www.kidney-international.

org commentary

H2-Aa, Cd74, C1qa, Cxcl16, Hexb,

The transcriptomic signature of Cd81, C1qb, and Cd72) were enriched
in younger mice. Macrophages with
the aging podocyte M1-phenotype signature (expressing
genes such as Hp, Itgal, Msrb1, and
Dhanunjay Mukhi1 and Katalin Susztak1
Gngt2) were enriched in kidneys of
older mice, suggesting that aging is
Aging is the strongest independent risk factor for chronic kidney
associated with proinflammatory
disease. Glomerular epithelial cells are unable to proliferate and have phenotype. This atlas will serve as
limited ability to renew; therefore, podocytes must maintain a delicate important reference to understand
intracellular homeostasis that enables them to function and adapt to aging-associated changes.
stress endured during the human life span. Here, Wang et al. Podocytes are highly differentiated
performed unbiased transcriptomic analysis of aging podocytes and epithelial cells that wrap around the
identified important novel regulators. glomerular capillaries. Podocytes are
connected by a protein complex called
Kidney International (2020) 98, 1079–1081; https://doi.org/10.1016/j.kint.2020.08.004
the slit-diaphragm and have a highly
Copyright ª 2020, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
sophisticated cytoskeleton to adjust to
see basic research on page 1160 mechanical stress and fully cover the
basement membrane.3 Podocyte loss is
the cardinal feature of proteinuria

C
hronic kidney disease dispropor-
tionately affects older in-
dividuals. Such as, using the
standard kidney function (estimated
glomerular filtration rate) calculations,
only 47% of people who are older than
70 have normal kidney function. Several
conserved key pathways have been
shown to affect aging in multiple cell
types, and they are also conserved be-
tween organisms, such as anabolic
hormone signaling (insulin and insulin-
like growth factor), chronic overactiva-
tion of the mechanistic target of rapa-
mycin (mTOR), AMP-activated protein
kinase (AMPK), nicotinamide adenine
dinucleotides (NAD), FOXO/sirtuins,
the resulting endoplasmic reticulum
and mitochondrial stress, cellular senes-
cence, and associated inflammation.1
The exact molecular pathways associated
with aging-associated kidney disease
have not been fully characterized.
The Tabula Muris Senis project per-
formed a comprehensive unbiased
1
Renal Electrolyte and Hypertension Division,
Department of Medicine, Department of Genetics,
Institute of Diabetes Obesity and Metabolism,
University of Pennsylvania, Philadelphia, Penn-
sylvania, USA
Correspondence: Katalin Susztak, Perelman
School of Medicine, University of Pennsylvania,
12-123 Smilow Translational Research Center,
3400 Civic Center Boulevard, Philadelphia,
Pennsylvania 19104, USA. E-mail: ksusztak@
pennmedicine.upenn.edu
transcriptomic analysis of close to 20
organs at 6 different time points to
capture the proteomics and transcrip-
tional signature of aging both at organ
and single-cell levels in mice.2 Not
surprisingly, the kidney showed one of
the most significant age-related
changes. In the kidney, there was a
strong age-associated decline in the
number of endothelial, mesangial, and
loop of Henle cells. These findings
correlate with the decline observed with
glomerular filtration rate and urine-
concentrating capacity during aging.
The investigators identified common
gene expression changes observed in
almost every organ during aging. For
example, both tissue expression and
blood levels of vascular cell adhesion
molecule 1 (Vcam1), periostin (Postn),
and fibroblast growth factor 10 (Fgf10)
showed a strong correlation with age.
Genes from specific ontology groups
such as extracellular matrix regulation,
unfolded protein binding, mitochon-
drial function, and inflammatory and
immune response showed the strongest
association with aging. The in-
vestigators, however, noted mild differ-
ences in the amplitude of these changes
in different organs. The kidney was one
of the organs that showed the most
significant changes in immune cell
diversity. For example, macrophages
with M2 subtype signature (expressing
genes such as Il10, H2-Eb1, H2-Ab1,
leading to glomerulosclerosis and end-
stage renal disease. Podocytes are un-
able to proliferate and have limited
ability to renew; therefore, >20%
podocyte loss results in an irreversible
glomerulosclerosis and decline in
glomerular filtration rate.4 Decrease in
podocyte number provokes residual
podocytes to compensate, such as
enlarging and changing their cytoarch-
itecture. Given similarities between ag-
ing and glomerular disease, it is likely
that shared pathways could play roles in
both.
Despite the critical role of aging in
chronic kidney disease development,
limited attention has been paid to this
important area in nephrology. Recent
reports suggested the roles of
endothelin-1 and renin-angiotensin
system. Both pathways act via G-pro-
tein–coupled receptors, raise intracel-
lular calcium, and could alter the
podocyte cytoskeleton and slit-
diaphragm. Global transcriptome anal-
ysis of aging whole kidney samples
provided limited information on the
precise transcriptome changes in
podocytes, due to cell heterogeneity.
Earlier unbiased single-cell sequencing
has been published for healthy adult
glomeruli, but did not include aging
samples.5 The Tabula Muris Senis
project now includes aged kidney sam-
ples; however, the coverage for
glomerular cells remains poor.2 Bulk

