Abstract
Introduction: Buffalo/Mna rats spontaneously develop nephrotic syndrome (NS) which recurs after renal transplantation. The immunosuppressive drug LF15-0195 can promote regression of the initial and post-transplantation nephropathy via induction of regulatory T cells. We investigate if this drug has an additional effect on the expression and localization of podocyte specific proteins. Methods: Buffalo/Mna kidney samples were collected before and after the occurrence of proteinuria, and after the remission of proteinuria induced by LF15-0195 treatment and compared by quantitative RT-PCR, Western blot, electron, and confocal microscopy to kidney samples of age-matched healthy rats. Cytoskeleton changes were assessed in culture by stress fibers induction by TNFα. Results: We observed, by electron microscopy, a restoration of foot process architecture in the LF15-0195-treated Buff/Mna kidneys, consistent with proteinuria remission. Nephrin, podocin, CD2AP, and α-actinin-4 mRNA levels remained low during the active disease in the Buff/Mna, in comparison with healthy rats which increase, while podocalyxin and synaptopodin transcripts were elevated before the occurrence of the disease but did not differ from healthy animals after. No difference in the mRNA and protein expression between the untreated and the LF15-0195-treated proteinuric Buff/Mna were seen for these 6 proteins. No changes were observed by confocal microscopy in the protein distribution at a cellular level, but a more homogenous distribution similar to healthy rats, was observed within the glomeruli of LF15-0195-treated rats. In addition, LF15-0195 could partially restore actin cytoskeleton of endothelial cells in TNFα-induced-cell stress experiment. Conclusion: The effect of LF15-0195 treatment appears to be mediated by 2 mechanisms: an immunomodulatory effect via regulatory T cells induction, described in our previous work and which can act on immune cell involved in the disease pathogenesis, and an effect on the restoration of podocyte cytoskeleton, independent of expression levels of the proteins involved in the slit diaphragm and podocyte function, showed in this article.
References
1.
Kato
F
, Watanabe
M
, Matsuyama
M
. Nephrotic syndrome in spontaneous thymoma rats, Buffalo/Mna
. Biomed Res
. 1983
;4
(1
):105
–10
. 2.
Nakamura
T
, Oite
T
, Shimizu
F
, Matsuyama
M
, Kazama
T
, Koda
Y
, et al. Sclerotic lesions in the glomeruli of Buffalo/Mna rats
. Nephron
. 1986
;43
(1
):50
–5
. 3.
Matsuyama
M
, Ogiu
T
, Kontani
K
, Yamada
C
, Kawai
M
, Hiai
H
, et al. Genetic regulation of the development of glomerular sclerotic lesions in the BUF/Mna rat
. Nephron
. 1990
;54
(4
):334
–7
. 4.
Akiyama
K
, Morita
H
, Suetsugu
S
, Kuraba
S
, Numata
Y
, Yamamoto
Y
, et al. Actin -related protein 3 (Arp3) is mutated in proteinuric BUF/Mna rats
. Mamm Genome
. 2008
;19
(1
):41
–50
. 5.
Le Berre
L
, Godfrin
Y
, Günther
E
, Buzelin
F
, Perretto
S
, Smit
H
, et al. Extrarenal effects on the pathogenesis and relapse of idiopathic nephrotic syndrome in Buffalo/Mna rats
. J Clin Invest
. 2002
;109
(4
):491
–8
. 6.
Le Berre
L
, Hervé
C
, Buzelin
F
, Usal
C
, Soulillou
JP
, Dantal
J
. Renal macrophage activation and Th2 polarization precedes the development of nephrotic syndrome in Buffalo/Mna rats
. Kidney Int
. 2005
;68
(5
):2079
–90
. 7.
Le Berre
L
, Bruneau
S
, Naulet
J
, Renaudin
K
, Buzelin
F
, Usal
C
, et al. Induction of T regulatory cells attenuates idiopathic nephrotic syndrome
. J Am Soc Nephrol
. 2009
;20
(1
):57
–67
. 8.
