The Protective Role of KANK1 in Podocyte Injury

Approximately 30% of steroid-resistant nephrotic syndromes are attributed to monogenic disorders that involve 27 genes. Mutations in KANK family members have also been linked to nephrotic syndrome; however, the precise mechanism remains elusive. To investigate this, podocyte-specific Kank1 knockout mice were generated to examine phenotypic changes. In the initial assessment under normal conditions, Kank1 knockout mice showed no significant differences in the urinary albumin-creatinine ratio, blood urea nitrogen, serum creatinine levels, or histological features compared to controls. However, following kidney injury with adriamycin, podocyte-specific Kank1 knockout mice exhibited a significantly higher albumin-creatinine ratio and a significantly greater sclerotic index than control mice. Electron microscopy revealed more extensive foot process effacement in the knockout mice than in control mice. In addition, KANK1-deficient human podocytes showed increased detachment and apoptosis following adriamycin exposure. These findings suggest that KANK1 may play a protective role in mitigating podocyte damage under pathological conditions.


Introduction
Nephrotic syndrome (NS) is characterized by massive proteinuria, hypoalbuminemia, and systemic edema [1] and is categorized into steroid-sensitive NS (SSNS) and steroidresistant NS (SRNS).Notably, approximately 50% of SRNS cases progress to end-stage renal disease within 15 years [2].Clinically, SRNS often presents as focal segmental glomerulosclerosis (FSGS), with genetic factors implicated in 5-10% of adult cases.[3].Recent studies have identified that nearly 30% of SRNS cases result from mutations in 1 of 27 specific genes [4].The proteins encoded by 27 genes act as functional proteins (e.g., glomerular slit membrane components, laminin/integrin signaling components, actin-binding proteins, actin-regulating small GTPases, lysosomal proteins, transcription factors, and proteins of coenzyme Q10 biosynthesis) [5].With advancements in next-generation sequencing (NGS), the discovery of additional related genes is anticipated.For instance, KANK1, KANK2, and KANK4 have been associated with NS [6], despite not being among the 27 previously identified genes [4].KANK family proteins play a role in actin regulation, which might be similar to ARHGDIA, the mutation of which is reported to cause SRNS [5].
KANK1, known as the KN motif and ankyrin repeat domain 1, has been recognized as a tumor suppressor gene in renal cell carcinoma [7].The KANK family, which encompasses KANK1 through KANK4, is characterized by the presence of KN motifs, coiled-coil domains, and ankyrin repeat domains (ANKRDs) [8,9].KANK1 is predominantly cytoplasmic and plays a pivotal role in the organization of the actin cytoskeleton [10], cell polarity [11], and focal adhesions [12][13][14].KANK1 exists in two isoforms; the longer variant is tissuespecific, with predominant expression in the heart and kidneys [15].KANK1 has been identified as 1 of the 677 genes significantly enriched in human glomeruli [16].
Recent research has linked the KANK1 gene with nephrotic syndrome [6].Although Kank1 RNAi mice exhibited a tendency towards proteinuria, the results were not statistically significant [17].Given the conservation of the gene between humans and zebrafish, a kank1 knockout zebrafish model was developed, featuring a C-to-T point mutation in exon 3 of the kank1 gene.The dye filtration assay in zebrafish has proven to be an effective tool for examining glomerular permeability [18,19].In preliminary experiments using this model, 500 kDa fluorescein isothiocyanate (FITC)-and 10 kDa rhodamine-conjugated dextran were injected into kank1 knockout or wild-type (WT) zebrafish embryos.The kank1 knockout group displayed an uptake of 500 kDa dextran in the proximal tubule, unlike the WT group.Based on this observation, we hypothesized that podocyte-specific Kank1 inactivation in mice might manifest as a kidney phenotype.

KANK1 Protein Is Expressed in Both Human and Mouse Podocytes
Co-immunofluorescence studies on frozen human kidney sections revealed that KANK1 was localized in the glomeruli, exhibiting co-localization with the podocyte-specific protein NPHS1 (Figure 1a).In addition, KANK1 expression was observed in the tubular basement membranes, albeit with minimal co-localization with SGLT2, a protein present in the brush borders of the proximal tubules (Supplementary Figure S1).In mouse models, KANK1 expression was similarly observed in the glomeruli, where it co-localized with the podocyte-specific protein SYNPO (Figure 1b).

