ABSTRACT Focal segmental glomerulosclerosis (FSGS) shares podocyte damage as an essential pathological finding. Several mechanisms underlying podocyte injury have been proposed, but many

important questions remain. Rho-associated, coiled-coil-containing protein kinase 2 (ROCK2) is a serine/threonine kinase responsible for a wide array of cellular functions. We found that

ROCK2 is activated in podocytes of adriamycin (ADR)-induced FSGS mice and cultured podocytes stimulated with ADR. Conditional knockout mice in which the ROCK2 gene was selectively disrupted

in podocytes (PR2KO) were resistant to albuminuria, glomerular sclerosis, and podocyte damage induced by ADR injection. In addition, pharmacological intervention for ROCK2 significantly

ROCK2-INDUCED METABOLIC REWIRING IN DIABETIC PODOCYTOPATHY Article Open access 08 April 2022 PODOCYTE-SPECIFIC CRB2 KNOCKOUT MICE DEVELOP FOCAL SEGMENTAL GLOMERULOSCLEROSIS Article Open

access 15 October 2021 CDGAP MAINTAINS PODOCYTE FUNCTION AND MODULATES FOCAL ADHESIONS IN A SRC KINASE-DEPENDENT MANNER Article Open access 04 November 2022 INTRODUCTION Focal segmental

glomerulosclerosis (FSGS) is a histologic pattern of glomerular injury with heterogenic causes. Experimental studies have illuminated podocyte injury as a common feature of FSGS that

eventually leads to effacement of the foot processes from the surface of the capillaries and impaired glomerular filtration in which larger molecules, such as proteins, pass into the urine.

Despite the global health burden, current pharmacological therapies cannot fully prevent or treat podocyte damage, and a number of patients with FSGS still progress toward kidney failure.

Achieving a thorough understanding of podocyte biology and kidney care provision is thus becoming increasingly important. Rho-associated, coiled-coil-containing protein kinase (ROCK) is a

ubiquitously expressed serine/threonine kinase that governs a wide range of physiologic or pathologic responses that vary based on the cell lineage and upstream stimulus. For example, ROCK

is a regulator of cell migration and motility through modulation of cytoskeletal rearrangement. Recent studies have shown that kidney ROCK activity is elevated in both animal models of

diabetes and patients with diabetes1,2. Furthermore, the pharmacological suppression of ROCK can prevent or treat diabetic kidney damage by attenuating inflammation and hypoxic

reactions2,3,4. ROCK has two distinct isoforms: ROCK1 and ROCK2. Findings in isoform-specific gene deletion mice argue for the roles of ROCK1 and ROCK2 in the organization of actomyosin

bundles and blood coagulation, respectively5,6. With respect to the kidney, we previously demonstrated that ROCK1 deletion is protective maintaining the glomerular function by promoting

AMP-activated protein kinase-regulated metabolism7. In addition, hyperglycemia alters podocyte homeostasis by inducing dynamin-related protein 1-mediated mitochondrial fission through ROCK1,

which leads to an abnormal kidney function8. In contrast, ROCK2 inhibits peroxisome proliferator-activated receptor α, thereby regulating fatty acid utilization9. Furthermore, ROCK2 has

been shown to mediate vascular inflammation10. However, the role of ROCK2 in FSGS attributed to podocyte damage remains unclear. To address this question and in turn uncover potential

therapeutic targets against FSGS, we investigate the functional aspects of podocyte ROCK2 in vivo and in vitro. ROCK2 is upregulated in ADR-induced glomerular injury, a rodent model of FSGS

characterized by podocyte damage followed by glomerulosclerosis, tubulointerstitial inflammation, and fibrosis. We show that ablation of ROCK2 in podocytes prevents podocyte damage and

albuminuria induced by ADR injection. In addition, we performed pharmacological intervention for ROCK2 to demonstrate protection against podocyte loss and glomerulosclerosis in ADR-injected

mice. RESULTS INDUCTION OF ROCK2 EXPRESSION IN FSGS To gain insight into the potential role of ROCK2 in FSGS, we first assessed its distribution in kidney cell types using the kidney

single-cell RNA sequencing database, Kidney Interactive Transcriptomics (https://humphreyslab.com/SingleCell). As demonstrated in Fig. 1a and b, ROCK2 was distributed in various types of

human and murine kidney cells, including podocytes, mesangial cells, endothelial cells, and tubules. We next examined the ROCK2 expression in patients with FSGS and in ADR-induced FSGS mice.