Kidney International (2020) 98, 1079–1097 1079

commentary
RNA-sequencing using isolated cells
offers an alternative, which provides
more in-depth information for a spe-
cific cell type in addition to
affordability.
In this issue, Wang et al.6 investigated
global transcriptome changes in young
(8- to 12-week-old) and aged (88- to 96-
week-old) podocytes. The team used 2
complementary approaches, including
isolating fluorescent-labelled podocytes
from the NPHS2Cre-tdTomato mice and
magnetic bead-based sorting method.
They showed that the transcriptome of
young mice is enriched for canonical
podocyte markers, such as nephrin and
podocin, while the expression of these
markers is decreased in the aged mice.
Gene set enrichment analysis by various
methods identified multiple biological
processes altered in aged podocytes,
which included inflammation, apoptosis,
PI3K-mTOR and p53 pathways, while
fatty acid metabolism and tight junction
proteins are decreased in aged podocytes
(Figure 1). PI3K-mTOR pathway is a
classic mechanism associated with
organismal aging. mTOR is an evolu-
tionary conserved signaling hub that
senses and integrates environmental and
intracellular nutrient and growth factor
signals and coordinates responses such
as cell growth, autophagy, proliferation,
apoptosis, and inflammation depending
on the individual cell and tissue. Rapa-
mycin that targets the mTOR pathway
has shown to extend the life span of
mice; however, rapamycin also has sig-
nificant renal side effects, limiting it
widespread use.7
Genes associated with development
and morphogenesis, such as Wnt, Bmp,
Fgf, and Slit/Robo2, were found to have
lower expression in aging podocytes.
These pathways are very tightly regu-
lated and both lower and higher levels
will lead to glomerular pathology.
While increased expression of Wnt/
Ctnnb1 in podocytes can drive cellular
dedifferentiation, it also associated with
an increase in resistance to apoptosis.
Bmp7 is important for podocyte sur-
vival and it counteracts Tgfb-induced
epithelial-to-mesenchymal transition of
podocytes. Expression of Tgfb was
elevated in aging podocytes altering the
1080
Figure 1 | Podocyte transcriptomic profiling was performed in young and aged
podocytes. Expression of genes associated with development metabolism and endocytosis
was lower in aged mice. Expression of genes associated with inflammation, apoptosis, and
p53 and mTOR was higher. Aging-associated signature genes were regulated by the
transcription factor Grainyhead 2 (Grhl2). FACS, fluorescent activated cell sorter; VIPER,
virtual inference of protein activity by enriched regulon.
Bmp/Tgfb balance. These results sug- gained significant attention as senescent
gest the role of dedifferentiation, cells secrete high levels of inflammatory
apoptosis, and hypertrophy by which cytokines, immune modulators, growth
podocyte number declines during factors, and proteases. The role of SASP
glomerular aging. is to activate the immune system and
Ligand receptor interactions showed target SASP cells for clearance. The
important autocrine and paracrine transcription factor C/EBPb has been
interaction in healthy podocytes. shown to be the major driver of SASP.
Because podocytes likely sense their Recent reports indicate that podocyte
neighboring cells, loss of neighboring specific deletion of C/Ebpb protects
podocytes can activate autocrine cells from glomerular injury and
signaling contributing to decline in senescence (Figure 1). The increase in
kidney function. This study defined inflammatory phenotype, specifically
manually curated podocyte-specific the increase in the numbers of B and T
autocrine and paracrine ligand- lymphocytes, was also observed in the
receptor pair from single-cell RNA- Tabula Muris Senis study.2
sequencing data of healthy glomeruli Wang et al.6 also performed virtual
previously published by Karaiskos et al.8 inference of protein activity by enriched
Using this criterion, the team has regulon (VIPER) analysis. This analysis
identified 55 ligand-receptor pairs as provided some very interesting insight
podocyte-specific and a significant into upstream transcription factors that
decrease in the autocrine signaling in globally regulated gene expression
aged podocytes. Most genes, except Ccl5 changes. The top 10 transcription fac-
and Fn, showed lower expression in tors that may regulate normal aging of
aging animals. podocytes included Hnf1b, Grhl2,
Aging was associated with an in- Sim1, Lrx1, Creb3l1, Zbtb16, Osr2,
crease of senescence-associated secre- Foxo4, Pax8, and Trerf1. The regulation
tory phenotype (SASP) genes, including and expression of some of these tran-
Ccl2, Ccl3, Ccl5, and Ccl8. SASP recently scription factors such as Hnf1b and
Kidney International (2020) 98, 1079–1097
 commentary