Kank1 Was Successfully Knocked Out in Mouse Podocytes
Kank1 was engineered to contain loxP sites in the 5th and 9th introns of Kank1 (Figure 2a).To specifically target and remove exons 6-9 in podocytes, we used mice carrying the podocin-Cre (pod-Cre Tg/+ ) gene.By crossing Kank1 flox/flox (Kank1 fl/fl ) mice with pod Cre Tg/+ mice, we generated offspring using various combinations of these genes, including Kank1 fl/fl , Kank1 fl/fl pod-Cre Tg/+ , and Kank1 +/+ pod-Cre Tg/+ mice.The resulting genotypes were discerned by distinct band patterns during genotyping: Kank1 +/+ pod-Cre Tg/+ mice exhibited a 459-bp band, while Kank1 fl/fl and Kank1 fl/fl pod-Cre Tg/+ mice displayed a 587-bp band (Fig ure 2b).Notably, a 102-bp band was present in Kank1 +/+ pod-Cre Tg/+ and Kank1 fl/fl pod-Cre Tg/ mice but absent in Kank1 fl/fl mice (Figure 2b).Subsequent real-time polymerase chain reac tion (PCR) revealed a marked reduction in Kank1 mRNA expression in the glomeruli o 0.26, p = 0.0001, Figure 2c).Western blotting corroborated these findings, showing undetectable KANK1 protein levels in the Kank1 fl/fl pod-Cre Tg/+ group (Figure 2d).Supplementary Figure S2 shows full blots.Immunofluorescence of frozen mouse kidney sections confirmed the diminished expression of KANK1 in the glomeruli of Kank1 fl/fl pod-Cre Tg/+ mice, in stark contrast to the clear expression observed in the Kank1 +/+ pod-Cre Tg/+ and Kank1 fl/fl groups (Figure 2e).Genotyping revealed a 459-bp band in Kank1 +/+ pod-Cre Tg/+ mice, whereas a 587-bp band was present in both Kank1 fl/fl and Kank1 fl/fl pod-Cre Tg/+ mice using Kank1 primers.A 102-bp band was observed in both Kank1 +/+ pod-Cre Tg/+ and Kank1 fl/fl pod-Cre Tg/+ mice, but it was absent in Kank1 fl/fl mice.(c) Kank1 mRNA expression was significantly reduced in the glomeruli of Kank1 fl/fl pod-Cre Tg/+ mice compared to Kank1 fl/fl mice at 6 months (*** p < 0.001).(d) A Western blot analysis showed that KANK1 protein expression was not detectable in the glomeruli isolated from Kank1 fl/fl pod-Cre Tg/+ mice, in contrast to Kank1 fl/fl mice at six months.β-actin expression was similar between the Kank1 fl/fl and Kank1 fl/fl pod-Cre Tg/+ groups.(e) At two months, KANK1 protein expression was significantly reduced in the glomeruli of Kank1 fl/fl pod-Cre Tg/+ mice when compared to the Kank1 +/+ pod-Cre Tg/+ group or Kank1 fl/fl group.Scale bars indicate 50 µm.Genotyping revealed a 459-bp band in Kank1 +/+ pod-Cre Tg/+ mice, whereas a 587-bp band was present in both Kank1 fl/fl and Kank1 fl/fl pod-Cre Tg/+ mice using Kank1 primers.A 102-bp band was observed in both Kank1 +/+ pod-Cre Tg/+ and Kank1 fl/fl pod-Cre Tg/+ mice, but it was absent in Kank1 fl/fl mice.(c) Kank1 mRNA expression was significantly reduced in the glomeruli of Kank1 fl/fl pod-Cre Tg/+ mice compared to Kank1 fl/fl mice at 6 months (*** p < 0.001).(d) A Western blot analysis showed that KANK1 protein expression was not detectable in the glomeruli isolated from Kank1 fl/fl pod-Cre Tg/+ mice, in contrast to Kank1 fl/fl mice at six months.β-actin expression was similar between the Kank1 fl/fl and Kank1 fl/fl pod-Cre Tg/+ groups.(e) At two months, KANK1 protein expression was significantly reduced in the glomeruli of Kank1 fl/fl pod-Cre Tg/+ mice when compared to the Kank1 +/+ pod-Cre Tg/+ group or Kank1 fl/fl group.Scale bars indicate 50 µm.

