptors in mammals, Notch1–4, and five different ligands, Delta-like1, 3, 4 (Dll1, 3, 4) and Jagged1 and 2 (Jag1, 2)

Both receptors and ligands are single-pass type I transmembrane receptor proteins

Canonical Notch signaling

1) signal-sending cell presents the ligand (Delta/Jagged family) to the signal receiving cell

2) sequential cleavage of Notch through ADAM metalloproteinases at site S2 and the γ-secretase complex at site S3.

3) released extracellular domain is endocytosed along with the ligand

4) cleaved intracellular Notch (NICD) translocates to the nucleus, where it interacts with its binding partner Rbpj

5) this complex (including MAML)drives target gene transcription (Hes, Hey).

6) signal termination usually occurs by ubiquitin-ligase-mediated NICD degradation.

Notch and development

Is involved in the development of diverse tissues in almost all species

By providing an ‘inductive signal’ it can induce a specific cell fate in neighboring cells

By providing an ‘inhibitory signal’ it can restrict a specific cell fate:

‘Mutual’ inhibitory Notch signalling: when initially cells that share the same cell fate both send and receive notch signals

‘Lateral’ inhibitory Notch signalling: when later one cell commits to a specialized fate and inhibits surrounding cells from adopting this fate.

In this situation, failure of Notch signalling results in cells adopting the special cell fate, while excessive Notch signalling prevents differentiation of these cells.

Notch and kidney development

Notch and kidney development

Studies suggesting a Notch mediated proximal fate determination of the proximal tubular epithelium and podocytes:

Mouse metanephroi cultured in the presence of a γ-secretase inhibitor, caused a severe deficiency in proximal tubules and glomeruli

Mice with genetic deletions for subunits of γ-secretase complex have no comma shaped and S shaped bodies or mature glomeruli.

Mice with hypomorphic allele for Notch2 or mice with a targeted deletion of Notch2 from the MM showed a similar phenotype. Notch 1 ectopic expression was able to rescue the Notch2 null phenotype but Notch1 deletion from the MM did not cause developmental abnormalities.

Notch and kidney development

Cells can adopt a podocyte fate in the absence of Notch signalling. The lack of podocyte development might have been observed secondary to a lack of proximal tubule specification.

The initial proximo-distal axis can still be formed in the absence of Notch2 through an Lhx1 dependant mechanism

Notch2 activation does not directly lead to proximal fate specification, but rather is necessary for the stabilization of the proximal fate, and by a Notch-controlled progenitor cell maintenance.

Notch and kidney development

Notch has been implicated in the collecting duct development.

Notch signalling in the developing collecting duct is involved in determining the ratio of principal and intercalated cells

Inactivation of Mind-bomb1(E3 ligase in the ligand –expressing cell and required for efficient Notch activation) led to an increase in intercalated cell number and a decrease in principal cell number in the developing collecting ducts.

Clinically, mice had increased UO, decreased Uosm, Na wasting and a severe urinary concentrating defect, similar to nephrogenic diabetes insipidus

Mutations of JAGGED1 or NOTCH2 have been described to be causally linked to Alagille syndrome (AGS). AGS is a multi-organ disorder, renal abnormalities includes renovascular disease, renal tubular acidosis, tubulointerstitial nephritis, and renal dysplasia/hypoplasia. In addition, a peculiar pattern of glomerular lesions, called ‘mesangiolipidosis’, has been described.

Role of Notch in AKI, repair, and regeneration

The expression of Notch receptors and ligands is much decreased in the mature kidney.

In rat ischemia and reperfusion model there was increase expression of Delta1, Notch2, and the downstream target Hes1.

Co-culture of Delta-1 expressing cells and renal tubular epithelial cells showed increase in proliferation of the TE cells suggesting a role of Notch2 in TE cell regeneration in AKI.

In the folic acid precipitation-induced AKI, Jaged1/Notch1/HeyL genes were activated. Treatment with γ-secretase did not affect rise or decline of serum BUN/Cr.

Notch and CKD In mouse models of fibrosis,

Jagged1/Notch1 was upregulated.

In human kidneys with diabetic nephropathy, there was an increase of Jagged1 and Hes1 expression in tubular cells.

Increased levels of Notch1 pathway protein expression in diabetic nephropathy and other CKD associated with kidney fibrosis

The severity of fibrosis correlated with the expression level of cleaved Notch1 in the tubulointerstitium.

Notch and kidney fibrosis

Role of Notch in glomerular disease

Analysis of kidney samples from glomerulus of ten proteinuric nephropathies groups showed increased expression of cleaved Notch1, Notch2, and Jagged1.

Degree of kidney disease (albuminuria/glomerulosclerosis) correlated with the level of Notch1, 2, and Jagged1 expression.

In mouse model, ectopic NICD expression in developing podocytes caused glomerulosclerois and proteinuria but if simultaneous inactivation of Rbpj the phenotype was rescued.