Pax8 are surprising as their expression
are considered highly specific to prox-
imal tubule cells. Expression of
Grainyhead-like2 (Grhl2) was especially
interesting, as it is an important regu-
lator of epithelial-to-mesenchymal
transition and apical and basal polar-
ity, including a large number of clau-
dins and cadherins (Figure 1), and
therefore it could play an important
role in aging podocytes.
The study has several caveats,
including the potential activation of
stress response genes induced by the
harsh digestion needed for podocyte
isolation. Decrease in podocyte number
can potentially mask the true transcrip-
tional drivers of aging. Such as Fu et al.9
performed bulk RNA-sequencing on
isolated glomeruli and sorted podocytes
from inducible tagged NPHS2-Cre-GFP
mice and showed that podocyte marker
gene expression was decreased in disease
state. In addition, the fluorescent acti-
vated cell sorter and magnetic isolation
might have introduced a bias in cellular
gene expression, such as cells that did
not fit the usual podocyte prototype, for
example, hypertrophied or dying cells
might not have been used for the anal-
ysis. Single-cell analysis could be a
powerful tool to resolve such changes.
Despite these weaknesses, publicly
available datasets from aging kidneys
showed good correlation with the results
reported by Wang et al.6
The next steps should include func-
tional validation of the identified path-
ways, which will necessitate the
generation of novel genetically modified
animal models and correlate changes in
gene expression and phenotype devel-
opment such as albuminuria and
glomerular filtration rate. Correlation
between mouse models and human
samples will be important to under-
for reduced kidney function and 2. Tabula Muris Consortium. A single-cell
transcriptomic atlas characterizes ageing
chronic kidney disease. tissues in the mouse. Nature. 2020;583:590–
595.
DISCLOSURE 3. Ronco P. Pathophysiology of the glomerulus:
KS serves on the scientific advisory board of KI tells the story. Kidney Int. 2020;97:5–9.
4. Rutkowski JM, Wang ZV, Park AS, et al.
Jnana Therapeutics. The other author
declared no competing interests. Adiponectin promotes functional recovery
after podocyte ablation. J Am Soc Nephrol.
2013;24:268–282.
5. Park J, Liu CL, Kim J, et al. Understanding the
ACKNOWLEDGMENTS
Work in the Susztak lab is supported by kidney one cell at a time. Kidney Int. 2019;96:
862–870.
National Institutes of Health grants R01
6. Wang Y, Eng DG, Kaverina NV, et al. Global
DK076077, R01 DK087635, and R01 transcriptomic changes occur in aged mouse
DK105821, and by funding from the Juvenile podocytes. Kidney Int. 2020;98:1160–1173.
Diabetes Research Foundation Ltd, 7. Huber TB, Edelstein CL, Hartleben B, et al.
Foundation for the National Institutes of Emerging role of autophagy in kidney
Health, GlaxoSmithKline, Gilead, Regeneron, function, diseases and aging. Autophagy.
Boehringer Ingelheim, Merck, and Novo 2012;8:1009–1031.
Nordisk. These funders and KS’s activities 8. Karaiskos N, Rahmatollahi M, Boltengagen A,
have no influence on the presented work. et al. A single-cell transcriptome atlas of the
mouse glomerulus. J Am Soc Nephrol. 2018;29:
2060–2068.
REFERENCES 9. Fu J, Akat KM, Sun Z, et al. Single-cell RNA
1. Nihalani D, Susztak K. Sirt1-Claudin-1 crosstalk profiling of glomerular cells shows dynamic
regulates renal function. Nat Med. 2013;19: changes in experimental diabetic kidney
1371–1372. disease. J Am Soc Nephrol. 2019;30:533–545.
Moore’s law for membranous
nephropathy
Norifumi Hayashi1,2 and Laurence H. Beck Jr2,3
The identification of target antigens in membranous nephropathy has
accelerated since the report of M-type phospholipase A2 receptor 1
(PLA2R1). One could say that technological advances have allowed for
the demonstration of Moore’s law (a doubling every 2 years in the
number of transistors that can be fit onto a computer chip) in the field
of membranous nephropathy, and that even more antigens can be
expected in the near future. In this issue of Kidney International, Sethi
et al. describe semaphorin-3B as a novel target antigen, defining a type
of membranous nephropathy with onset in the pediatric population.
Kidney International (2020) 98, 1081–1084; https://doi.org/10.1016/j.kint.2020.06.020
Copyright ª 2020, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
see clinical investigation on page 1253

