Selective kidney targeting increases the efficacy of mesenchymal stromal/stem cells for alleviation of murine stenotic-kidney senescence and damage

Abstract

Chronic ischemia triggers senescence in renal tubules and at least partly mediates kidney dysfunction and damage through a p16^Ink4a-related mechanism. We previously showed that mesenchymal stromal/stem cells (MSCs) delivered systemically do not effectively decrease cellular senescence in stenotic murine kidneys. We hypothesized that selective MSC targeting to injured kidneys using an anti-KIM1 antibody (KIM-MSC) coating would enhance their ability to abrogate cellular senescence in murine renal artery stenosis (RAS). KIM-MSC were injected into transgenic INK-ATTAC mice, which are amenable for selective eradication of p16^Ink4a+ cells, 4 weeks after induction of unilateral RAS. To determine whether KIM-MSC abolish p16^Ink4a-dependent cellular senescence, selective clearance of p16^Ink4a+ cells was induced in a subgroup of RAS mice using AP20187 over 3 weeks prior to KIM-MSC injection. Two weeks after KIM-MSC aortic injection, renal senescence, function, and tissue damage were assessed. KIM-MSC delivery decreased gene expression of senescence and senescence-associated secretory phenotype (SASP) factors, and improved micro-MRI-derived stenotic-kidney glomerular filtration rate and perfusion. Renal fibrosis and tubular injury also improved after KIM-MSC treatment. Yet, their efficacy was slightly augmented by prior elimination of p16^Ink4a+ senescent cells. Therefore, selective targeting of MSC to the injured kidney markedly improves their senolytic potency in murine RAS, despite incomplete eradication of p16⁺ cells. KIM-MSC may constitute a useful therapeutic strategy in chronic renal ischemic injury.

Introduction

Chronic renal ischemia causes tissue hypoxia, inflammation, microvascular rarefaction, and fibrosis, leading to kidney damage and dysfunction (Kwon & Lerman, 2015). We have recently shown that cellular senescence contributes to stenotic kidney (STK) injury (Kim, Puranik, et al., 2021; Kim, Zou, et al., 2021). Cellular senescence-associated growth arrest arises from activation of p16^Ink4a/Rb, p53/p21^Cip1, and/or cytokine-signaling pathways related cellular stress (Kirkland & Tchkonia, 2017). To identify the contribution of p16^Ink4a-dependent cellular senescence to STK injury, we previously induced renal artery stenosis (RAS) in INK-ATTAC (p16^Ink4a-Apoptosis Through Targeted Activation of Caspase-8) transgenic mice (Kim, Puranik, et al., 2021). In this model, a p16^Ink4a promoter drives expression of a conditional, synthetically fatal transgene to remove senescent cell populations. High expression of p16^Ink4a is linked to expression of the membrane-bound myristoylated FK506-binding-protein-caspase-8 fusion protein, which can be selectively dimerized by delivery of AP20187 (AP) (Pajvani et al., 2005), which induces activation of caspase-8 (Baker et al., 2011) and thereby apoptosis of senescent cells. We showed that RAS triggers prominent senescence in renal epithelial cells, which at least partly mediates kidney dysfunction and damage (Kim, Puranik, et al., 2021).

Mesenchymal stroma/stem cells (MSCs) decrease STK damage in renovascular disease (Abumoawad et al., 2020; Saad et al., 2017). Contrarily, we have shown that while systemic MSC delivery alleviates murine STK injury, it only modestly suppresses renal senescence (Kim, Zou, et al., 2021). Yet, recent studies have demonstrated the therapeutic potential of MSC for decreasing cell senescence and inflammation in mice with aging (Wu, He, Zhu, Pu, & Zhou, 2021) or acute kidney injury (AKI) (Rodrigues et al., 2017). Possibly, limited homing and retention of systemically delivered MSC might impede their ability to inhibit STK senescence in RAS.

Kidney injury molecule (KIM)-1, a marker of kidney injury, is a type-1 transmembrane protein, the expression of which is markedly upregulated in damaged renal tubules (Han, Bailly, Abichandani, Thadhani, & Bonventre, 2002). We have previously successfully amplified MSC homing to injured murine kidneys by coating them with anti-KIM1 antibody (KIM-MSC) (Zou et al., 2018). Engineered KIM-MSC had increased retention in injured kidneys, and thereby confer superior therapeutic efficacy compared to native MSC (Zou et al., 2018). We therefore hypothesized that these engineered MSC are also capable of achieving vigorous attenuation of tubular cell senescence to an extent that would benefit little from further selective elimination of p16^Ink4a+ cells from the STK.