In vitro experiments on renal progenitor cells showed that Notch activation induced cell proliferation, whereas Notch pathway silencing was required for progenitor cell differentiation towards the podocyte lineage.

Prolonged activation of Notch signalling induced cell death.

Prolonged Notch pathway inhibition throughout the regenerative phases of glomerular injury resulted in increased proteinuria and severe glomerulosclerosis.

Loss of nephrin is associated with SD malformation or destabilization and results in proteinuria in humans and rodents.

Raft-mediated endocytosis of nephrin is upregulated after podocyte injury

Unclear role of dynamin dependent, clathrin-mediated endocytosis of non-raft, membrane-associated nephrin in podocyte injury

Podocyte-specific Cre recombinase (CRE) was used to induce conditional expression of myc-tagged Notch1-IC (MYC NOTCH-IC) first detected in embryonic mouse podocytes and persisting in mature glomeruli throughout postnatal life (CRE[+];NOTCH-IC mice).

Newborn CRE[+];NOTCH-IC mice exhibited normal glomerular architecture on light microscopy and normal mRNA expressions of podocyte-specific markers Nphs1 , Nphs2 , and Wt1, but developed proteinuria at 2 weeks of life.

MYC NOTCH-IC was detected in less than one third of podocytes in CRE[+];NOTCH-IC mouse glomeruli within 2 weeks of birth

• No significant decrease in total nephrin ptn levels of glomerular lysates isolated from newborn CRE[+];NOTCH-IC mice compared with CRE negative control

• Western blot may not be sensitive enough to detect changes in nephrin levels that occur in subset of podocytes

Western Blot analysis of total Nephrin protein levels in glomeruli isolated from kidneys of newborn CRE[+];NOTCH-IC mice and control littermates.

Nephrin immunostaining patterns in glomeruli of newborn CRE[+];NOTCH-IC mice. Shown are images of mouse glomeruli after triple indirect immunofluorescence labeling of newborn kidney tissue sections with antinephrin (green), anti-WT1 (red), and anti-MYC (blue) antibodies.

Immunostaining patterns for ZO-1, Neph1 and Podocin in glomeruli of newborn CRE[+];NOTCH-IC mice and control littermates

• Analysis of anti nephrin

staining in MYCNotch1C expressing transgenic mouse glomeruli at single cell resolution

• A score was assigned to

each [MYC, WT1]– double positive cell based on the pattern of anti-nephrin antibody staining along the cell’ s basolateral surface (1=linear,2=granular, 3=absent)

• As a point of reference: 79% of podocytes in 21 glomeruli of three newborn CRE[-];NOTCH-IC (control) mice were scored as 1, 16% were scored as 2, and 3% were scored as 3.

Cell surface nephrin localization in transfected HEK293T cells. Shown are representative, merged images of HEK293T cells stained with anti-nephrin (green), anti-FLAG (red), and WGA (blue) to detect NephrinMYC, NOTCH1-ICFLAG, and cell membrane, respectively

Merged images of HEK293T cells stained with anti-nephrin (green), anti-FLAG (red), and WGA (blue) to detect NephrinMYC, NOTCH1-ICFLAG, and cell membrane, respectively. Cells were preincubated with either DMSO vehicle (A, A’, D, andD’), Dynasore (C,C’, D, and D’), or MbCD (E, E’, F, and F’). (A, C, and E)

Conclusions

Results suggest that ectopic Notch signaling activates a dynamin-dependent mechanism, such as clathrin-mediated endocytosis, that promotes nephrin internalization

Notch-IC– independent effect on nephrin internalization may be present, which caused a 7% decrease in surface expression in MβCD-treated cells that did not express Notch-IC.

Notch signaling has also been found to regulate endocytosis in HeLa cells.

Notch signaling may be involved in regulating transcription of genes encoding endocytic proteins.

Demonstration that nephrin staining was not attenuated in CRE[+]; NOTCH-IC;RBPKO podocytes supports a Notch-dependent transcriptional mechanism for controlling nephrin trafficking in this context.

Need for studies to define molecular responses downstream of Notch-IC/RBPJk complexes

Future directions

Notch signalling is known to be involved in many different oncological diseases. Recently, Notch signalling has been an attractive therapeutic target.

Ligand binding andγ-secretase-mediated cleavage is an important step at which Notch pathway activity that can be pharmacologically modified.

γ-secretase complex inhibitors are being tested for the treatment of Alzheimer’s disease.

Since Notch is a key player in the developing and adult kidney, more studies are needed for understanding the activation and the effect of specific ligands and receptors in development and disease.

Notch cross-talks with other pathways needs to be understood to efficiently manipulate Notch signalling.

Thank you

Bielesz B et al. J Clin Invest. 2010 Nov;120(11):4040-54

Cheng HT et al. Development. 2007 Feb;134(4):801-11.

Jeong HW et al. J Clin Invest. 2009 Nov;119(11):3290-300.

Sirin Y, Susztak K. J Pathol. 2012 Jan;226(2):394-403.