Kestilä
M
, Lenkkeri
U
, Männikkö
M
, Lamerdin
J
, McCready
P
, Putaala
H
, et al. Positionally cloned gene for a novel glomerular protein: nephrin—is mutated in congenital nephrotic syndrome
. Mol Cell
. 1998
;1
(4
):575
–82
. 9.
Putaala
H
, Soininen
R
, Kilpeläinen
P
, Wartiovaara
J
, Tryggvason
K
. The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death
. Hum Mol Genet
. 2001
;10
(1
):1
–8
. 10.
Boute
N
, Gribouval
O
, Roselli
S
, Benessy
F
, Lee
H
, Fuchshuber
A
, et al. NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome
. Nat Genet
. 2000
;24
(4
):349
–54
. 11.
Kaplan
JM
, Kim
SH
, North
KN
, Rennke
H
, Correia
LA
, Tong
HQ
, et al. Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis
. Nat Genet
. 2000
;24
(3
):251
–6
. 12.
Kos
CH
, Le
TC
, Sinha
S
, Henderson
JM
, Kim
SH
, Sugimoto
H
, et al. Mice deficient in alpha-actinin-4 have severe glomerular disease
. J Clin Invest
. 2003
;111
(11
):1683
–90
. 13.
Shih
NY
, Li
J
, Karpitskii
V
, Nguyen
A
, Dustin
ML
, Kanagawa
O
, et al. Congenital nephrotic syndrome in mice lacking CD2-associated protein
. Science
. 1999
;286
(5438
):312
–5
. 14.
Doyonnas
R
, Kershaw
DB
, Duhme
C
, Merkens
H
, Chelliah
S
, Graf
T
, et al. Anuria, omphalocele, and perinatal lethality in mice lacking the Cd34-related protein podocalyxin
. J Exp Med
. 2001
;194
(1
):13
–27
. 15.
Asanuma
K
, Kim
K
, Oh
J
, Giardino
L
, Chabanis
S
, Faul
C
, et al. Synaptopodin regulates the actin-bundling activity of alpha-actinin in an isoform-specific manner
. J Clin Invest
. 2005
;115
(5
):1188
–98
. 16.
Bitzan
M
, Babayeva
S
, Vasudevan
A
, Goodyer
P
, Torban
E
. TNFα pathway blockade ameliorates toxic effects of FSGS plasma on podocyte cytoskeleton and β3 integrin activation
. Pediatr Nephrol
. 2012
;27
(12
):2217
–26
. 17.
Wright
RD
, Beresford
MW
. Podocytes contribute, and respond, to the inflammatory environment in lupus nephritis
. Am J Physiol Ren Physiol
. 2018
;315
(6
):F1683
–94
. 18.
Matsui
I
, Ito
T
, Kurihara
H
, Imai
E
, Ogihara
T
, Hori
M
. Snail, a transcriptional regulator, represses nephrin expression in glomerular epithelial cells of nephrotic rats
. Lab Invest
. 2007
;87
(3
):273
–83
. 19.
Furness
PN
, Hall
LL
, Shaw
JA
, Pringle
JH
. Glomerular expression of nephrin is decreased in acquired human nephrotic syndrome
. Nephrol Dial Transplant
. 1999
;14
(5
):1234
–7
. 20.
Koop
K
, Eikmans
M
, Baelde
HJ
, Kawachi
H
, de Heer
E
, Paul
LC
, et al. Expression of podocyte-associated molecules in acquired human kidney diseases
. J Am Soc Nephrol
. 2003
;14
(8
):2063
–71
. 21.
Dai
S
, Wang
Z
, Pan
X
, Wang
W
, Chen
X
, Ren
H
, et al. Functional analysis of promoter mutations in the ACTN4 and SYNPO genes in focal segmental glomerulosclerosis
. Nephrol Dial Transplant
. 2010
;25
(3
):824
–35
. 22.