As shown in Fig. 1c, transcript datasets obtained from Nephroseq (https://www.nephroseq.org) demonstrated a positive correlation between the glomerular expression of ROCK2 and urinary

protein excretion in patients with FSGS, which suggests the association of activated ROCK2 signaling with an abnormal kidney function. In addition, kidney ROCK2 protein levels were elevated

in ADR-injected mice compared to vehicle-injected sham mice (Fig. 1d). We next performed immunohistochemistry on kidney sections of ADR-injected mice to determine the distribution of ROCK2.

In ADR-injected mice, ROCK2 was strongly detected in glomerular cells including podocytes (Fig. 1e). Furthermore, glomeruli obtained from these mice exhibited a significant elevation in

transcript levels of ROCK2 compared with samples isolated from sham mice (Fig. 1f). We then confirmed the expression of ROCK2 in cultured podocytes stimulated with ADR. The expression of

ROCK2 was significantly induced at the mRNA level after ADR exposure (Fig. 1g). Consistent with the transcript levels of ROCK2, its protein expression was elevated following ADR treatment

(Fig. 1h). Taken together, these results support the notion that ROCK2 expression is augmented during ADR-induced podocyte injury. EFFECTS OF PODOCYTE-SPECIFIC ROCK2 DELETION ON GLOMERULAR

INTEGRITY AND PROGRESSION OF KIDNEY FIBROSIS IN MICE To begin testing for functional links between ROCK2 expression and ADR-induced kidney damage, we crossed Rock2-flox mice with mice

expressing Cre recombinase under the control of the Nphs2/Podocin promoter to generate mice with podocyte-specific disruption of ROCK2. Podocyte-specific ROCK2 ablation was confirmed by

immunohistochemistry (Supplementary Fig. 1). For our experiments, we used Nphs/Podocin-Cre+ Rock2fl/fl mice (PR2KO) and Nphs/Podocin-Cre- Rock2fl/fl (WT) littermates. PR2KO mice were viable,

fertile, and without discernable defects in phenotype. To determine the function of podocyte ROCK2 in FSGS, WT and PR2KO mice were administered ADR intravenously (20 mg/kg) 4 weeks before

morphological and biochemical analyses (Fig. 2a). Saline-injected mice served as controls. In the setting of ADR nephropathy, the serum creatinine levels tended to be reduced in PR2KO mice

compared to WT mice, but this difference was not statistically significant (Fig. 2b). However, the selective ablation of ROCK2 in podocytes resulted in significant prevention of the increase

in urinary albumin excretion (Fig. 2c). Kidney sections were then analyzed for histological injuries typically observed in FSGS. In contrast to WT mice, those lacking ROCK2 exclusively in

podocytes were completely impervious to glomerular sclerosis (Fig. 2d). We next counted the number of podocytes by investigating WT1-positive cells in glomeruli. As demonstrated in Fig. 2e,

healthy glomeruli in sham mice showed uninterrupted WT1 staining. While a significant reduction in podocytes was observed in ADR-injected WT mice, PR2KO mice were protected from the loss of

podocytes. The ultrastructure of glomeruli was visualized by TEM. As shown in Fig. 2f and g, TEM micrographs of ADR-injected mice showed widespread foot process effacement and diffuse

thickening of GBM. In contrast, PR2KO mice demonstrated a significant reduction in foot process width and GBM thickness in the setting of FSGS. Because urinary albumin can be reabsorbed in