To test this hypothesis, we treated mice with RAS with KIM-MSC that target damaged tubular cells, which are conceivably senescent. We compared the effects of KIM-MSC on STK senescence, function, and tissue injury in murine RAS with and without prior selective clearance of p16^Ink4a+ senescent cells.

Materials and Methods

All procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee. Twenty three INK-ATTAC transgenic mice on a C57BL/6 background (Baker et al., 2011) (heterozygotes, male, 20–24 weeks of age) were randomly assigned to four groups (sham, RAS, RAS+KIM-MSC, and RAS+AP+KIM-MSC). RAS (n=18) or sham (n=5) groups underwent respective surgeries. One week later, intraperitoneal injections of AP20187 (AP, B/B homodimerizer, Clontech, Mountain-View, CA, total 45mg/kg) or vehicle (x3/week for 3 weeks) were administered to 12 RAS mice (n=6 per group). At the end of the regimen, one month after surgeries, allogeneic KIM-MSCs (5X10⁵ in 200μL PBS) were injected into the aorta of 2 groups (RAS+KIM-MSC and RAS+AP+KIM-MSC) through a carotid cannula. PBS vehicle (200μL) was injected into the sham and remaining RAS mice. Two weeks later, micro-magnetic resonance tomography (MRI) scanning was performed to assess renal function parameters (Jiang, Ponzo, et al., 2018). Urine samples were then collected via cystocentesis and mice euthanized by terminal blood sampling. The kidneys were harvested, weighed, and cut into two halves to be fresh frozen or preserved in formalin.

MSC preparation

We isolated allogenic MSC from abdominal adipose tissue of adult donor mice (C57BL/6, 12 weeks of age) (Zou et al., 2018). Fresh adipose tissue (~1g) was chopped and digested in collagenase-H for 45 minutes. The supernatant was collected, filtered, and cultured for approximately 2 weeks in Advanced MEM media containing with 5% PLTMax (Mill Creek Life Science, Rochester, MN). After three passages, cells were stored in cell recovery medium at −80°C. Collected cells were characterized by flow cytometry (FlowSight and Ideas®, Millipore) to determine the abundance of Sca‐1⁺, CD73⁺, and CD90⁺ cells and to exclude CD34⁺ and CD45⁺ cells and by an MSC Functional Identification Kit to assay tri-lineage differentiation capacity (Eirin et al., 2015). MSC were coated prior to injection as previously described (Zou et al., 2018). Briefly, MSC were washed with serum-free DMEM and incubated sequentially with palmitated protein-G and allophycocyanin (APC)-conjugated monoclonal rat anti-mouse anti-KIM1 antibody (100 mg/ml, R&D, FAB1817A) for 1 hour at 37⁰C each time. The binding efficiency of KIM-MSC to the KIM1 protein was assessed as previously described (Zou et al., 2018).

Mouse studies

Systemic measurements.

Systolic blood pressure (SBP) was measured biweekly by tail-cuff (XBP1000 system Kent Scientific, Torrington, CT) (Cheng et al., 2009). Plasma creatinine was assayed using the DetectX® Serum Creatinine kit (Arbor assays, Ann Arbor, MI)(Kim, Jiang, et al., 2019) and plasma activin-A by a Quantikine ELISA kit (R&D) (Kim, Puranik, et al., 2021).

In vivo renal function.

To assess renal oxygenation, perfusion, blood flow, and glomerular filtration rate, micro-MRI experiments were performed on a vertical 16.4T animal scanner equipped with a 38mm inner diameter birdcage coil (Bruker, Billerica, MA) (Jiang, Ponzo, et al., 2018). Mice were anesthetized and maintained on 1.0–2.0% isoflurane in the supine position, with ECG, respiration, and body temperature monitored (SA Instruments, Stony Brook, NY). Renal volume was measured from coronal images acquired using a respiration-gated 3D Fast Imaging with Steady Precession sequence. Renal (whole kidney and cortex) perfusion and blood flow were measured by arterial spin labeling with the flow-sensitive alternating inversion-recovery sequence. Renal oxygenation was assessed by blood oxygen level-dependent (BOLD) MRI using a respiration-gated 3D multi-echo gradient echo sequence. GFR was measured using our previously developed contrast-enhanced MRI technique (Jiang, Tang, Mishra, Macura, & Lerman, 2018). Renal volumes were quantified from the images using ANALYZE^™ (version 12.0, Biomedical Imaging Resource, Mayo Clinic, MN) and all other MRI images were reconstructed and analyzed off-line using in-house developed software packages in MATLAB® (The Mathworks, Natick, MA) (Jiang, Ponzo, et al., 2018; Jiang, Tang, Mishra, Macura, & Lerman, 2019).

