1. Role of podocyte for integrity of glomerulus and glomerular diseasesDr

2. Introduction Podocytes
Highly differentiated cells with a unique architecture that includes a
cell body, major processes and foot processes bridged by slit diaphragms
Most differentiated cell type in the glomerulum, which forms a crucial component of the glomerular filtration barrier

3. Introduction
In the past decade, podocyte research has been greatly aided by
the development of powerful new molecular, cellular and animal tools, leading to
elucidation of an increasing number of proteins involved in podocyte function and identification of mutated genes in hereditary glomerulopathies
The International Journal of Biochemistry & Cell Biology 2010; 42:1380–7.

4. Introduction Accumulating evidence indicates that
Podocyte disorders may not only underlie these hereditary glomerulopathies but also play crucial role in a broad spectrum of acquired glomerular diseases
Genetic susceptibility, environmental influence and systemic responses are all involved in the mediation of the pathogenesis of podocytopathies
The International Journal of Biochemistry & Cell Biology 2010; 42:1380–7.

5. RENAL CORPUSCLE Afferent arteriole Efferent arteriole
Mesangial cells & matrix between roots of capillaries
Visceral epithelium of podocytes
Glomerular capillary tuft
Capsular space
Bowman’s capsule with simple squamous parietal epithelium
Start of Proximal tubule

6. Podocyte
Podocytes (or visceral epithelial cells) are cells in the Bowman's capsule in the kidneys that wrap around the capillaries of the glomerulus
The long processes, or "foot projections," of the podocytes wrap around the capillaries, and leave slits between them.
Blood is filtered through these slits, known as a slit diaphragm

7. RENAL CORPUSCLE 2 VASCULAR POLE URINARY POLE Afferent arteriole
Efferent arteriole
Mesangial cells & matrix between roots of capillaries
Glomerular capillary tuft
Bowman’s capsule
Capsular space
URINARY POLE
Start of Proximal tubule

8. Podocyte proteins
Several proteins are required for the foot projections to wrap around the capillaries and function
When infants are born with certain defects in these proteins, such as nephrin and CD2AP, their kidneys cannot function

9. RENAL CORPUSCLE 3
Efferent arteriole
Blood pressure drives the ultrafiltration
Afferent arteriole
Mesangial cells & matrix between roots of capillaries
Visceral epithelium of podocytes
Glomerular capillary tuft
Capsular space
Bowman’s capsule with simple squamous parietal epithelium
Start of Proximal tubule
The process puts at risk of loss from the body large quantities of water and valuable materials, so that the priority of the proximal tubule is to recover most of these

10. Podocytes and Renal Disease

11. Inherited Mutations of Podocyte Proteins
Congenital NS of the Finnish Type (CNF)
Nephrin mutations
AR Nephrotic Syndrome
Podocin mutations

12. Inherited Mutations of Podocyte Proteins
AD Nephrotic Syndrome (FSGS)
Alpha-actinin-4 mutations
Altered mechanical properties of the podocytes (impaired cytoskeletal fxn)
Penetrance=high
Subnephrotic proteinuria and progressive renal insufficiency

13. FILTRATION BARRIER Podocytes with Fenestrated endothelium Basal lamina
The charged proteoglycans of the BL help control what passes through
Podocytes with
Capillary lumen
Capsular space
Filtration slits between feet
Capsular space
Fenestration
Basal lamina
Filtration slit closed by a diaphragm

14. } } FILTRATION BARRIER Detail Filtration diaphragm
comprising nephrin molecules interlocked, but spaced, in a zipper-like array, determining the size of the molecules that can pass } } BL
Foot/pedicel of PODOCYTE
Podocyte’s foot can be more or less spread on the basal lamina by actin-based mechanisms to regulate flow

15. Variations
People have variations in these proteins, and some variations may predispose them to kidney failure later in life.
Nephrin is a zipper-like protein that forms the slit diaphragm, with spaces between the teeth of the zipper, big enough to allow sugar and water through, but too small to allow proteins through.
Nephron defects are responsible for congenital kidney failure. CD2AP forms the podocyte cytoskeleton, and stabilizes the slit diaphragm

16. Function of Podocytes
Adjacent podocytes interdigitate to cover the basal lamina which is intimately associated with the glomerular capillaries, but the podocytes leave gaps or thin filtration slits
The slits are covered by slit diaphragms which are composed of a number of cell-surface proteins including nephrin, podocalyxin, and P-cadherin, which ensure that large macromolecules such as serum albumin and gamma globulin remain in the bloodstream.