the tubules, leading to tubulointerstitial injury, we investigated whether or not the ablation of ROCK2 in podocytes influenced damage in the tubular compartment. To this end, we quantified

the area of interstitial fibrosis in WT mice and PR2KO mice subjected to ADR. As in the glomerulus, tubulointerstitial fibrosis was attenuated in PR2KO mice (Fig. 2h). To discern the

molecular mechanisms underlying these beneficial effects of ROCK2 inhibition, we analyzed the gene expression of profibrotic genes in kidney tissues. Concordant with the functional and

microscopic data, quantitative PCR of kidney tissue identified the upregulation of Tgfb1, Pai1, Acta2, and Fn1, which encode transforming growth factor β-1, plasminogen activator

inhibitor-1, α-smooth muscle actin, and fibronectin-1, respectively. These inductions were significantly prevented in the kidneys of PR2KO mice (Fig. 2i). Taken together, these findings show

that specific deficiency of ROCK2 rescued podocyte damage, and as a consequence, glomeruli and interstitial lesions were protected from sclerotic changes. EFFECTS OF PHARMACOLOGICAL

INTERVENTION FOR ROCK2 ON KIDNEY DAMAGE IN ADR-INJECTED MICE As the above results establish the importance of ROCK2 in the progression of FSGS, we next sought to determine the therapeutic

effects of ROCK2 blockade. ADR-injected mice were treated with either SLx-2119, a ROCK2 inhibitor, or vehicle for 2 weeks (Fig. 3a). Treatment with SLx-2119 significantly ameliorated

ADR-induced albuminuria compared to vehicle treatment without affecting serum creatinine levels (Fig. 3b, c). Furthermore, glomerular fibrosis was significantly reduced in

SLx-2119-administered ADR mice compared with vehicle-treated mice (Fig. 3d). Consistent with the results obtained from PR2KO mice, the loss of glomerular podocytes was attenuated in mice

treated with SLx-2119 (Fig. 3e). The ultrastructures of podocytes (Fig. 3f) and GBM (Fig. 3g) were also protected from ADR injury in these mice. As demonstrated in Fig. 3h,

tubulointerstitial fibrosis was significantly inhibited by the pharmacological intervention for ROCK2. To analyze the downstream effects of podocyte ROCK2 on fibrosis mediators, we measured

the mRNA expression of a panel of factors relevant to kidney fibrosis by quantitative PCR (Fig. 3i). We found that SLx-2119 treatment significantly suppressed the induction of profibrotic

genes in the kidney (Fig. 3i). Taken together, these findings indicate that pharmacological ROCK2 inhibition can attenuate kidney histological and functional damage by, at least in part,

suppressing fibrotic regulators in the ADR-induced mouse model. ROLE OF ROCK2 IN CYCLIC NUCLEOTIDE SIGNALING PATHWAYS As a complementary approach, we next investigated the molecular basis by

which ROCK2 blockade provides protective actions on podocytes. As shown in Fig. 4a, transcriptomic profiles were assessed in ADR-stimulated podocytes. The criteria for the detection of

differentially expressed genes in this experiment were an absolute fold-change value of > 2 and high statistical significance (_p_ value < 0.05). We found that 412 mRNAs were

significantly upregulated, while 239 mRNAs were downregulated in ROCK2-inhibited podocytes (Fig. 4b, c). When a gene enrichment analysis was used to identify key pathways driving these

transcriptional changes, the top differentially expressed pathways in these cells included the cyclic GMP (cGMP)-dependent protein kinase (PKG) and cyclic AMP (cAMP) signaling pathways (Fig.

 4d). The upregulated genes involved in cyclic nucleotide signaling included the gene encoding regulator of G protein signaling 2 (RGS2), which prevents the progression of kidney fibrosis11.

Induction of RGS2 was confirmed by quantitative PCR in podocytes treated with ROCK2 inhibitor (Fig. 4e), siRNA against ROCK2 (Fig. 4f), and primary podocytes obtained from PR2KO mice (Fig. 