The Inactivation of Kank1 in Podocytes at Two or Six Months Did Not Result in Albuminuria
At two months, the urinary albumin-creatinine ratio (ACR) was consistent across the Kank1 +/+ pod-Cre Tg/+ , Kank1 fl/fl, and Kank1 fl/fl pod-Cre Tg/+ groups (Figure 3a).Similarly, no notable differences were observed in the blood urea nitrogen (BUN) or serum creatinine (Cr) levels among the groups (Figure 3b).However, the sclerotic index was significantly higher in the Kank1 fl/fl pod-Cre Tg/+ group than in the Kank1 fl/fl group (0.59 ± 0.15 vs. 0.34 ± 0.08, p = 0.0201), with no marked difference when compared to the Kank1 +/+ pod-Cre Tg/+ group (Figure 3c).The fibrotic index showed no significant differences among the three groups (Figure 3c).In addition, an electron microscopic analysis revealed no significant disparities between the groups at this age (Figure 3d).At six months, the urinary ACR remained stable in the Kank1 fl/fl pod-Cre Tg/+ group compared to that in the Kank1 fl/fl group (Supplementary Figure S3a).Similarly, there was no significant difference in BUN or Cr levels between Kank1 fl/fl and Kank1 fl/fl pod-Cre Tg/+ mice at six months (Supplementary Figure S3b).However, both sclerotic (0.86 ± 0.41 vs. 0.31 ± 0.25, p = 0.0181) and fibrotic (0.19 ± 0.11 vs. 0.08 ± 0.03, p = 0.0433) indices were significantly higher in the Kank1 fl/fl pod-Cre Tg/+ group than in the Kank1 fl/fl group (Supplementary Figure S3c).At one year, the urinary ACR showed no significant difference between the Kank1 fl/fl and Kank1 fl/fl pod-Cre Tg/+ groups (Figure 4a).Similarly, BUN and Cr levels were comparable between the two groups at this age (Figure 4b).There was no significant difference in sclerotic or fibrotic indices between the two groups (Figure 4c).Furthermore, electron microscopic analysis revealed no notable disparities in the results between the groups (Figure 4d).At one year, the urinary ACR showed no significant difference between the Kank1 fl/fl and Kank1 fl/fl pod-Cre Tg/+ groups (Figure 4a).Similarly, BUN and Cr levels were comparable between the two groups at this age (Figure 4b).There was no significant difference in sclerotic or fibrotic indices between the two groups (Figure 4c).Furthermore, electron microscopic analysis revealed no notable disparities in the results between the groups (Fig- ure 4d).albumin-creatinine ratio (ACR) at one year between the Kank1 fl/fl group and the Kank1 fl/fl pod-Cre Tg/+ group.(b) The blood urea nitrogen (BUN) and serum creatinine (Cr) levels were comparable between the two groups at one year.(c) There was no significant difference in sclerotic or fibrotic index between the two groups.Scale bars indicate 20 µm.(d) There was no significant difference in electron microscopic results between the two groups at one year.Scale bars indicate 4 µm.

Compensation by other Members of the Kank Family Was Not Evident in Podocyte-Specific Kank1 Knockout Mice
One possible explanation for the observed outcomes under normal conditions is that compensatory mechanisms from other members of the Kank family, specifically Kank2, Kank3, and Kank4, may mitigate phenotypic effects in the Kank1 fl/fl pod-Cre Tg/+ group.However, real-time PCR analyses conducted on the glomeruli of Kank1 fl/fl pod-Cre Tg/+ mice yielded results consistent with those obtained from Kank1 fl/fl mice (Figure 5a-c).

Albuminuria and Glomerular Sclerosis Evident in Podocyte-Specific Kank1 Knockout Mice under Pathological Conditions
To investigate potential phenotypic differences under disease conditions, we induced kidney injury in adriamycin-treated mice.Following a two-week period post-administration, we observed a significant increase in the urinary ACR in the Kank1 fl/fl pod-Cre Tg/+ group compared to the Kank1 fl/fl group.The logarithmic urinary ACR was also significantly higher in the Kank1 fl/fl pod-Cre Tg/+ group than in the Kank1 fl/fl group (7.4 ± 1.1 vs. 5.5 ± 0.8, p = 0.0157; Figure 6a).No significant differences were detected in BUN or Cr levels between the two groups (Figure 6b).Post-adriamycin treatment, the sclerotic index was significantly elevated in the Kank1 fl/fl pod-Cre Tg/+ group compared to the Kank1 fl/fl group (0.46 ± 0.09 vs. 0.29 ± 0.04, p = 0.0056), although the fibrotic index showed no significant difference (Figure 6c).An electron microscopic analysis revealed a marked decrease in the density of foot processes per micrometer of glomerular basement membranes in the Kank1 fl/fl pod-Cre Tg/+ group relative to the Kank1 fl/fl group (1.00 ± 0.18 vs. 1.50 ± 0.15, p = 0.0051; Figure 6d).