Histologic analysis.

Each microscopic analysis was performed in 10–15 random fields per slide digitized using ZEN (Carl Zeiss, Oberkochen, Germany). Positive areas were semi-automatically quantified using a masking algorithm based on color thresholding and edge detection in a computer-aided image-analysis program AxioVision© (Carl Zeiss) (Kim, Eirin, Zhang, Lerman, & Lerman, 2019). To track APC-labeled KIM-MSC ex-vivo, renal cryosections were studied by fluorescence microscopy and retention was calculated as percent APC-positive cells in the kidneys out of total injected MSC (# of MSC in the whole kidney were converted from # of MSC/ field area volume). To assess kidney injury (indexed by endogenous KIM1 expression), immunohistochemical staining using an anti-KIM1 antibody (R&D, MAB1750) was performed on renal paraffin sections and quantified as percent KIM1-positivity to total field area. For kidney fibrosis, Masson Trichrome (MT)-staining was performed and expressed as percent of total field area. Tubular injury was scored in Periodic Acid-Schiff (PAS) stained sections, as described (Zhang et al., 2016). All slides were analyzed in a blinded manner.

Real time PCR.

Senescence and senescence-associated secretory phenotype (SASP) genes were studied in frozen kidney tissue. Relative quantitative PCR utilized the following Taqman probes (Thermo-Fisher): Cdkn2a (mm00494449), Cdkn1a (mm00432448), Activin-a (mm00434339), Hif1α (mm00468869), Il1α (mm00439620), Il6 (mm00446190), C-C motif chemokine-ligand (Ccl)2 (mm00441242), matrix metallopeptidase (Mmp)3 (mm00440295), Tnfα (mm00443258), Serpine1 (Mm00435858), and Gapdh (mm99999915) as internal control. Fold changes of each target gene relative to corresponding sham groups were calculated using the 2-ΔΔCT method (Livak & Schmittgen, 2001).

Statistical analysis

Data were statistically analyzed using JMP software version 13.0 (SAS Institute, Cary, NC). Normally distributed variables were expressed as mean±standard deviation and non-normally-distributed data as median (interquartile range). Parametric (ANOVA and Tukey-Kramer HSD) and non-parametric (Kruskal-Wallis and Steel-Dwass) tests were used for comparisons among normally- and non-normally distributed groups, respectively. p<0.05 was considered to be statistically significant.

Results

Delivery of KIM-MSCs and mouse characteristics

To enhance tubular homing of MSC, we delivered KIM-MSC to INK-ATTAC mice and compared their effect with or without prior removal of p16^Ink4a+ cells using AP. Prior to KIM-MSC delivery, RAS mice were pre-treated for 3 weeks by either AP (RAS+AP+KIM-MSC) or vehicle (RAS+KIM-MSC) injections starting 1 week after RAS induction (Figure 1a). Body weights and contralateral kidney weights were unchanged among the groups (p=0.29 and p=0.87, respectively, Table 1). SBP was not different at baseline (p=0.72), but ultimately increased in all RAS groups compared to sham (all p≤0.009) as well as compared to their own baseline (RAS, p=0.02; RAS+KIM-MSC, p=0.003; RAS+AP+KIM-MSC, p=0.0001). The STKs were atrophied in all RAS groups compared to sham (all p<0.05), but heavier in RAS+KIM-MSC and RAS+AP+KIM-MSC than in RAS (p=0.0009 and p<0.0001, respectively). On renal cryosections, retention of APC-labeled KIM1-MSC was not different between RAS+KIM-MSC and RAS+AP+KIM-MSC (p=0.32) (Fig. 1b).

Fig. 1.

(a) Experimental design. INK-ATTAC transgenic mice were treated with kidney injury molecule-coated mesenchymal stem cells (KIM-MSC) after AP20187 or vehicle injection from the 7^th to 28^th days after renal artery stenosis (RAS) surgery. (b) KIM1 retention originating from KIM-MSC. (c) KIM1 staining and the quantification of endogenous KIM1-positive area (pink). Blue: DAPI. *p<0.05 vs. sham, †p<0.05 vs. RAS.

Table 1.