Kawachi
H
, Koike
H
, Kurihara
H
, Yaoita
E
, Orikasa
M
, Shia
MA
, et al. Cloning of rat nephrin: expression in developing glomeruli and in proteinuric states
. Kidney Int
. 2000
;57
(5
):1949
–61
. 23.
Doublier
S
, Ruotsalainen
V
, Salvidio
G
, Lupia
E
, Biancone
L
, Conaldi
PG
, et al. Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome
. Am J Pathol
. 2001
;158
(5
):1723
–31
. 24.
Kim
BK
, Hong
HK
, Kim
JH
, Lee
HS
. Differential expression of nephrin in acquired human proteinuric diseases
. Am J Kidney Dis
. 2002
;40
(5
):964
–73
. 25.
Patrakka
J
, Ruotsalainen
V
, Ketola
I
, Holmberg
C
, Heikinheimo
M
, Tryggvason
K
, et al. Expression of nephrin in pediatric kidney diseases
. J Am Soc Nephrol
. 2001
;12
(2
):289
–96
. 26.
Schmid
H
, Henger
A
, Cohen
CD
, Frach
K
, Gröne
HJ
, Schlöndorff
D
, et al. Gene expression profiles of podocyte-associated molecules as diagnostic markers in acquired proteinuric diseases
. J Am Soc Nephrol
. 2003
;14
(11
):2958
–66
. 27.
Hingorani
SR
, Finn
LS
, Kowalewska
J
, McDonald
RA
, Eddy
AA
. Expression of nephrin in acquired forms of nephrotic syndrome in childhood
. Pediatr Nephrol
. 2004
;19
(3
):300
–5
. 28.
Schwarz
K
, Simons
M
, Reiser
J
, Saleem
MA
, Faul
C
, Kriz
W
, et al. Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin
. J Clin Invest
. 2001
;108
(11
):1621
–9
. 29.
Jones
N
, Blasutig
IM
, Eremina
V
, Ruston
JM
, Bladt
F
, Li
H
, et al. Nck adaptor proteins link nephrin to the actin cytoskeleton of kidney podocytes
. Nat
. 2006
;440
(7085
):818
–23
. 30.
Benzing
T
. Signaling at the slit diaphragm
. J Am Soc Nephrol
. 2004
;15
(6
):1382
–91
. 31.
Sellin
L
, Huber
TB
, Gerke
P
, Quack
I
, Pavenstädt
H
, Walz
G
. NEPH1 defines a novel family of podocin-interacting proteins
. FASEB J
. 2003
;17
(1
):115
–7
. 32.
Shih
NY
, Li
J
, Cotran
R
, Mundel
P
, Miner
JH
, Shaw
AS
. CD2AP localizes to the slit diaphragm and binds to nephrin via a novel C-terminal domain
. Am J Pathol
. 2001
;159
(6
):2303
–8
. 33.
Lehtonen
S
, Zhao
F
, Lehtonen
E
. CD2-associated protein directly interacts with the actin cytoskeleton
. Am J Physiol Ren Physiol
. 2002
;283
(4
):F734
–43
. 34.
Lynch
DK
, Winata
SC
, Lyons
RJ
, Hughes
WE
, Lehrbach
GM
, Wasinger
V
, et al. A cortactin-CD2-associated protein (CD2AP) complex provides a novel link between epidermal growth factor receptor endocytosis and the actin cytoskeleton
. J Biol Chem
. 2003
;278
(24
):21805
–13
. 35.
Kim
JM
, Wu
H
, Green
G
, Winkler
CA
, Kopp
JB
, Miner
JH
, et al. CD2-Associated protein haploinsufficiency is linked to glomerular disease susceptibility
. Sci
. 2003
;300
(5623
):1298
–300
. 36.
Patrie
KM
, Drescher
AJ
, Welihinda
A
, Mundel
P
, Margolis
B
. Interaction of two actin-binding proteins, synaptopodin and alpha-actinin-4, with the tight junction protein MAGI-1
. J Biol Chem
. 2002
;277
(33
):30183
–90
. 37.