17. PODOCYTE’S ROLES Podocytes with Create size barrier to protein passage
Interact with endothelial cells
Create charge barrier to protein passage
Counterbalance pressure
Help keep capillary-loop shape
Produce & maintain basal lamina
As listed by Shankland SJ. The podocyte’s response to injury: role in proteinuria and glomerulosclerosis. Kidney Int 2006;69:
Capillary lumen
Fenestrated endothelium
Basal lamina
Podocytes with
Filtration slits between feet
Capsular space

18. Small molecules such as water, glucose, and ionic salts are able to pass through the slit diaphragms and form an ultrafiltrate which is further processed by the nephron to produce urine Podocytes are also involved in regulation of glomerular filtration rate (GFR). When podocytes contract, they cause closure of filtration slits. This decreases the GFR by reducing the surface area available for filtration

19. Structural features
Indicate a high rate of vesicular traffic in these cells. Many coated vesicles and coated pits can be seen along the basolateral domain of the podocytes
Podocytes possess a well-developed endoplasmic reticulum and a large Golgi apparatus, indicative of a high capacity for protein synthesis and post-translational modifications
A large number of multivesicular bodies and other lysosomal indicating a high endocytic activity.
"Pedicels" (or "foot processes") extend from the podocyte and increase the surface area

20. Pathology
Disruption of the slit diaphragms or destruction of the podocytes can lead to massive proteinuria where large amounts of protein are lost from the blood
An example of this occurs in the congenital disorder Finnish-type nephrosis, which is characterised by neonatal proteinuria leading to end-stage renal failure
This disease has been found to be caused by a mutation in the nephrin gene

21. Possible mechanisms of proteinuria reduction
Reduction in glomerular capillary hydrostatic pressure
Restoring glomerular filtration barrier
- Cytoprotection: podocyte, endothelium
Restoration of glomerular basement membrane pore size distribution
Restoring proximal tubule protein reabsorption: cytoprotection

22. Podocyturia
Evidence that podocyte depletion characterizes most progressive CKD
Direct counting of urinary podocytes is impractical
Enumeration with FACS has proven difficult
Podocyte proteins: total, exosomes
Kuusniemi, KI

23. Podocyturia correlates more closely than proteinuria with disease activity in animal models
PAN
Thy-1
5/6 Nx
Yu JASN 2005

24. The structure of the glomerulus.
Urinary space
The normal glomerulus (right) is a capillary tuft supported on a basement membrane lined by podocytes on the urine-space aspect. Along its route from the blood space to the urine space, the plasma ultrafiltrate passes sequentially through the fenestrated glomerular capillary endothelium, the collagenous network of the GBM and the filtration slits and slit diaphragm formed by the podocyte foot processes. The foot processes show effacement and fusion in nephrotic syndrome (left). GBM, glomerular basement membrane; MC, mesangial cells; US, urine space.
Nature Genetics  24, (2000)

25. Scanning electron micrograph
Physiol Rev Apr;88(2):451-87

26. Interdigitating Foot Processes
Development 135, (2008)

27. Schematic diagram of the slit diaphragm and other important proteins involved in maintaining foot process assembly.
Development 135, (2008)

28. The Slit Diaphragm of Podocytes
Figure 2. The slit diaphragm of podocytes is a specialized cell junction with signaling properties. The slit diaphragm connects interdigitating foot processes of the podocyte (FP) and is an essential part of the glomerular filter of the kidney. Slit diaphragm proteins (nephrin and neph1) recruit cytoplasmic adaptor proteins to initiate signal transduction events that lead to the regulation of complex biologic programs.
J Am Soc Nephrol 15: , 2004

