Gender and progression of chronic kidney diseases: insights from

cellular studies
Giacomo Garibotto, Daniela Verzola, Fanny Tosetti & Maria Teresa
Gandolfo
Department of Internal Medicine, Nephrology Division, Genoa University,
Genoa Italy
Address for correspondence:
Giacomo Garibotto MD
Department of Internal Medicine
Division of Nephrology
Viale Benedetto V,!
"!"#$ Genoa, Italy
%h&&#'("(#)#*'*'
+a,: &&#'("(#)#*')*'
e-mail:gari./nige0it

Men e,hibit a more rapid age-related decline in renal f/nction than 1omen
and several renal diseases appear to be gender-dependent 2", $30
4o1ever, the mechanisms /nderlying are not completely yet /nderstood0
Gonadal steroids have an important infl/ence on sodi/m handling and
renal hemodynamics0 It has been fo/nd that androgens increase pro,imal
sodi/m reabsorption and intraglomer/lar press/re by mod/lating afferent
and efferent arteriolar ton/s via angiotensin II, endothelin and o,idative
stress 2#30 In contrast, female se, hormones lead to renal vasodilation and
decrease filtration fraction0
5eceptors for estradiol and testosterone have been demonstrated in
several renal str/ct/res, incl/ding glomer/li, vessels and renal t/b/les0
N/clear receptors for both androgens and estrogens have been observed
in mesangial cells from male rats 2630 A role for se, hormones to ind/ce
renal damage stems from st/dies in 1hich aging male rats develop
protein/ria and glomer/losclerosis, 1hereas female and estrogen-treated
male rats are resistant to disease progression 2)-730 8e, hormones affect
the str/ct/re of renal t/b/les and glomer/li and vario/s aspects of renal
f/nctions in e,perimental animals and h/mans0 N/mero/s st/dies have
indicated that se, hormones affect mammalian 9idneys, ca/sing se,/al
dimorphism in a variety of morphological and:or f/nctional properties0 Most
of these data 1ere obtained from e,periments in animals, largely rats and
mice, and in in vitro systems; only a limited amo/nt of data is from
h/mans0 <here st/died, se,/al dimorphism 1as fo/nd to be mediated by
the relevant se, hormone receptors, b/t this 1as not a /niversal
concl/sion0 Gender differences in the 9idney mass 1ere first described in
mice and rats abo/t *( years ago and 1ere later also proven for the
h/man 9idneys0 =he ca/se of the higher organ mass in males is not a
different n/mber of nephrons 2the n/mber of glomer/li per 9idney in males
and females is similar3, b/t the anabolic action of androgens largely
affects gro1th 2hypertrophy3 of pro,imal t/b/le cells, so that the main
morphological differences are locali>ed in the 9idney corte,0
2
<hen foc/sing on the specific effects of se,/al hormones on the biology
of the different 9idney cells, estrogens have sho1ed to red/ce mesangial
proliferation and type I and IV collagen synthesis 2*3, 1hile promoting the
activity of metalloproteinases 2'30 =hese observations contrib/te to e,plain
the red/ced prevalence of glomer/losclerosis and the slo1er 9idney
disease progression, observed in the animal models and h/man s/b?ects0
5ecently, apoptosis 2programmed cell death3 has been proposed as a
mechanism by 1hich androgens can ca/se accelerated nephron loss in
men 2"(30 Apoptosis is a energy-dependent process mediated by the
activation of specific genes and it is no1 recogni>ed to play an important
role in reg/lating the n/mber of renal cells in health and disease0
@ells /ndergoing apoptosis present cromatin condensation, n/clear
fragmentation, generation of cytoplasmatic blebs, loss of adhesion and
cell/lar shrin9age, follo1ed by formation of apoptotic bodies that are
capt/red by phagocytes0 At a cell/lar level, apoptosis is ind/ced thro/gh
the seA/ential activation of specific en>ymes, named caspases 2aspartic