4g). The protein levels of RGS2 were analyzed using ELISA. The results in Fig. 4h demonstrate that RGS2 protein levels increased with SLx treatment under ADR-stimulated conditions. However,

kidney tissue samples did not yield similar results (Fig. 4i). These findings suggest that ROCK2-mediated regulation of RGS2 may be restricted to podocytes. To assess the role of RGS2 in

mediating ROCK2’s function, we next conducted a TUNEL assay under ADR-stimulated conditions. As demonstrated in Fig. 4j, the number of TUNEL-positive cells was decreased by the treatment of

SLx. However, the protective action of ROCK2 inhibition was partially canceled by the treatment of siRNA against RGS2. These data support the idea that the beneficial actions of ROCK2

inhibition are dependent, at least in part, on RGS2 upregulation. Of the molecules selectively regulated by ROCK2, genome-scale integrated analysis of gene networks in tissues (GIANT)

indicated that cadherin 13 (CDH13) provided the strongest predicted functional connection between ROCK2 and RGS2 selectively in podocytes (Fig. 4g). DISCUSSION Podocytes are highly

specialized epithelial cells that cover the outer layer of the GBM. Since the podocyte loss is a major determinant of both experimental and human FSGS12,13, the identification of potential

therapeutic targets for preventing podocyte damage has clinical importance for the treatment of FSGS. Using genetic and biochemical approaches, we identified ROCK2 as an important mediator

of albuminuria in a murine model of FSGS. We have now provided proof of concept that ROCK2 inhibition attenuated glomerular sclerosis, GBM abnormalities, and subsequent tubulointerstitial

fibrosis. In the present study, we showed that podocyte ROCK2, the expression of which is increased under ADR-stimulated conditions, is a key mediator of histological and functional

abnormalities in FSGS. In earlier loss-of-function studies, we demonstrated the crucial roles of ROCK2 in podocyte health in several proteinuric kidney disease models (i.e. diabetic

nephropathy, obesity-induced glomerulopathy). In addition, ROCK2 inhibition has been shown to be protective against tubulointerstitial fibrosis in a unilateral ureteral obstruction model14.

When considered alongside these previous observations, the current work implicates ROCK2 as a key molecule for the development of a wide range of kidney diseases and thus adds important

public health-related findings. Given the importance of ROCK2 in the podocyte function, elucidating the upstream stimuli governing its gene expression and activation is of particular

interest. Our data indicate that ROCK2 gene expression is upregulated in human and murine models of FSGS. However, the mechanism regulating the elevation of ROCK2 in FSGS remains unclear.

Several circulating factors have been considered pathogenic feed-forward enhancers of ROCK2 in FSGS because these have been detected in the sera of patients and experimental models of

FSGS15. Among these, Hiroki et al. demonstrated that inflammatory stimuli (e.g., angiotensin II, interleukins) increase ROCK2 expression at both mRNA and protein levels16. Inflammatory

cytokines and transforming growth factor β have been shown to upregulate ROCK2 function as evaluated by the extent of a substrate of ROCK217. In addition, ROCK2 can be activated by cleavage

of the auto-inhibitory C-terminus, mediated by caspase 2 or granzyme B18,19. Of note, ROCK2 expression and activity were found to be related16 and regulated by the peripheral clock gene

BMAL120. BMAL1 directly binds to the promoter of ROCK2 in a time-of-day-dependent manner, thereby modulating the time-of-day variation in ROCK2 activity; however, evidence is scant, so

further research is needed. The breadth of our approach, including the analysis of podocyte-specific ROCK2 deletion models in the context of ADR injury, is a strength of this study, which

yielded several clear findings. We found ROCK2 signaling inhibition to be sufficient to prevent or attenuate podocyte loss, along with interstitial fibrosis. However, several studies have

provided compelling evidence supporting the importance of the ROCK1 isoform in podocytes under physiological or pathological conditions. For instance, ROCK1 has been shown to regulate