29. Molecular anatomy of the podocyte foot process cytoskeleton.
The podocyte foot processes contain a contractile system composed of actin, myosin-II, -actinin-4, talin, vinculin and synaptopodin that is connected to the GBM via 3 1 integrin. The linkage of the actin cytoskeleton to the slit diaphragm components, nephrin and P-cadherin, may be mediated by CD2AP or by a complex of ZO-1, -, - and -catenin. The localization of podocin in the podocyte cell membrane remains to be established. The actin cytoskeleton is well suited to integrate different signalling pathways from the matrix:GBM interface, the slit diaphragm or the cell surface. Disruption of any of these pathways may lead to reorganization of the actin cytoskeleton and foot process effacement as seen with nephrotic syndrome. N, nephrin; P-C, P-cadherin; , -catenin; , -catenin; , -catenin; Z, ZO-1; 3, 3-integrin; 1, 1-integrin; V, vinculin; T, talin; P, paxillin; -act-4, -actinin-4; synpo, synaptopodin.
Nature Genetics  24, (2000)

30. . Signaling at the slit diaphragm
Signal transduction from cellular receptors requires intracellular adaptor proteins (Figure 3). Adaptor proteins are noncatalytic polypeptides that contain one or more protein interaction modules that mediate protein interactions (47). As described above, many of the protein interactions are regulated through phosphorylation and dephosphorylation of crucial protein and lipid substrates (40,48). By screening phosphotyrosine-binding proteins for their interaction with the tyrosine-phosphorylated cytoplasmic tail of nephrin, the protein p85 was the first SH2 domain-containing protein to be identified that binds to the tyrosine-phosphorylated cytoplasmic tail of nephrin in vivo (36). p85 is the regulatory subunit of class Ia phosphoinositide 3-OH (PI3) kinase. PI3 kinase activity is responsible for the phosphorylation of lipids at the inner leaflet of the plasma membrane (49). Recruitment of the regulatory p85 subunit to the cytoplasmic tail of nephrin induced the activation of the p110 catalytic subunit, which converts the membrane lipid phosphatidylinositol-4,5-biphosphate to phosphatidylinositol-3,4,5-triphosphate. Thus, lipids at the cytoplasmic side of the filtration slit are phosphorylated by nephrin-activated PI3 kinase, which may initiate a cascade of events in the podocyte foot process (36). Signaling proteins with pleckstrin-homology (PH) domains accumulate at sites of PI3 kinase activation by directly binding to these phosphorylated lipids. These proteins themselves regulate a variety of crucial cellular programs such as cell survival, actin cytoskeletal dynamics, endocytosis, and cell metabolism (49). Of particular interest in the podocyte is the PH domain–containing serine-threonine kinase AKT (36). Other PH domain proteins that are activated by PI3 kinase and could play a role in podocyte biology include GDP-GTP exchange factors for Rac and ARF6 and protein tyrosine kinases of the Bruton’s tyrosine kinase (Btk) and Tec family. Binding of PI3 kinase–generated phospholipids to the PH domain of AKT leads to the translocation and activation of AKT. Among a wealth of effects, AKT activity has been found to be required for the growth factor–dependent survival of a wide variety of cell types ranging from fibroblasts to neurons by blocking apoptosis (50). Consequently, nephrin-mediated activation of PI3 kinase and AKT activity has been shown to inhibit podocyte apoptosis and to increase the threshold for podocyte cell death induced by apoptotic stimuli (36).
However, nephrin is not the only slit diaphragm protein to associate with p85 and activate PI3 kinase. Recently, it was demonstrated that CD2AP directly interacts with p85 (36). Together with nephrin, CD2AP strongly activates PI3 kinase in podocytes. Targeted disruption of the cd2ap gene dramatically reduces AKT activity in podocytes and is associated with an increased susceptibility to podocyte apoptosis (36). These findings are particularly interesting in the context of the pathogenic steps that lead to glomerulosclerosis (51–54). Podocyte death and podocyte depletion have been proposed as hallmarks of both primary and secondary forms of glomerulosclerosis for many years and are now considered a key step in the development of progressive renal disease (51,55–57). Thus, the structural and functional integrity of the slit diaphragm proteins and signaling at the slit diaphragm may be required for the inhibition of apoptosis and for cell survival in podocytes. In support of this hypothesis, cd2ap+/– heterozygous mice are haploinsufficient and develop severe glomerular changes at 9 mo of age with a histologic pattern similar to that in human focal segmental glomerulosclerosis (32,33).
Although the underlying mechanism of CD2AP-mediated PI3 kinase activity at the molecular level is not completely understood, recent evidence suggests that by directly interacting with several target proteins, CD2AP may orchestrate PI3 kinase effectors to amplify efficient PI3 kinase signaling (T. Benzing, unpublished data). In analogy to CIN85, CD2AP may also be involved in the recycling and endocytosis of transmembrane receptors (e.g., nephrin), thereby regulating signal transduction from the slit diaphragm (32,58). An additional function of the PI3 kinase downstream effector AKT is to repress collagenase expression and to induce the synthesis of laminin and type IV collagen chains (59,60); both are key components of the glomerular basement membrane. Because basement membrane abnormalities are involved in the development of several forms of proteinuria, it is tempting to speculate that AKT activation may contribute to the synthesis and/or maintenance of an intact glomerular basement membrane. These data further support the concept that signal transduction at the slit diaphragm is critical for podocyte function, viability, and integrity of the glomerular filter.
Benzing, T. J Am Soc Nephrol 2004;15:
Copyright ©2004 American Society of Nephrology