Characteristics of mice with renal artery stenosis (RAS) treated with KIM-MSC

	Sham	RAS	RAS+KIM-MSC	RAS+AP+KIM-MSC
Systolic blood pressure (mmHg)
Baseline	118.0±16.9	113.5±29.3	109.3±17.0	105.3±6.9
Final	119.8±12.7	151.8±16.7*†	150.9±22.9*†	150.3±13.8*†
Body weight (g)
Baseline	26.7±0.7	29.0±4.0	27.2±1.9	27±2.1
Final	28.6±2.6	26.8±2.7	25.9±2.0	27.5±1.6
Kidney weight (mg)
Right or stenotic	244±42.4	67.5±14.3*	158.5±42.4*‡	186.3±30.0*‡
Left or contralateral	235.2±26.6	254.3±75.6	233.7±35.1	251.2±28.7

Data are mean±SD,

^*p<0.05 vs. sham,

^†p<0.05 vs. baseline,

^‡p<0.05 vs. RAS,

AP: AP20187, KIM: kidney injury molecule-1, MSC: mesenchymal stem cells

KIM-MSC reduce STK damage in murine RAS.

On renal paraffin sections stained with anti-KIM1 antibody, the percentage of endogenous KIM1⁺ area was increased in RAS (p=0.04 vs. sham) but decreased similarly in both MSC-injected groups (RAS+KIM-MSC, p=0.04, and RAS+AP+KIM-MSC, p=0.03 vs. RAS) (Fig. 1c).

Plasma creatinine level strongly tended to be elevated in RAS compared to sham (p=0.06) and decreased in RAS+AP+KIM-MSC (p=0.04 vs. RAS) but not in RAS+KIM-MSC (which showed high variability), although it was not different from normal (Fig. 2a).

Fig. 2.

Kidney injury molecule-coated mesenchymal stem cells (KIM-MSC) reduce renal dysfunction in murine renal artery stenosis (RAS). (a) Plasma levels of creatinine. (b, c). Single-kidney renal blood flow and glomerular filtration rate by MRI. (d, f, g). Renal perfusion by arterial spine labeling-MRI (color bar: mL/100g/min). (e,h) Hypoxia by blood oxygen-level-dependent (BOLD) MRI (red: greater hypoxia; color-bar:s⁻¹). The improvement in renal function was slightly enhanced by pre-elimination of p16⁺ cells using AP. RK, right kidney; STK, stenotic kidney. *p<0.05 vs. sham, †p<0.05 vs. RAS.

MRI studies showed that STK-RBF was decreased 6 weeks after RAS induction (all p≤0.04 vs. sham), but improved by MSC with AP pre-treatment (p=0.04 vs. RAS), although it remained lower than sham in both MSC-treated RAS groups (Fig. 2b). STK-GFR was also reduced in RAS (p=0.0006 vs. sham), but no longer decreased after MSC delivery, regardless of pre-treatment (RAS+KIM-MSC, p=0.13; RAS+AP+KIM-MSC, p=0.10 vs. sham) (Fig. 2c). While STK cortical perfusion in RAS was not different from sham due to high variability, it was higher in RAS+AP+KIM-MSC than in RAS (p=0.03) (Fig. 2d, f). Whole kidney perfusion was significantly blunted in RAS compared to sham (p=0.003), but not in either MSC-treated group (RAS+KIM-MSC, p=0.17; RAS+AP+KIM-MSC, p=0.29 vs. sham) (Fig. 2g). STK cortical R₂* tended to be different among the groups (ANOVA p=0.06) due to seemingly greater hypoxia in RAS, but did not reach statistical significance (Fig. 2e,h).

Masson-Trichrome-stained sections showed evident STK fibrosis in RAS (p=0.04 vs. sham), but not in MSC-treated RAS. In particular, MSC+AP pre-treatment efficiently reduced fibrosis compared to RAS (p=0.03) (Fig. 3a,c). Tubular injury in PAS-stained slides showed a similar pattern (p=0.04 RAS vs. sham, p=0.04 RAS+AP+MSC vs. RAS) (Fig. 3b,d).

Fig. 3.

Kidney injury molecule-coated mesenchymal stem cells (KIM-MSC) reduce renal damage in murine renal artery stenosis (RAS). Neither was different from sham in RAS+KIM-MSC, yet RAS+AP+KIM-MSC also decreased renal fibrosis and tubular injury compared to RAS. (a) Masson’s trichrome staining. (b) Periodic acid-Schiff (PAS) staining. (c, d) Quantifications. *p<0.05 vs. sham, †p<0.05 vs. RAS.