Albuminuria and Glomerular Sclerosis Evident in Podocyte-Specific Kank1 Knockout Mice under Pathological Conditions
To investigate potential phenotypic differences under disease conditions, we induced kidney injury in adriamycin-treated mice.Following a two-week period post-administration, we observed a significant increase in the urinary ACR in the Kank1 fl/fl pod-Cre Tg/+ group compared to the Kank1 fl/fl group.The logarithmic urinary ACR was also significantly higher in the Kank1 fl/fl pod-Cre Tg/+ group than in the Kank1 fl/fl group (7.4 ± 1.1 vs. 5.5 ± 0.8, p = 0.0157; Figure 6a).No significant differences were detected in BUN or Cr levels between the two groups (Figure 6b).Post-adriamycin treatment, the sclerotic index was significantly elevated in the Kank1 fl/fl pod-Cre Tg/+ group compared to the Kank1 fl/fl group (0.46 ± 0.09 vs. 0.29 ± 0.04, p = 0.0056), although the fibrotic index showed no significant difference (Figure 6c).An electron microscopic analysis revealed a marked decrease in the density of foot processes per micrometer of glomerular basement membranes in the Kank1 fl/fl pod-Cre Tg/+ group relative to the Kank1 fl/fl group (1.00 ± 0.18 vs. 1.50 ± 0.15, p = 0.0051; Figure 6d).

Generation of the Human Immortalized KANK1 Knockout (KANK1KO) Podocyte Cell Line
The observation of a link between Kank1 deficiency and kidney dysfunction in a mouse model prompted us to establish an in vitro human KANK1KO podocyte model.We investigated whether or not similar effects occurred in KANK1-deficient human podocytes.Rhodamine-phalloidin staining revealed KANK1 protein localization in the podocyte cytoplasm, specifically at the actin fiber edges (Figure 7a).Despite the nonspecific presence of nuclear stain, even without the primary KANK1 antibody, CRISPR-Cas9 technology facilitated the development of a KANK1KO podocyte cell line.This revealed a homozygous insertion (c.110 ins A) in exon 2 of KANK1, leading to a frameshift, premature stop codon, and truncated p.Y37* protein (Figure 7b).In KANK1KO podocytes, KANK1 protein expression at approximately 180 kDa was undetectable, unlike the visible band in WT podocytes (Figure 7c).Supplementary Figure S4 shows the full blot.Following adriamycin treatment, KANK1KO podocytes exhibited a significant reduction in cell attachment and an increase in apoptosis compared to WT podocytes (Figure 7d,e), emphasizing the protective role of the gene against podocyte injury.

Generation of the Human Immortalized KANK1 Knockout (KANK1KO) Podocyte Cell Line
The observation of a link between Kank1 deficiency and kidney dysfunction in a mouse model prompted us to establish an in vitro human KANK1KO podocyte model.We investigated whether or not similar effects occurred in KANK1-deficient human podocytes.Rhodamine-phalloidin staining revealed KANK1 protein localization in the podocyte cytoplasm, specifically at the actin fiber edges (Figure 7a).Despite the nonspecific presence of nuclear stain, even without the primary KANK1 antibody, CRISPR-Cas9 technology facilitated the development of a KANK1KO podocyte cell line.This revealed a homozygous insertion (c.110 ins A) in exon 2 of KANK1, leading to a frameshift, premature stop codon, and truncated p.Y37* protein (Figure 7b).In KANK1KO podocytes, KANK1 protein expression at approximately 180 kDa was undetectable, unlike the visible band in WT podocytes (Figure 7c).Supplementary Figure S4 shows the full blot.Following adriamycin treatment, KANK1KO podocytes exhibited a significant reduction in cell attachment and an increase in apoptosis compared to WT podocytes (Figure 7d,e), emphasizing the protective role of the gene against podocyte injury.protein was not detectable at approximately 180 kDa in KANK1KO podocytes, while there was a visible band in wild-type (WT) immortalized podocytes.The expression of β-actin was comparable between the two groups.(d) Attached cells were significantly decreased from 24 h after adriamycin treatment in KANK1KO podocytes compared to WT podocytes (94.7% ± 13.6% vs. 61.1% ± 24.6% after 24 h, * p < 0.05, 78.2% ± 13.8% vs. 29.2%± 29.5% after 48 h, ** p < 0.01).(e) The percentage of apoptotic cells was significantly increased in KANK1KO podocytes compared to WT podocytes (** p < 0.01), and the percentage was increased significantly after adriamycin treatment (** p < 0.01).