31. Secondary FSGS 1. Familial
á-actinin 4
nephrin
Podocin
TRPC6
Mutations in WT-1
Mutations in CD2-associated protein
Mitochondrial cytopathies

32. Stages of glomerular development and progression with emphasis on the role of the podocyte. The S-shaped stage. Developing podocytes acquire podocyte markers during the S-shaped stage of nephron development at which time blood vessels and mesenchyme invade (arrow). The podocytes (round nuclei) separate from the parietal epithelial cells (triangular nuclei) forming what will become Bowman's space. The head-shaped stage: at this stage of glomerular development, the glomerulus consists of a ball of cell surrounded by developing podocytes. The capillary loop stage: this stage of glomerular development includes the infolding of the surface layer in order to enlarge the area available for filtration as well as the development of foot processes that interdigitate between podocytes and abut the underlying GBM which is being synthesized as a collaboration between the podocyte and underlying endothelial and mesangial cells. The mature glomerulus: the mature glomerulus has maximized filtration surface area by developing intertwining finger-like projections coated by fenestrated endothelial cells on the inside, a specialized strong thin GBM in the middle, and interdigitating podocyte foot processes connected by slit diaphragms on the outer surface. Mesangial expansion: loss of some podocytes (20%) is associated with mesangial expansion possibly as an attempt to reduce the filtration surface area. Adhesion formation: loss of podocytes resulting in appearance of bare areas of filtration surface results in adhesion of the bare surface to Bowman's capsule (synechia). Segmental sclerosis: loss of podocytes beyond a critical level results in a fibrotic glomerular response in that part of the glomerulus (segmental sclerosis). Global sclerosis: loss of podocytes beyond a critical level results in widespread scarring of that glomerulus (global sclerosis).

33. Capillary wall thickening
Capillary Wall Thickening - GBM Thickening: diabetic glomerulosclerosis
Capillarv Wall Thickening - Immune Deposits: capillary loops have a rigid, accentuated spherical appearance with normal mesangium

34. The International Journal of Biochemistry & Cell Biology 2010; 42:1380–7.

35. The International Journal of Biochemistry & Cell Biology 2010; 42:1380–7.

36. Regardless of the etiology/pathologic classification,
Four major patterns of podocyte morphology alteration during glomerular diseases
A. Foot process effacement
B. Apoptosis
C. Arrested development:
D. Dedifferentiation
The International Journal of Biochemistry & Cell Biology 2010; 42:1380–7.