KIM-MSCs decrease STK senescence.

Plasma activin-A level was increased in RAS and RAS+KIM-MSC compared to sham p=0.0001, and p=0.03, respectively), and lower than RAS in RAS+AP+KIM-MSC (p=0.02) (Fig. 4a). Six weeks after RAS induction, STK senescence and SASP gene expression were strikingly upregulated in RAS compared to sham (all p≤0.04), but no longer increased in either MSC-treated groups. Gene expression of Cdkn2A (p16^Ink4a), Cdkn1A (p21^Cip1), and ActivinA was also further downregulated in RAS+AP+KIM-MSC compared to RAS (all p≤0.04), as was gene expression of Il-6, Mmp3, Tnfα, and Serpine1 (all p≤0.04) (Fig. 4b).

Fig 4.

Kidney injury molecule-1-coated mesenchymal stem cells (KIM-MSC) decrease senescence in the stenotic kidney (STK) of mice with renal artery stenosis (RAS). (a) Plasma levels of activin-A decreased significantly only by AP+KIM1-MSC. (b) Renal gene expression of senescence and SASP genes was mostly decreased by both interventions, but more significantly by AP-pretreatment before MSC delivery. Gene expression was quantified by RT-PCR (relative to Gapdh). *p<0.05 vs. sham, †p<0.05 vs. RAS.

Discussion

This study shows that KIM-MSC delivery decreased kidney damage and markedly downregulated expression of genes related to senescence and the SASP. It improved renal function, including stenotic-kidney glomerular filtration rate and perfusion, and attenuated renal fibrosis and tubular injury. Interestingly, prior elimination of p16^INK4a+ senescent cells slightly augmented the benefits of KIM-MSC on both renal function and cellular senescence, underscoring the link between the two. Therefore, kidney targeting to the STK results in a meaningful intensification of the potency and efficacy of MSC and may represent a useful therapeutic strategy in chronic renal injury.

KIM1 is a tubular transmembraine protein that is cleaved by renal hypoxia or nephrotoxic insults, and constitutes a biomarker for AKI, but is also upregulated in patients with chronic tubulointerstitial diseases (Waikar et al., 2016). Sustained KIM1 expression is linked to renal fibrosis and thus progressive CKD in mice (Humphreys et al., 2013). We have previously found that STK KIM1 expresssion peaked in 48 hours, decreased to 50% by 2 weeks, but remained significantly upregulated (Zou et al., 2018). Increased KIM1 expression in the STK as observed in the current study augments homing of MSC coated with an anti-KIM1 antibody to the injured kidney. We previously showed that KIM-MSC delivery in RAS mice exhibited enhanced STK retention compared to native MSCs (Zou et al., 2018). Moreover, we now found that injection of KIM-MSC decreased STK endogenous KIM1 expression, suggesting recovery of damaged tubules, underscored by decreased tubular injury observed by PAS staining. KIM-MSC also improved STK cortical perfusion, which was linked to attenuation of renal atrophy, although it did not reattain a normal volume after treatment.

This study showed that KIM-MSC is an effective strategy to mitigate renal senescence. In contrast with our previous findings of a merely partial effect of native MSC delivery to blunt renal senescence (Kim, Zou, et al., 2021), in the current study KIM-MSC alleviated senescence and SASP gene expression, despite comparable doses of injected cells as in our previous studies and ongoing RAS. A recent study also demonstrated that MSCs attenuate premature renal senescence in AKI. While the methodology was different, employing double-doses of human umbilical cord‐derived MSC early after damage in a rat model with temporary kidney hypoxic damage (Rodrigues et al., 2017), taken together these studies highight the ability of MSC as a class to attenuate renal ischemia-induced senescence.