Faul
C
, Donnelly
M
, Merscher-Gomez
S
, Chang
YH
, Franz
S
, Delfgaauw
J
, et al. The actin cytoskeleton of kidney podocytes is a direct target of the antiproteinuric effect of cyclosporine A
. Nat Med
. 2008
;14
(9
):931
–8
. 38.
Orlando
RA
, Takeda
T
, Zak
B
, Schmieder
S
, Benoit
VM
, Mcquistan
T
, et al. The glomerular epithelial cell anti-adhesin podocalyxin associates with the actin cytoskeleton through interactions with Ezrin
. J Am Soc Nephrol
. 2001
;12
(8
):1589
–98
. 39.
Schmieder
S
, Nagai
M
, Orlando
RA
, Takeda
T
, Farquhar
MG
. Podocalyxin activates RhoA and induces actin reorganization through NHERF1 and Ezrin in MDCK cells
. J Am Soc Nephrol
. 2004
;15
(9
):2289
–98
. 40.
Fan
Q
, Xing
Y
, Ding
J
, Guan
N
, Zhang
J
. The relationship among nephrin, podocin, CD2AP, and alpha-actinin might not be a true “interaction” in podocyte
. Kidney Int
. 2006
;69
(7
):1207
–15
. 41.
Wada
T
, Pippin
JW
, Marshall
CB
, Griffin
SV
, Shankland
SJ
. Dexamethasone prevents podocyte apoptosis induced by puromycin aminonucleoside: role of p53 and bcl-2–related family proteins
. J Am Soc Nephrol
. 2005
;16
(9
):2615
–25
. 42.
Yoo
TH
, Fornoni
A
. Nonimmunologic targets of immunosuppressive agents in podocytes
. Kidney Res Clin Pract
. 2015
;34
(2
):69
–75
. 43.
Ransom
RF
, Lam
NG
, Hallett
MA
, Atkinson
SJ
, Smoyer
WE
. Glucocorticoids protect and enhance recovery of cultured murine podocytes via actin filament stabilization
. Kidney Int
. 2005
;68
(6
):2473
–83
. 44.
Li
X
, Ding
F
, Wang
S
, Li
B
, Ding
J
. Cyclosporine A protects podocytes by regulating WAVE1 phosphorylation
. Sci Rep
. 2015
;5
(1
):17694
. 45.
Yoo
TH
, Pedigo
CE
, Guzman
J
, Correa-Medina
M
, Wei
C
, Villarreal
R
, et al. Sphingomyelinase-Like phosphodiesterase 3b expression levels determine podocyte injury phenotypes in glomerular disease
. J Am Soc Nephrol
. 2015
;26
(1
):133
–47
. 46.
Perosa
F
, Favoino
E
, Caragnano
MA
, Dammacco
F
. Generation of biologically active linear and cyclic peptides has revealed a unique fine specificity of rituximab and its possible cross-reactivity with acid sphingomyelinase-like phosphodiesterase 3b precursor
. Blood
. 2006
;107
(3
):1070
–7
. 47.
Fornoni
A
, Sageshima
J
, Wei
C
, Merscher-Gomez
S
, Aguillon-Prada
R
, Jauregui
AN
, et al. Rituximab targets podocytes in recurrent focal segmental glomerulosclerosis
. Sci Transl Med
. 2011
;3
(85
):85ra46
. 48.
Liang
P
, MacRae
TH
. Molecular chaperones and the cytoskeleton
. J Cell Sci
. 1997
;110
(Pt 13
):1431
–40
. 49.
Andrés
D
, Alvarez
AM
, Díez-Fernández
C
, Zaragoza
A
, Cascales
M
. HSP70 induction by cyclosporine A in cultured rat hepatocytes: effect of vitamin E succinate
. J Hepatol
. 2000
;33
(4
):570
–9
. 50.
Shin
YC
, Choi
KY
, Kim
WG
. Cyclosporin A has a protective effect with induced upregulation of Hsp70 and nNOS on severe spinal cord ischemic injury in rabbits
. J Invest Surg
. 2007
;20
(2
):113
–20
. 