Discussion
In this study, we successfully demonstrated the suppression of the Kank1 gene in podocytes at both the mRNA and protein levels using podocyte-specific Kank1 knockout mice.The KANK1 protein, which co-localizes with podocyte-specific markers in humans, was confirmed to be expressed in podocytes.Its localization coincided with that of actin fiber markers, consistent with previous findings of its association with the actin cytoskeleton [9].Under normal conditions, no notable phenotypic alterations were detected in podocyte-specific Kank1 knockout mice for up to one year, which is consistent with another report of the Kank1 knockout mouse model [20].Compensation by other members of the Kank family was not evident in the present study.However, following adriamycin-induced renal injury, the mice manifested a pronounced elevation in the urinary ACR and sclerotic index relative to controls.In addition, human KANK1KO podocytes displayed an increased propensity for detachment and apoptosis following adriamycin exposure.Overall, these findings highlight KANK1's potential as a protective factor against podocyte injury in disease states.
In the mouse models developed in this study, the omission of exon 6-9 in Kank1 led to a frameshift mutation, resulting in a premature stop codon.Consequently, the KANK1 protein was truncated, reducing its length from 1360 to 1052 amino acids and lacking 5 ANKRDs.Recent studies have elucidated the function of ANKRDs [21], and loss of domain function can lead to the results in the present study.Previous research has indicated that a singular rare variant in KANK1 and three in KANK2 were associated with minimal change nephrotic syndrome (MCNS), while two rare variants in KANK4 were linked to FSGS [6], aligning with the mild phenotype observed in podocyte-specific Kank1 knockout mice.In addition, a study on Kank1 RNAi mice reported no significant increase in proteinuria [17], corroborating our findings.Although KANK1 may not be a direct causative factor, the results of the present study suggest that KANK1 can contribute to albuminuria.
In light of previous research suggesting that adriamycin exposure results in certain phenotypes [22], we administered this compound to both Kank1 fl/fl pod-Cre Tg/+ and Kank1 fl/fl mice.Following adriamycin-induced injury, Kank1 fl/fl pod-Cre Tg/+ mice exhibited a notable increase in the urinary ACR and sclerotic index relative to Kank1 fl/fl mice.Considering that elevated serum hemopexin levels have been implicated as a permeability factor in patients with MCNS [23][24][25], we assessed these levels in both mouse models before and after adriamycin administration.The results indicated a significant elevation in serum hemopexin levels in Kank1 fl/fl pod-Cre Tg/+ mice post-treatment, whereas no such increase was observed in Kank1 fl/fl mice (Supplementary Figure S5).
There are several limitations associated with the current study.First, we did not perform experiments involving alternative inducible mouse models other than the Adriamycin model.Second, the gene expression analyses were not performed in Kank1 knockout mice or control mice in a healthy state or during renal damage.Third, other urinary biomarkers were not tested in the current mouse model.Fourth, the effects of drugs used for nephrotic syndrome, such as angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, sodium-glucose cotransporter-2 inhibitors, and steroids, were not tested in the current mouse model.Fifth, only one KANK1KO podocyte cell line was generated; however, the knockout of KANK1 was observed at the genomic and protein levels, while artifacts in other genes could not be excluded.Finally, primary glomerular culture could not be performed due to technical problems.
In summary, our study revealed that podocyte-specific Kank1 knockout mice, following adriamycin-induced kidney injury, display a significant rise in ACR, an augmented sclerotic index, and enhanced foot process effacement compared to their control counterparts.Furthermore, KANK1KO podocytes exhibited an increased susceptibility to detachment after adriamycin treatment, correlating with a greater propensity for apoptosis.Taken together, these results suggest that KANK1 may play a protective role in mitigating podocyte damage under pathological conditions.Therapeutic strategies for KANK1 gene mutations have not yet been established, and further research is required.As it is predicted that those with KANK1 gene mutations are more likely to develop kidney diseases, it is important for people with KANK1 gene mutations to be aware of the risk of developing kidney diseases and to strive to protect their kidneys.

Mice
C57BL/6 Kank1 fl/fl mice were generated by inserting loxP sites in introns 5 and 9 of the Kank1 gene using Cyagen (Santa Clara, CA, USA).C57BL/6 pod-Cre Tg/+ mice were purchased from the Jackson Laboratory (Sacramento, CA, USA).All experimental protocols were approved by the Animal Care and Use Committee of Mie University (No. 27-32), and all experiments were performed in accordance with approved guidelines.