37. Clin J Am Soc Nephrol 2007;2:529-42.

38. Am J Nephrol 2003;23:353–60

39. Four major patterns of glomerular injury in podocytopathies.
MCN: No changes are present on light microscopy. FSGS: FSGS is characterized by segmental solidification of the tuft with accumulation of extracellular matrix. Synechiae form between the tuft and Bowman’s capsule. Podocytes are lost in the areas of sclerosis. DMS: Mesangial expansion as a result of accumulated extracellular matrix is the characteristic feature and is accompanied by hypertrophy and mild cobblestone hyperplasia of overlying podocytes. CG: The characteristic features of CG include wrinkling and folding of the glomerular basement membranes (collapse) and proliferation of overlying podocytes forming pseudocrescents. Numerous protein reabsorption droplets are present in the podocyte cytoplasm. Magnifications: 40 in MCN and FSGS; 60 in DMS and CG (Silver stain for all).
Clin J Am Soc Nephrol 2007;2:

40. Can podocytes be repaired?
Recent studies have raised the possibility that a population of stem cells
Might differentiate into, and replace, podocytes lost during injury or with normal aging
The International Journal of Biochemistry & Cell Biology 2010; 42:1380–7.

41. Hydrostatic mechanisms
Reducing efferent arteriolar tone
- ACEI, ARB
Treating systemic hypertension
- all agents

42. Podocyte injury Mitochondrial dysfunction
Loss of filtration slits and slit diaphragms
- Mutations
Transcription
- ER processing
Signaling
Actin cytoskeleton
Detachment, loss of adhesion
Apoptosis
Loss of anionic charge: podocalyxin (glucose)
Replenishment failure (?)
Dysregulation (collapsing glomerulopathy)
IC, C5b-9

43. Protecting and restoring podocyte phenotype
Preventing IC deposition
Glucocorticoids
Transcription
Actin stabilization
Ransom KI 2005
Anti-apoptotic
Wada JASN 2005
Transport from ER
Fuji KI 2006
Mizoribine
- Transport from ER via energetics
Nakajo JASN 2007
Retinoids
reverse FPE
­nephrin, podocin
Vaughan KI 2005
Cyclosporine

44. Endothelium
Haraldsson, Physiol Rev 2008

45. Cathepsin L expression increases in PAN
Podocyte foot process effacement and disruption of the slit diaphragm are typically associated with glomerular proteinuria and can be induced in rats by the injection of puromycin aminonucleoside. Here, we show that the induction of puromycin aminonucleoside nephrosis involves podocyte migration conducted by a coordinated interplay between the cysteine protease cathepsin L and 3 integrin. Puromycin aminonucleoside treatment up-regulates cathepsin L expression in podocytes in vivo as well as expression and enzymatic activity of cathepsin L in podocytes in vitro. Isolated podocytes from mice lacking cathepsin L are protected from cell puromycin aminonucleoside-induced cell detachment. The functional significance of cathepsin L expression was underscored by the observation that puromycin aminonucleoside-induced cell migration was slowed down in cathepsin L-deficient podocytes and by the preservation of cell-cell contacts and expression of vital slit diaphragm protein CD2AP. Cathepsin L expression and activity were induced in podocytes lacking 3 integrin. Similarly, acute functional inhibition of 3 integrin in wild type podocytes with a blocking antibody increased the expression of cathepsin L activity. Down-regulation of 3 integrin protected against puromycin aminonucleoside-induced podocyte detachment. In summary, these data establish that podocyte foot process effacement is a migratory event involving a novel interplay between cathepsin L and 3 integrin.
, Western blot showing low levels of cathepsin L expression in untreated wild type podocytes (control (con)). PAN treatment up-regulates both cathepsin L and procathepsin L expression. Blot membranes were probed with anti-tubulin antibody and showed comparable signal intensity in both lanes. b, in untreated podocytes, cathepsin shows a perinuclear localization consistent with a lysosomal distribution (left). Exposure to PAN increases cathepsin L staining throughout the entire cell (right). c, total cathepsin L activity in PAN-treated podocytes is significantly increased at 30 and 45 min (see "Results" for details). d, visualized cathepsin L activity is localized in perinuclear lysosomes in untreated podocytes (left) and shifted of cathepsin L cleavage into podocyte processes after PAN treatment (right).
Upon an insult, stationary podocytes transform into migratory podocytes. This migratory response leads to the up-regulation of cathepsin L expression and activity as well as SD remodeling, FP effacement, and proteinuria. Down-regulation of 3 integrin causes an increased attachment of podocytes to the GBM. Thereafter, podocytes can either recover with restoration of the normal filtration barrier or, if the insult persists, detach and promote progression of glomerular disease.
Reiser, J. et al. J. Biol. Chem. 2004;279:

46. GBM
GBM: 3 layers of GBM; central dense layer, lamina densa, lamina rara interna and lamina rara externa in which foot processes of podoctyes are embedded
GBM: GBM has type 4collagen, laminin, HSPG. HSPG mostly in lamina rara interna and externa.

47. GBM
GBM responsible for ultrafiltration and is size/charge selective barrier.
HSPG plays major role in establishing charge barrier properties of GBM
High anionic sites rich in HSPG throughout lamina rara interna.

48. Capillary Wall Thickening - GBM Thickening:
GBM in diabetes is thickened in width several fold

49. Capillary Wall Thickening - Immune Deposits:
Subepithelial electron-dense immune deposits (D) surrounded by spikes of GBM projecting between them.
Capillary wall is thickened and deformed

50. Minimal Change Disease:
Effacement of podocyte foot processes-this is the MAJOR lesion in minimal change disease, present in every glomerulus
No electron dense deposits
Extensive cell swelling
Degree of effacement correlates w/ amt of proteinuria
Dx can only be made in absence of other pathology by LM, IF, EM
Damage is reversible after coricosteroids

51. Focal Segmental Glomerulosclerosis
“Foam cells” are within sclerotic loop
Podocytes are degenerated with prominent cytoplasmic vacuolation and are partially detached from GBM (arrow)
GBM is irregular but no electron dense deposits
Non-involved glomerular capillary loops show changes identical to minimal change disease

52. Focal Segmental Glomerulosclerosis
Adhesion between segmental sclerotic lesion and Bowman’s capsule
This lesion involves some but not all glomeruli (focal) and only a portion of capillary tuft (segmental)
Hyalinosis present (arrow)
Can lead to globally sclerosed glomeruli, atrophy and fibrosis

53. Alport Syndrome: Characteristic lesion only observed by EM
Thickened irregular GBM with splitting of lamina densa into layers
Occurs in absence of other findings

54. Membranous Glomerulopathy:
STAGE I: Sub-epithelial electron-dense deposits containing IgG and C3
Spikes have not yet developed
STAGE II: underlying GBM and spikes

55. Membranous Glomerulopathy:
STAGE III: spikes fuse over deposits making a thick GBM
Thus deposits become intramembranous
Prominent foot process obliteration indicates severe proteinuria

56. Membranous Glomerulopathy
Global thickening of capillary walls “spherical” rigid appearance of the glomerular capillary walls due to homogenous eosinophilic thickening (“spiked cheerios”)
Projections of silver positive GBM extending perpendicular from cap wall into Bowman’s space

57. Membranous Glomerulopathy:
Stage1: subepithelial immune deposits
Stage2: GBM spikes. Foot process effacement
Stage3: the spikes encircle the deposits as they become sequestered within thickened GBM
Stage4: resorption of deposits and distortion of GBM by irregular thickening

58. Conclusions
In the last decade our knowledge about the role of podocytes in the onset and progression of proteinuric kidney disease has increased tremendously
Podocytes are no longer considered to be simply a passive target but can also be a mediator of continuing glomerular injury

59. Conclusions
Although highly differentiated podocytes have limited proliferative capacity under normal conditions, new experimental findings suggest a
Potential role in podocyte regeneration by progenitor cells residing within the Bowman’s capsule, which may open a new era for the treatment of chronic glomerular diseases

60. Thank You!