specific cysteine proteases30 =1o principal path1ays can lead to the
activation of caspases: the intrinsic one, that involves partecipation of
mitochondria and recr/itment of the Bcl-$ family of proteins, and the
estrinsic one, that follo1s the activation of a death receptor, i0e0 +as, by its
ligand, +asB 2+ig/re "30
Apoptosis is involved in physiologic processes of remodeling both d/ring
development in the immat/re 9idney and d/ring healing after damage in
the mat/re 9idney 2+ig/re $30
Apoptosis may mediate beneficial deletion both of le/9ocytes and of
e,cess resident cells from the inflamed glomer/l/s0 In glomer/lar capillary
repair, apoptosis is necessary in reg/lating the n/mber of intrinsic
endothelial cells in a model of =hy-" nephritis in rats 2""30 An important
role for macrophage apoptosis has also been s/ggested in the resol/tion
3
of fibrocell/lar crescents to an acell/lar fibrotic str/ct/re, in a rat model of
crescentic glomer/lonephritis 2"$30
Apoptosis can also contrib/te to the cell loss and str/ct/ral damage that
1e observe in chronic 9idney diseases 2"#30 Gro1ing evidence s/ggests
that inappropriate engagement of the cell death CprogramC contrib/tes to
the progression of renal diseases s/ch as polycystic 9idney disease and
glomer/lonephritis, mediating the loss of CdesirableC cells and event/al
development of the hypocell/lar end-stage 9idney 2"6,")30 8everal st/dies
have emphasi>ed the cr/cial role of t/b/lointerstitial in?/ry in the
progression of 9idney diseases, indipendently from the ca/se and site of
initial damage0 =/b/lar cell apoptosis is no1adays considered one
important mechanism in the progression to Dnd 8tage 5enal Disease
2D85D30
@yclosporine A, Angiotensin II and hyperglycemia can promote apoptosis
in t/b/lar cells in vitro, by the activation of specific path1ays 2"!-"*30
Moreover apoptosis of podocytes and mesangial cells has been
demonstrated in cell c/lt/re /nder the e,pression of gl/cose-ind/ced
apoptotic signals 2"',$(30 Diabetic db:db mice presented increased
apoptotic inde, in the cortical t/b/lar epitheli/m 2$"3 and enhanced
apoptotic loss of podocytes 2$$30 Moving from in vitro and animal st/dies
to h/mans, apoptosis of 9idney cells has been recently proposed as a
relevant mechanism of glomer/lar and t/b/lar cell loss in patients affected
by type $ diabetic nephropathy and protein/ria 2$#30 Apoptosis 1as abo/t
!- and #-fold higher, respectively, in glomer/li and t/b/les in 9idneys of
patients 1ith early nephropathy compared to normal 9idney and there 1as
no f/rther increase in advanced diabetic nephropathy, s/ggesting that
apoptosis co/ld be an early activated mechanism in the progression of
diabetic nephropathy 2+ig/re #3 2$#30
Androgens are 9no1n to promote apoptosis in h/man vasc/lar endothelial
cells, derived from male /mbilical vein 2$630 In t/rn, endothelial apoptosis
contrib/tes to generation of atherogenesis0 8e, hormones li9ely infl/ence
mesangial cell s/rvival, even if conflicting res/lts derive from different
4
st/dies0 In rat primary c/lt/res, estrogens promoted apoptosis, b/t this
effect 1as also obtained 1ith minimal doses of testosterone 2$)30
<e recently demonstrated that testosterone, at phisiological
concentrations 2"-"( nmol:l3, 1as able to promote apoptosis in a h/man
pro,imal t/b/lar cell line 24E-$3 in a dose dependent 1ay 2"(30 8imilar
res/lts 1ere also obtained in primary c/lt/res of h/man t/b/lar pro,imal
cells, obtained from normal portions of 9idneys s/rgically removed for
renal cell carcinoma0 In both c/lt/re types, apoptosis 1as triggered by
ser/m deprivation for $6-6* ho/rs0
Apoptosis 1as eval/ated by imm/nofl/orescence 2apoptotic cells 1ere