Isolation of Mouse Glomeruli
Glomeruli were isolated from Kank1 fl/fl and Kank1 fl/fl pod-Cre Tg/+ mice at six months by a perfusion method involving magnetic beads with collagenase digestion for RNA and without collagenase digestion for protein experiments [27].

Western Blotting
Glomeruli were isolated from six-month-old Kank1 fl/fl or Kank1 fl/fl pod-Cre Tg/+ mice without collagenase digestion.The samples were separated on NuPAGE 4-12% Bis-Tris gels (ThermoFisher Scientific) and transferred to polyvinylidene difluoride membranes.After blocking with Tris-buffered saline containing 0.1% Tween 20 and 5% milk, the upper membrane was incubated with anti-KANK1 antibody (1:1000) (ATLAS ANTIBOD-IES, Bromma, Sweden) at 4 • C overnight.The membrane was then incubated for 1 h at room temperature with horseradish peroxidase (HRP)-labeled donkey anti-rabbit antibody (1:3000) (GE Healthcare, Chicago, IL, USA) and developed using Amersham ECL Prime (GE Healthcare) according to the manufacturer's instructions.The lower membrane (<100 kDa) was incubated with anti-β-actin antibody (1:2000) (Cell Signaling, Danvers, MA, USA) at 4 • C overnight, incubated for 1 h at room temperature with HRP-labeled sheep anti-mouse antibody, and developed in the same way.

Immunofluorescence Studies in Mice
Snap-frozen kidney blocks from two-month-old animals were prepared in Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan) and sliced into 10 µm sections, and thereafter, they were made on silane-coated microslides (MUTO PURE CHEMICALS, Tokyo, Japan).The sections were fixed in acetone for 10 min at −20 • C.After blocking with 10% normal goat serum in PBS for 60 min, the sections were incubated with anti-KANK1 antibody (1:500; ATLAS ANTIBODIES) or no primary antibody overnight at 4 • C.After washing with PBS, the sections were incubated with goat anti-rabbitAlexaFluor488 (1:500) at room temperature for 1 h and DAPI (1:3000) for 5 min and then examined using an FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).

Urine and Blood Analyses
Urinary ACR, BUN, and Cr were measured in the Kank1 +/+ pod-Cre Tg/+ , Kank1 fl/fl , and Kank1 fl/fl pod-Cre Tg/+ groups at 2 months (n = 6) and the Kank1 fl/fl group, and the Kank1 fl/fl pod-Cre Tg/+ group at 6 months or 1 year (n = 6 each).The urinary ACR was measured using an immunoturbidimetric method for urinary albumin (Shibayagi, Gunma, Japan) and an enzymatic assay for urinary creatinine (Wako, Tokyo, Japan).BUN and Cr levels were measured as previously described [29].

Transmission Electron Microscopic Analyses
Mouse kidneys were cut into 1-2 mm blocks, pre-fixed in 2.5% glutaraldehyde and 2% PFA in 0.1 M phosphate buffer (pH 7.4), and post-fixed in 2% osmium tetra-oxide for 2 h at 4 • C. The samples were dehydrated in graded ethanol and embedded in epoxy resin.Ultrathin sections stained with uranyl acetate for 10 min and lead staining solution for 5 min were examined using a HITACHI H-700 (Hitachi, Tokyo, Japan).To assess the extent of podocyte foot process effacement, foot processes along at least 400 µm of the total glomerular basement membrane length were quantified in 4 glomeruli of the Kank1 fl/fl or Kank1 fl/fl pod-Cre Tg/+ group after adriamycin treatment (each n = 4) using the ImageJ software (version 1.54) program (National Institutes of Health, Bethesda, MD, USA), as previously reported [31,32].

Detachment Assays
WT and KANK1KO podocyte cell lines were separately cultured in 24-well plates.The number of cells per field was counted to establish a baseline number.The cells were treated with 0.5 µg/mL adriamycin for a total of 48 h.The detachment assay was modified according to a previous study [34].Specifically, the number of detached cells in 5 wells was evaluated at pre-treatment, 24 h after treatment, and 48 h after treatment in 3 independent experiments.