mitochondrial dynamics in podocytes8. Previous work from our group has shown that ROCK1 regulates AMP-activated protein kinase-mediated fatty acid metabolism. These studies indicate

essential roles of ROCK in mediating the kidney function and structure; however, the fundamental question is whether or not inhibition of both ROCK isoforms provides additional benefits for

proteinuric kidney disease. Future studies aimed at elucidating the interdependency between ROCK1 and ROCK2, through the generation of compound mutant models, will therefore prove

beneficial. The present findings will further expand our current understanding of the role of ROCK2 in podocyte biology. Several attempts have been made to explain the evolution from ROCK2

activation to progressive podocyte injury. In this study, we proposed that the molecular mechanism behind the effect of ROCK2 inhibition on the regulation of podocyte injury is most likely

related to its ability to modulate cyclic nucleotide signaling, a mechanism implicated in the pathogenesis of kidney fibrosis. Our mechanistic investigations exploring the ROCK2-cyclic

nucleotide signaling axis are in agreement with a recent observation showing antifibrotic effects of cGMP activation in unilateral ureteral obstruction models21. In addition, a large body of

literature has reported drugs that elevate the cellular concentration of cGMP to inhibit kidney fibrosis. One gene that stood out as selectively upregulated in podocytes treated with ROCK2

inhibitor was RGS2, which is activated by PKG. Consistent with these findings, RGS2 deficiency has been demonstrated to accelerate kidney inflammation and fibrosis in unilateral ureteral

obstruction models11. The GIANT indicated CDH13 as a mediator of ROCK2-dependent regulation of RGS2; however, whether or not the observed mechanism is due to the direct actions of ROCK2

remains unclear. The present study showed that glomerular podocytes highly expressed ROCK2 in the setting of FSGS. Furthermore, we found that the genetic deletion of ROCK2 in podocytes

prevented histological and functional abnormalities in FSGS mice. This finding prompted us to test whether or not pharmacological intervention for ROCK2 could attenuate kidney abnormalities

seen in FSGS. Chemical inhibition of ROCK2 rescued podocyte damage and in turn kidney fibrosis. In addition to the known beneficial effects of ROCK2 inhibition in diabetic nephropathy and a

unilateral ureteral obstruction model14,17, this study identified novel effects on FSGS. Experiments using selective ROCK2 inhibitors in rodent models of kidney disease generally have

preventive actions on the disease process, including reducing albuminuria and mesangial sclerosis, as well as decreasing GBM thickness. Thus, ROCK2 appeared to be a comprehensive therapeutic

target for preventing or curing an abnormal kidney function, regardless of the etiology. With respect to translating experimental findings into clinical trials, the therapeutic benefits of

ROCK2 inhibition must be carefully weighed against the potential risk of toxicity. However, it should be noted that the safety and feasibility of ROCK2 inhibition in humans has already been

established with belumosudil, an orally available selective ROCK2 inhibitor, in patients with chronic graft-versus-host disease22. In addition, clinical trials with ROCK2 inhibitors

targeting diffuse cutaneous systemic sclerosis are ongoing23. Further trials involving ROCK2 inhibitors with the goal of reducing the risk of an abnormal kidney function and death would

engender additional confidence. We acknowledge the limitation of the mouse background used in this study. In general, C57/BL6 mice are considered resistant to ADR-induced kidney injury as

indicated by serum creatinine levels in our study. The serum levels of creatinine tended to be increased by ADR injection, but this increase was not statistically significant, which is

consistent with a previous report24. An additional issue we could not dissect was the biological significance of ROCK2 as a regulator of chronic kidney disease due to other causes, such as

hypertension, immune dysregulation, and others (e.g. nonproteinuric kidney disease). Because ROCK2 is expressed in a broad range of kidney cells, further studies using other models of

glomerular and tubular disease are warranted. In addition, how podocyte damage evolves into progressive glomerular diseases remains unclear. These limitations should be considered when