identified as positive for Anne,in V and negative for %ropidi/m Iodide3 and
by imm/nostaining 2=UNDB positive cells30 %re-treatment 1ith fl/tamide
2androgen receptor antagonist3 or "7-estradiol co/ld prevent
testosterone-ind/ced apoptosis at 6* ho/rs0 "7-estradiol per se didnFt
affect cell s/rvival, if added at phisiological concentrations to the c/lt/re
system d/ring ser/m deprivation0
As a follo1ing step 1e tried to investigate the path1ay involved in
apoptosis promotion by testosterone0 =he e,pression of +as, +asB and
+ADD 2+as associating death domain containing protein3 proteins 1ere
increased 1ithin 6* ho/rs of testosterone treatment0 =his effect 1as
prevented by pre-treatment 1ith "7-estradiol0 @aspase-#, the 9ey
en>yme in the apoptotic cascade, 1as activated in testosterone-treated
cells after 6* ho/rs, as assessed by the dectection of the p"7 active
fragment, by imm/nofl/orescence and 1estern blot, and by the cleavage
of its s/bstrate %A5%-I, by imm/nohistochemistry and 1estern blot0
=estosterone-ind/ced apoptosis after 6* ho/r treatment 1as inhibited by
adding inhibitors of caspase -# and -* to the c/lt/re medi/m0
All these data s/ggest that promotion of apoptosis, initiated by
testosterone, involves the activation of the estrinsic path1ay0 4o1ever,
testosterone treatment 1as also associated 1ith changes in e,pression of
proteins belonging to the Bcl-$ family; in fact, increased e,pression of the
pro-apoptotic Ba, 2prevented by fl/tamide3 and red/ction of anti-apoptotic
Bcl-$ 1as observed in treated cells0 Besides, the /se of an inhibitor of
5
caspase-', involved in the intrinsic path1ay of apoptosis, 1as able to
inhibit the pro-apoptotic effect of testosterone0
In concl/sion, these data s/ggest that, in c/lt/red cells from h/man
pro,imal t/b/le, testosterone acts principally by a death- receptor
dependent path1ay, 1ith activation of +ADD and caspase-*0 =his en>yme
can cleave procaspase-#, generating the effective form of the protein, or,
alternatively, activate the intrinsic path1ay, by cleavage of Bid 21hich
belongs to the Bcl-$ protein family3, that enters the mitochondria and
ind/ces release of citochrome @ and caspase-'0
<e have demonstrated that androgens are able to increase s/sceptibility
of h/man t/b/lar cells to apoptosis, activating an apoptotic path1ay that
principally involves increased e,pression of +as and +asB and caspase
activation0 =his apoptotic path1ay can be already activated by different
stim/li in several chronic renal diseases0 8o, the path1ays leading to
programmed death that are activated by androgens are shared by several
conditions, 1ith res/lting cell and nephron loss in chronic diseases0 =hese
observations s/pport the hypothesis that testosterone plays a role in
promoting renal chronic damage in male gender0
References
1. 8ilbiger 85, Ne/garten G0 =he impact of gender on the progression of
chronic renal disease0 Am G Eidney Dis "'');$!:)")-##0
2. Ne/garten G, Acharya A, 8ilbiger 850 Dffect of gender on the
progression of non diabetic renal disease: a metaanalysis0 G Am 8oc
Nephrol $(((;"":#"'-$'0
#0 5ec9elhoff G+, Granger G%0 5ole of androgens in mediating
hypertension and renal in?/ry0 @lin D,p %harmacol %hysiol
"''';$!:"$7-#"0
60 Ne/rgarten G, 8ilbiger 80 Dffects of se, hormones on mesangial cells0
Am G Eidney Dis "'');$!:"67-)"0
)0 Baylis @0 Age-dependent glomer/lar damage in the rat0 Dissociation
bet1een glomer/lar in?/ry and both glomer/lar hypertension and
6
hypertrophy0 Male gender as a primary ris9 factor0 G @lin Invest
"''6;'6:"*$#-'0
6. Goles GA, van Goor 4, Braam B, <ille9es-Eoolschi?n N, Gansen D4,
van =ol A et al. %rotein/ria, lipoproteins and renal apolipoprotein