Figure 2 .
Figure 2. Successful knockout of podocyte-specific Kank1.(a) LoxP sites were strategically inserted into introns 5 and 9 of the Kank1 gene.(b)Genotyping revealed a 459-bp band in Kank1 +/+ pod-Cre Tg/+ mice, whereas a 587-bp band was present in both Kank1 fl/fl and Kank1 fl/fl pod-Cre Tg/+ mice using Kank1 primers.A 102-bp band was observed in both Kank1 +/+ pod-Cre Tg/+ and Kank1 fl/fl pod-Cre Tg/+ mice, but it was absent in Kank1 fl/fl mice.(c) Kank1 mRNA expression was significantly reduced in the glomeruli of Kank1 fl/fl pod-Cre Tg/+ mice compared to Kank1 fl/fl mice at 6 months (*** p < 0.001).(d) A Western blot analysis showed that KANK1 protein expression was not detectable in the glomeruli isolated from Kank1 fl/fl pod-Cre Tg/+ mice, in contrast to Kank1 fl/fl mice at six months.β-actin expression was similar between the Kank1 fl/fl and Kank1 fl/fl pod-Cre Tg/+ groups.(e) At two months, KANK1 protein expression was significantly reduced in the glomeruli of Kank1 fl/fl pod-Cre Tg/+ mice when compared to the Kank1 +/+ pod-Cre Tg/+ group or Kank1 fl/fl group.Scale bars indicate 50 µm.

Figure 2 .
Figure 2. Successful knockout of podocyte-specific Kank1.(a) LoxP sites were strategically inserted into introns 5 and 9 of the Kank1 gene.(b)Genotyping revealed a 459-bp band in Kank1 +/+ pod-Cre Tg/+ mice, whereas a 587-bp band was present in both Kank1 fl/fl and Kank1 fl/fl pod-Cre Tg/+ mice using Kank1 primers.A 102-bp band was observed in both Kank1 +/+ pod-Cre Tg/+ and Kank1 fl/fl pod-Cre Tg/+ mice, but it was absent in Kank1 fl/fl mice.(c) Kank1 mRNA expression was significantly reduced in the glomeruli of Kank1 fl/fl pod-Cre Tg/+ mice compared to Kank1 fl/fl mice at 6 months (*** p < 0.001).(d) A Western blot analysis showed that KANK1 protein expression was not detectable in the glomeruli isolated from Kank1 fl/fl pod-Cre Tg/+ mice, in contrast to Kank1 fl/fl mice at six months.β-actin expression was similar between the Kank1 fl/fl and Kank1 fl/fl pod-Cre Tg/+ groups.(e) At two months, KANK1 protein expression was significantly reduced in the glomeruli of Kank1 fl/fl pod-Cre Tg/+ mice when compared to the Kank1 +/+ pod-Cre Tg/+ group or Kank1 fl/fl group.Scale bars indicate 50 µm.

Figure 3 .
Figure 3. Inactivation of Kank1 in podocytes at two months does not result in albuminuria.(a) The urinary albumin-creatinine ratio (ACR) was comparable across all three groups.(b) No significant differences were observed in blood urea nitrogen (BUN) or serum creatinine (Cr) levels among the groups.(c)The sclerotic index at two months was significantly higher in the Kank1 fl/fl pod-Cre Tg/+ group compared to the Kank1 fl/fl group (* p < 0.05).In contrast, the Kank1 +/+ pod-Cre Tg/+ group did not show a significant difference from the Kank1 fl/fl pod-Cre Tg/+ group.In addition, the fibrotic index remained consistent among the three groups.Scale bars indicate 20 µm.(d) Microscopic analysis confirmed these findings, with no significant differences in the electron microscopic studies across the groups.Scale bars indicate 4 µm.

Figure 3 .
Figure 3. Inactivation of Kank1 in podocytes at two months does not result in albuminuria.(a) The urinary albumin-creatinine ratio (ACR) was comparable across all three groups.(b) No significant differences were observed in blood urea nitrogen (BUN) or serum creatinine (Cr) levels among the groups.(c)The sclerotic index at two months was significantly higher in the Kank1 fl/fl pod-Cre Tg/+ group compared to the Kank1 fl/fl group (* p < 0.05).In contrast, the Kank1 +/+ pod-Cre Tg/+ group did not show a significant difference from the Kank1 fl/fl pod-Cre Tg/+ group.In addition, the fibrotic index remained consistent among the three groups.Scale bars indicate 20 µm.(d) Microscopic analysis confirmed these findings, with no significant differences in the electron microscopic studies across the groups.Scale bars indicate 4 µm.