interpreting the findings of this study. In conclusion, the present study demonstrates that podocyte ROCK2 is activated in the context of FSGS. Furthermore, genetic and pharmacological

inhibition of ROCK2 significantly attenuated albuminuria and histological abnormalities in ADR-induced nephropathy. Our work sheds light on the molecular regulation of podocyte damage and

provides a more complete understanding of ROCK2 as an orchestrator of kidney homeostasis. METHODS MICE Podocyte-specific ROCK2 knockout (PR2KO) mice were created on C57BL/6 background by

mating the Rock2flox/flox line with Nphs2-Cre mice obtained from The Jackson Laboratory. Rock2flox/flox mice were generated by transgenic insertion of the LoxP site flanking exon 3 of the

Rock2 gene and maintained at our facilities. Mice were kept in a temperature-controlled facility at 22 °C on a daily 12-h light/dark schedule and fed tap water and standard chow _ad

libitum_. In the first set of experiments, a single dose of 20 mg/kg doxorubicin hydrochloride (#D1515; Merck KGaA, Darmstadt, Germany) or vehicle (0.9% NaCl) was injected into the tail vein

of 8-week-old male wild-type (WT) or PR2KO mice to induce ADR nephropathy. It has been suggested that C57BL mice are resistant to ADR-induced kidney damage but tissue injury is inducible at

higher doses (13–25 mg/kg)25,26,27 than those required in BALB/c mice. In the second set of experiments, 6-week-old male C57BL/6J mice were randomly divided into 3 experimental groups as

follows: (1) vehicle; (2) ADR + vehicle; and (3) ADR + SLx-2119. ADR dissolved in 0.9% NaCl was injected intravenously once 2 weeks before SLx-2119 treatment. SLx-2119 (100 mg/kg) was

administered every 12 h via orogastric gavage for 2 weeks. Significant inhibition of kidney ROCK2 activity was confirmed at this concentration in mice as previously demonstrated17. At 12

weeks old, serum and urine samples were collected from individual mice under isoflurane anesthesia. Kidney tissues were snap-frozen in liquid nitrogen and stored at –80 °C. The mice were

observed at least once daily for signs of illness. Any signs of illness including lethargy, rapid breathing, skin discoloration, and paresis were reported in this study. Studies dealing with

animal use were conducted under protocols approved by the Committee on Ethical Animal Care and Use of the Jikei University School of Medicine. HISTOLOGY Kidney samples obtained from mice

were fixed overnight in 10% buffered formalin and embedded in paraffin. Sections 3 μm in thickness were stained with periodic acid-Schiff for microscopic evaluations. Glomerular and

mesangial areas were analyzed in 20 glomeruli per section. The area was quantified by the ImageJ software program (National Institutes of Health, Bethesda, MD). Kidney tissues were processed

for Masson’s trichrome staining to detect the levels of collagen deposition in the kidneys. Photographs were taken in 10 randomly selected areas with an EVOS M5000 Imaging System

(Invitrogen, Waltham, MA). The extent of interstitial and perivascular fibrosis in kidney sections was quantified by the ImageJ software program. Data are expressed as the ratio of stained

area per total tissue area. For immunofluorescence, 3-μm-thick paraffin-embedded sections were deparaffinized and subjected to antigen retrieval in citrate buffer. The sections were stained

with an anti-ROCK2 antibody (#ab71598; Abcam, Cambridge, UK) and an anti-nephrin antibody (#BP5030; OriGene, Rockville, MD). To determine the loss of glomerular podocytes, the sections were

stained with Wilms’ tumor 1 (WT1) antibody (#ab89901; Abcam), and the number of positive cells was counted at least in 20 glomeruli for each mouse. CELL CULTURE No cell lines used in this

study were found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI BioSample. A conditionally immortalized murine podocyte cell line (E11) was

obtained from Cell Line Services, but was not further authenticated after purchase. Mycoplasma-free E11 podocytes were propagated in 10 U/mL murine interferon-γ at 33 °C and then