In senescent cells, the SASP exerts noxious effects, facilitates pro-inflammatory intercellular communication with adjacent or remote cells, and promotes senescent cell accumulation, leading to organ dysfunction. Strategies to target senescence include small molecules that aim to block SASP, or ‘senomorphic’ drugs (Lagoumtzi & Chondrogianni, 2021). MSC may simulate senomorphics, which indirectly blunt senescence without necessarily killing senescent cells. MSCs release soluble factors and extracellular vesicles that mediate their paracrine activity, and in swine RAS successfully reduce renal vein levels of proinflammatory cytokines linked to SASP, including TNF-α and IL-6 (Eirin et al., 2017). The present study extends those previous studies to show that the expression of various SASP genes was also dramatically decreased after KIM-MSC treatment, which might mitigate propagation of senescence. KIM-MSC delivery also considerably downregulated p16^Ink4a and p21^Cip1 gene expression, although this effect was enhanced by prior eradication of p16^Ink4a+ cells, because gene downregulation became also significant compared to RAS. We had shown that a small number of senescent cells releasing SASP factors is sufficient to exert deleterious effects on normal neighboring tissue (Kim et al., 2020). Therefore, SASP blocking through MSC may be a potent therapeutic strategy in RAS. In addition, adult stem cell multipotency displayed by MSC can also facilitate renal regeneration in several forms of CKD (Eirin & Lerman, 2014). MSC also have immunomodulatory properties via secreted soluble factors or direct communication with immune cells (Li, Chen, & Sun, 2021) to guide them into an anti-inflammatory phenotype, restrain helper and cytotoxic T-lymphocytes, or promote regulatory T-cells (Li et al., 2021), possibly affecting the interaction of immune and senescent cells.

Notably, the therapeutic efficacy of KIM-MSC was slightly augmented by abolishing p16^Ink4a-dependent cellular senescence prior to treatment. These observations suggest that MSC selectively targeted to KIM-1-expressing injured renal tubules achieve a substantial effect on p16^Ink4a+ cells although they do not achieve their complete elimination. Indeed, while we have previously demonstrated prominent senescence of renal tubular cells in RAS (Kim, Puranik, et al., 2021; Santelli et al., 2019), the overlap between KIM1-expressing and senescent tubules may be incomplete. In fact, renal senescence in RAS is also mediated by other pathways, like p21^Cip1, which was also blunted by both interventions in the current study. We have previously demonstrated that p21^Cip1 gene positivity is upregulated in urinary exosomes from renovascular hypertensive patients (Kim, Zou, et al., 2021) and reduced by intra‐arterial infusion of autologous MSC (Kim, Zou, et al., 2021). We have also shown that eliminating a wide range of senescent cells using a broad senolytic strategy is more effective in improving ischemic kidney damage than targeting only p16^Ink4a+ cells (Kim, Puranik, et al., 2021). We now found that KIM-MSC efficaciously downregulated both Cdkn2a (p16^Ink4a) and Cdkn1a (p21^Cip1), as well as a host of SASP factors, the expression of which was no longer higher than in sham mice. Nevertheless, elimination of p16^Ink4a+-senescent cells prior to KIM-MSC delivery did elicit slightly greater improvement in plasma creatinine, STK-cortical perfusion and RBF, as well as senescence and SASP gene expression compared to untreated RAS, implying a small but noteworthy additional effect of selective p16^Ink4a+ senescent cell clearance.

The contribution of additional cell-cycle events like p53/p21^Cip1 to the effects of MSC on cellular senescence merits further study. Additional limitations of this study include small group sizes. Although the male hormonal microenvironment might aggravate renal ischemic injury (Hosszu, Fekete, & Szabo, 2020), we could not compare the effects of sex due to limited availability of female transgenic mice. KIM-MSC treatment was ineffective in reducing SBP, which might require higher or repeated doses. This may have also accounted for the relatively modest effect on systemic activin-A levels. While we could not perform longitudinal biological analysis of senescence and renal damage, our previous study demonstrated in detail the effect of AP alone on clearance of p16^Ink4a+ senescent cells.

In conclusion, KIM-MSC effectively blunted renal senescence, dysfunction, and structural damage that might contribute to progression of chronic kidney disease. Their senomorphic efficacy approached, although did not quite reach, that combined with prior purging p16^Ink4a+ senescent cells using AP. Therefore, KIM-MSC may represent a broad and potent therapeutic strategy in chronic renal ischemic injury, at least partly through decreasing senescent cell effects. Futures studies need to explore the efficacy of combining or engineering MSC with other senolytic strategies.

Abbreviation.

AKI

acute kidney injury

AP

AP20187

APC

allophycocyanin

BOLD

blood oxygen level-dependent

CKD

chronic kidney disease

GFR

glomerular filtration rate

INK-ATTAC

p16^Ink4a-Apoptosis Through Targeted Activation of Caspase-8

KIM

kidney injury molecule

MRI

magnetic resonance tomography

MSC

mesenchymal stem cell

MT

Masson Trichrome

PAS

Periodic Acid-Schiff

RAS

renal artery stenosis

RBF

renal blood flow

SASP

senescence-associated secretory phenotype

SBP

systolic blood pressure

STK

stenotic kidney