2. 4 .
The Inactivation of Kank1 in Podocytes at One Year Did Not Result in Abnormalities in Urine, Blood, or Histological Tests

16 2. 4 .
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 7 of The Inactivation of Kank1 in Podocytes at One Year Did Not Result in Abnormalities in Urine, Blood, or Histological Tests

Figure 4 .
Figure 4. Inactivation of Kank1 in podocytes at one year was not associated with any detectable abnormalities in urine, blood, or histological tests.(a) There was no significant difference in the

Figure 4 .
Figure 4. Inactivation of Kank1 in podocytes at one year was not associated with any detectable abnormalities in urine, blood, or histological tests.(a) There was no significant difference in the albumin-

2. 5 .
Compensation by other Members of the Kank Family Was Not Evident in Podocyte-Specific Kank1 Knockout Mice One possible explanation for the observed outcomes under normal conditions is that compensatory mechanisms from other members of the Kank family, specifically Kank2, Kank3, and Kank4, may mitigate phenotypic effects in the Kank1 fl/fl pod-Cre Tg/+ group.However, real-time PCR analyses conducted on the glomeruli of Kank1 fl/fl pod-Cre Tg/+ mice yielded results consistent with those obtained from Kank1 fl/fl mice (Figure5a-c).Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW 8 of 16

Figure 5 .
Figure 5. Compensation by the other Kank family members was not observed in podocyte-specific Kank1 knockout mice.(a-c) The mRNA levels of Kank2, Kank3, and Kank4 in the glomeruli of the Kank1 fl/fl pod-Cre Tg/+ group showed no significant difference when compared to the Kank1 fl/fl group.

Figure 5 .
Figure 5. Compensation by the other Kank family members was not observed in podocyte-specific Kank1 knockout mice.(a-c) The mRNA levels of Kank2, Kank3, and Kank4 in the glomeruli of the Kank1 fl/fl pod-Cre Tg/+ group showed no significant difference when compared to the Kank1 fl/fl group.

Figure 6 .
Figure 6.Mild glomerular sclerosis was observed in podocyte-specific Kank1 knockout mice after adriamycin-induced kidney injury.(a) Two weeks after adriamycin injection, the urinary albumincreatinine ratio (ACR) was significantly increased in the Kank1 fl/fl pod-Cre Tg/+ group compared to the Kank1 fl/fl group (log ACR; 5.5 ± 0.8 vs. 7.4 ± 1.1, * p < 0.05).(b) There was no significant difference in BUN or Cr levels between the Kank1 fl/fl group and the Kank1 fl/fl pod-Cre Tg/+ group.(c) The sclerotic index after adriamycin injection was significantly higher in the Kank1 fl/fl pod-Cre Tg/+ group than in the Kank1 fl/fl group (* p < 0.05), while the fibrotic index was not significant between the two groups.Scale bars indicate 20 µm.(d) Foot processes (FP) per micrometer of glomerular basement membranes (GBM) were significantly lower in the Kank1 fl/fl pod-Cre Tg/+ group than in the Kank1 fl/fl group (* p < 0.05).Scale bars indicate 2 µm.

Figure 6 .
Figure 6.Mild glomerular sclerosis was observed in podocyte-specific Kank1 knockout mice after adriamycin-induced kidney injury.(a) Two weeks after adriamycin injection, the urinary albumincreatinine ratio (ACR) was significantly increased in the Kank1 fl/fl pod-Cre Tg/+ group compared to the Kank1 fl/fl group (log ACR; 5.5 ± 0.8 vs. 7.4 ± 1.1, * p < 0.05).(b) There was no significant difference in BUN or Cr levels between the Kank1 fl/fl group and the Kank1 fl/fl pod-Cre Tg/+ group.(c) The sclerotic index after adriamycin injection was significantly higher in the Kank1 fl/fl pod-Cre Tg/+ group than in the Kank1 fl/fl group (* p < 0.05), while the fibrotic index was not significant between the two groups.Scale bars indicate 20 µm.(d) Foot processes (FP) per micrometer of glomerular basement membranes (GBM) were significantly lower in the Kank1 fl/fl pod-Cre Tg/+ group than in the Kank1 fl/fl group (* p < 0.05).Scale bars indicate 2 µm.

Figure 7 .
Figure 7. Generation of the human immortalized KANK1 knockout podocyte cell line.(a) The KANK1 protein was localized in the podocyte cytoplasm, co-localizing with the actin fibers' edges

Figure 7 .
Figure 7. Generation of the human immortalized KANK1 knockout podocyte cell line.(a) The KANK1 protein was localized in the podocyte cytoplasm, co-localizing with the actin fibers' edges as stained with rhodamine-phalloidin. Scale bars indicate 20 µm.(b) Sequence analysis identified a homozygous c.110insA mutation in exon 2 of KANK1 in the KANK1KO podocytes.(c) The expression of the KANK1