differentiated by culture for 10-14 days at 37 °C in the absence of interferon-γ28. ROCK2 knockdown podocytes were established by incubating cells with siRNA as described previously9. ROCK2

deletion did not affect the expression levels of ROCK1 in podocytes (Supplementary Fig. 2). GLOMERULAR ISOLATION Mouse glomeruli were isolated as described elsewhere17. In brief, mice were

perfused through the heart with magnetic Dynabeads (Invitrogen). After perfusion, the kidneys were removed, minced into small pieces, and digested by collagenase A in Hanks’ balanced salt

solution buffer. The digested tissue was then filtered through 100-μm and 70-µm cell strainers. Glomeruli containing Dynabeads were collected using a magnet. PRIMARY CULTURE OF MURINE

PODOCYTES Podocyte isolation was performed as previously described28. After removing erythrocytes with ammonium chloride potassium lysis buffer and depleting endothelial cells with CD31

antibody (#102504; Biolegend, San Diego, CA, USA), nephrin-positive cells were isolated from minced mouse kidneys using magnet-activated cell sorting with nephrin antibody (#PA5-25932;

Thermo Fisher Scientific, Waltham, MA, USA). RNA ISOLATION, QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (PCR), AND RNA SEQUENCING Total RNA was prepared from kidney tissues and cultured

podocytes using TRIzol reagent (Invitrogen). Next, 1 µg of total RNA was reverse-transcribed using the iScript RT Reagent Kit (Bio-Rad, Hercules, CA). To analyze the mRNA expression,

real-time quantitative PCR was performed using the Thermal Cycler Dice Real Time System TP800 (Takara Bio, Shiga, Japan) with SYBR Green I fluorescence signals. The transcript levels of

genes were normalized to β-actin and expressed as levels relative to the control. The primer sequences utilized for PCR are presented in Supplemental Table 1. RNA sequencing was performed by

the Illumina NovaSeq6000 platform by Macrogen (Seoul, Korea). PROTEIN ANALYSES ROCK2 protein abundance was detected by Western blot analyses as described previously9. The primary antibodies

used were anti-ROCK2 antibody (#ab71598; Abcam) and anti-β-actin antibody (#sc-47778; Santa Cruz Biotechnology). The molecular size estimation was performed by referencing size markers

present on the membrane during the experiment. RGS2 protein levels were measured using ELISA kit (#abx542128; Abbexa, Cambridge, UK). TRANSMISSION ELECTRON MICROSCOPY (TEM) Kidney samples

were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer overnight at 4 °C and processed at the Electron Microscopy Facility at The Jikei University School of Medicine. The specimens were

then postfixed with 1% osmium tetroxide in the same buffer at 4 °C for 2 h. Dehydration was performed using a graded series of ethanol washes, and then the sample was placed in propylene

oxide and embedded in Epok 812 (Oken, Tokyo, Japan). Ultrathin sections were prepared with a diamond knife, and stained with uranium acetate and lead citrate. The sections were analyzed by a

pathologist with a JEM-1400 Plus transmission electron microscope (JEOL, Tokyo, Japan) at 100 kV. Foot process width and glomerular basement membrane (GBM) thickness were examined in 20

positions of each mouse using the ImageJ software program. The foot process was defined as any connected epithelial segment butting on GBM between two neighboring filtration pores or slits.

The GBM thickness was determined as the distance between the outer limit of the endothelium and the cell membrane of the podocyte foot process. STATISTICS AND REPRODUCIBILITY For each

animal, different investigators were involved. An investigator administered ADR or SLx-2119. This investigator was the only person aware of the treatment group allocation. Other

investigators were unaware of the treatment. Confounders were not controlled as experiments were processed randomly and individually. The sample size was determined based on the literature2.