stand the relevance of changes observed
in mice for our patient samples.
In summary, this study is a step
forward to advance our understanding
of podocyte aging. The team has
discovered changes in developmental
pathways and metabolism and inflam-
mation. Future studies shall analyze
podocyte aging in patients, as aging
remains the most important risk factor
1
Division of Nephrology, Kanazawa Medical Uni-
versity, Uchinada, Ishikawa, Japan; 2Renal Sec-
tion, Department of Medicine, Boston University
School of Medicine, Boston, Massachusetts, USA;
and 3Department of Medicine, Boston Medical
Center, Boston, Massachusetts, USA
Correspondence: Laurence H. Beck, Jr., Renal
Section, Department of Medicine, Boston Univer-
sity School of Medicine and Boston Medical Cen-
ter, 650 Albany Street, X-536, Boston,
Massachusetts 02118, USA. E-mail: lhbeckjr@bu.
edu
I
t was not so long ago that there was a
disease “entity” known as idiopathic
membranous nephropathy (MN)
defined pathologically by the membra-
nous lesion in the absence of secondary
associations. But just as all histopatho-
logic lesions are now understood to
represent a spectrum of underlying
etiologies at the molecular level, so too
is MN. After a decades-long hunt for

Kidney International (2020) 98, 1079–1097 1081