deposits in /ninephrectomi>ed female analb/minemic rats0 Eidney Int
"'');67: 66$-)#0
70 Goles GA, van Goor 4, van der 4orst MB, van =ol A, <eening GG,
Eoomans 4A0 Hvariectomy decreases plasma triglyceride levels and
both prevents and alleviates glomer/lar disease in /ninephrectomi>ed
female analb/minemic rats0 G Am 8oc Nephrol "''!;7:""*'-'70
8. E1an G, Ne/garten G, 8herman M, Ding H, +otadar U, Bei G et al.
Dffects of se, hormones on mesangial cell proliferation and collagen
synthesis0 Eidney Int "''!; )(:""7#-'0
9. G/ccione M, 8ilbiger 85, Bei G, Ne/garten G0 Dstradiol /preg/lates
mesangial cell MM%-$ activity via transcription factor A%-$0 Am G
%hysiol 5enal %hysiol $(($; $*$:+"!6-'0
10. Ver>ola D, Gandolfo M=, 8alvatore +, Villaggio B, Gianiorio +,
=raverso % et al. =estosterone promotes apoptotic damage in h/man
renal t/b/lar cells0 Eidney Int $((6; !): "$)$-"$!"0
11. 8himi>/ A, Mas/da I, Eitam/ra 4, Ishi>a9i M, 8/gisa9i I, Iamana9a
N0 5ecovery of damaged glomer/lar capillary net1or9 1ith endothelial
cell apoptosis in e,perimental proliferative glomer/lonephritis0
Nephron "''*; 7': $(!-"60
12. Ban 4I, Mits/hashi 4, Ng II, Ni9olic-%aterson DG, Iang N, M/ < et
al. Macrophage apoptosis in rat crescentic glomer/lonephritis0 Am G
%athol "''7; ")": )#"-*0
13. Hrti> A, Bor> @, G/sto %, @atalan M%, Dgido G0 @ontrib/tion of
apoptotic cell death to renal in?/ry0 G @ell Mol Med $(("; ):"*-#$0
14. =ao I, Eim G, +a/bel 8, </ G@, +al9 8A, 8chrier 5< et al. @aspase
inhibition red/ces t/b/lar apoptosis and proliferation and slo1s
disease progression in polycystic 9idney disease0 %roc Natl Acad 8ci
U8A $((); "($: !')6-'0
7
15. Ma9ino 4, 8/giyama 4, Iamasa9i I, Maeshima I, <ada G,
Eashihara N0 Glomer/lar cell apoptosis in h/man l/p/s nephritis0
Vircho1s Arch $((#; 66#: !7-770
16. G/sto %, Bor> @, 8an> A, Dgido G, Hrti> A0 Intracell/lar mechanisms of
cyclosporin A-ind/ced t/b/lar cell apoptosis0 G Am 8oc Nephrol
$((#;"6: #(7$-*(0
17. Bhas9aran M, 5eddy E, 5adha9rishanan N, +ran9i N, Ding G, 8inghal
%@0 Angiotensin II ind/ces apoptosis in renal pro,imal t/b/lar cells0
Am G %hysiol $((#; $*6: + '))-!)0
18. Ver>ola D, Bertolotto MB, Villaggio B, Httonello B, Dallegri +,
+r/mento G et al. =a/rine prevents apoptosis ind/ced by high
ambient gl/cose in h/man t/b/le renal cells0 G Investig Med $(($; )(:
66#-)"
19. <olf G, @hen 8, Jiyadeh +N0 +rom the periphery of the glomer/lar
capillary 1all to1ard the center of disease: podocyte in?/ry comes of
age in diabetic nephropathy0 Daibetes $((); )6: "!$!-#60
20. Mishra 5, Dmancipator 8N, Eern =, 8imonson M80 4igh gl/cose
evo9es an intrinsic proapoptotic signaling path1ay in mesangial cells0
Eidney Int $((); !7: *$-'#0
21. Hrti> A, Jiyadeh +N, Neilson DG0 D,pression of apoptosis-reg/latory
genes in renal pro,imal t/b/lar epithelial cells e,posed to high
ambient gl/cose and in diabetic 9idneys0 G Investg Med "''7; 6):
)(-)!0
22. 8/s>ta9 E, 5aff A@, 8chiffer M, BKttinger D%0 Gl/cose-ind/ced
reactive o,ygen species ca/se apoptosis of podocytes and podocyte
depletion at the onset of diabetic nephropathy0 Diabetes $((!; )):
$$)-##0
23. Ver>ola D, Gandolfo M=, +errario +, 5astaldi M%, Villaggio B,
Gianiorio + et al. Apoptosis in the 9idneys of patients 1ith type II
diabetic nephropathy0 Eidney Int0 $((7 8ep "$0
24. Bing 8, Dai A, <illiams M5I, Myles E, Dilley 5G, Eomesaroff %A,
8/dhir E0 =estosterone enhances apoptosis-related damage in
8
h/man vasc/lar endothelial cells0 Dndocrinology $(($ "6#:
"""'-""$)0
25. 8inghai %@, 5eddy E, 8an1al V, Eapasi A, Gibbons N, Mattana G et
al. Age and se, mod/late renal e,pression of 8G%-$ and
transgl/taminase and apoptosis of splenocytes, thymocytes and
macrophages0 G Investig Med "''7; 6): )!7-7)0
9