No data were excluded from the analysis. All experiments were performed in at least three biological replicates (see figure legends), and results are represented as the mean ± standard

error of the mean (n as indicated in the figure legends). Statistical evaluations of two groups were performed by a two-tailed Student’s _t_-test. Data involving more than two groups were

evaluated by an analysis of variance and Bonferroni’s post hoc correction. Pearson’s correlation was used to analyze the associations between ROCK2 levels and urinary protein excretion. A

value of _p_ < 0.05 was considered to be statistically significant. REPORTING SUMMARY Further information on research design is available in the Nature Portfolio Reporting Summary linked

to this article. DATA AVAILABILITY Numerical source data for graphs in the manuscript can be found in Supplementary Data 1 file. RNA sequencing data are available at NCBI database under

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apoptosis by inhibiting the notch signaling pathway in diabetic nephropathy. _Int. J. Mol. Sci_. 18, (2017). https://doi.org/10.3390/ijms18081795. Download references ACKNOWLEDGEMENTS This

work was supported by Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (23K07709, 22K08347, 21K20914, 20K08645); the Astellas Foundation for Research

on Metabolic Disorders; the Japan Diabetes Foundation; the Mochida Memorial Foundation for Medical and the Pharmaceutical Research; and the Ichiro Kanehara Foundation. We thank Yuko Niikura

and Yuki Takemura for their excellent technical assistance with the electronic microscopy. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Division of Diabetes, Metabolism and Endocrinology,

Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan Keiichiro Matoba, Yosuke Nagai, Kensuke Sekiguchi, Shinji Ohashi, Etsuko Mitsuyoshi, Tamotsu

Yokota & Rimei Nishimura * Department of Pathology, The Jikei University School of Medicine, Tokyo, 105-8461, Japan Masayuki Shimoda * Core Research Facilities for Basic Science,

Research Center for Medical Science, The Jikei University School of Medicine, Tokyo, 105-8461, Japan Toshiaki Tachibana * Department of Endocrinology and Diabetes, Fukuoka University School

of Medicine, Fukuoka, 814-0180, Japan Daiji Kawanami * Nomura Hospital, Tokyo, 181-8503, Japan Kazunori Utsunomiya Authors * Keiichiro Matoba View author publications You can also search for

this author inPubMed Google Scholar * Yosuke Nagai View author publications You can also search for this author inPubMed Google Scholar * Kensuke Sekiguchi View author publications You can

also search for this author inPubMed Google Scholar * Shinji Ohashi View author publications You can also search for this author inPubMed Google Scholar * Etsuko Mitsuyoshi View author

publications You can also search for this author inPubMed Google Scholar * Masayuki Shimoda View author publications You can also search for this author inPubMed Google Scholar * Toshiaki

Tachibana View author publications You can also search for this author inPubMed Google Scholar * Daiji Kawanami View author publications You can also search for this author inPubMed Google

Scholar * Tamotsu Yokota View author publications You can also search for this author inPubMed Google Scholar * Kazunori Utsunomiya View author publications You can also search for this

author inPubMed Google Scholar * Rimei Nishimura View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS K.M. designed and performed the research,

analyzed the data, and wrote the manuscript. Y.N. helped with glomerular isolation. K.S., S.O. and E.M. assisted DNA genotyping. M.S. and T.T. helped with the histological examinations.

D.K., K.U., Y.T. and R.N. wrote the manuscript. All authors read and commented on the manuscript. CORRESPONDING AUTHOR Correspondence to Keiichiro Matoba. ETHICS DECLARATIONS COMPETING

INTERESTS The authors declare no competing financial interests. PEER REVIEW PEER REVIEW INFORMATION _Communications Biology_ thanks Joan Krepinsky and the other, anonymous, reviewer(s) for

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and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Matoba, K., Nagai, Y., Sekiguchi, K. _et al._ Deletion of podocyte Rho-associated, coiled-coil-containing protein kinase 2 protects mice

from focal segmental glomerulosclerosis. _Commun Biol_ 7, 402 (2024). https://doi.org/10.1038/s42003-024-06127-3 Download citation * Received: 19 July 2023 * Accepted: 29 March 2024 *

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