A heart derived-soluble factor which controls kidney and cardiovascular function after acute cardiorenal syndrome

doi:10.21203/rs.3.rs-3876449/v1

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A heart derived-soluble factor which controls kidney and cardiovascular function after acute cardiorenal syndrome

https://doi.org/10.21203/rs.3.rs-3876449/v1

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Heart and kidney are bi-directionally interacting organs. Because heart and kidney disease are amongst the most common human diseases, investigating disease-causing interactions is important. Here, we identified a new heart-derived endocrine mediator of kidney function, cardiac cysteine-and-glycine-rich protein 3 (CSRP3). We determined CSRP3's stimulus for release from the heart, plasma transit, and kidney disease-causing mechanism. We found that cardiac CSRP3 was upregulated after cardiac injury (modeled using cardiac arrest and cardiopulmonary resuscitation in the mouse), and released into the systemic circulation, subsequently undergoing megalin-dependent endocytosis in the renal proximal tubule and changing kidney cell phenotype. Administration of CSRP3 to mice experiencing focal kidney injury reproduced the kidney phenotype observed in cardiac arrest-exposed mice. Genetic deletion of cardiac CSRP3 or renal megalin ameliorated cardiac injury-induced chronic kidney injury. Lastly, pharmacologic megalin inhibition ameliorated CSRP3-mediated chronic renal injury. We describe the role of cardiac CSRP3 in a heart-kidney interaction which directs specific renal dysfunction and renovascular remodeling after injury. We describe a novel mechanism of the intricate coupling of heart and kidney which determines renal function. These investigations may eventually lead to novel therapy for heart-induced kidney disease.

Biological sciences/Physiology/Cardiovascular biology

Biological sciences/Physiology/Kidney

Organ crosstalk is the complex, bi-directional, and integrated communication between physically distant organs. Organ crosstalk is important to biological homeostasis but can also communicate acute or chronic dysfunction in one organ, causing injury in another organ in which the inflammatory response, metabolic dysregulation, and fibrotic changes are involved ^{1, 2}. Since the cardiovascular disease is the world’s leading cause of death and heart-kidney crosstalk is believed to be pathogenic in diseases of both organs, deeper understanding about the mechanisms of heart-kidney interaction, especially “cardiorenal syndrome (CRS)” is critically important ³⁴. For example, in acute cardiorenal syndrome (cardiorenal syndrome type 1, CRS) despite rapid application of flow-restoring therapies, kidney failure is still worsens outcomes⁵. This form of acute kidney injury (AKI) is characterized by rapid worsening renal function and decreased urine output after acute decline in heart function such as that caused by cardiac arrest, acute heart failure, or acute myocardial infarction⁶. 34–50% of patients with acute cardiovascular disease develop AKI ^{7, 8} and 13–38% of AKI is caused by acute cardiovascular disease and cardiovascular surgery ^{9, 10}, accounting for 3.9 million cases/year in the US, and costing $24.0 billion ^{11, 12}. AKI may incite chronic kidney disease (CKD, termed “AKI-CKD transition”)¹³. Clinical studies confirm that CRS-1 predisposes to CKD¹⁴. Thus, further investigation of the mechanism of this important heart-kidney crosstalk is imperative. To overcome limitations in modeling CRS1, we employed murine cardiac arrest and cardiopulmonary resuscitation^{15, 16}. CA/CPR successfully replicated CRS1 (cardiac arrest-induced AKI) and AKI-CKD transition ¹⁵. We previously identified a heart specific protein, cysteine and glycine rich protein 3 (CSRP3) in glomerular filtrate aspirated after CA/CPR and in the plasma of cardiac arrest survivors². This protein was previously identified as a candidate plasma biomarker of cardiac injury¹⁷ and because of its filterable size it could mediate renal function via megalin-mediated uptake into proximal tubular epithelial cells^{2, 18, 19}. Importantly, CSRP3 has developmental transcriptional and proliferative effects in cells of mesenchymal lineage, suggesting a fundamental role in cell development which could underpin disease^{20, 21}.

Therefore, in this investigation we hypothesized that heart-specific CSRP3 is delivered from injured heart to kidney, altering renal tubular epithelial cell function and driving AKI-CKD transition. To test the hypothesis we performed CA/CPR, a heart-induced kidney ischemia model, and AKI-equivalent renal ischemia-reperfusion injury (IRI), a kidney-only ischemia model, to identify the heart’s contribution to AKI-CKD transition. We characterized the dynamic plasma release and renal uptake of CSRP3. We then used mice with inducible, proximal tubule-specific deletion of megalin to determine the mechanism of CSRP3 uptake in the kidney, and confirmed human relevance in human proximal tubule epithelial cells (hPTEC). We then tested the role of CSRP3 in AKI-CKD transition using novel, inducible, cardiac-specific CSRP3 gene-deleted mice. Finally, we utilized an FDA-approved small molecule to inhibit renal CSRP3 uptake to evaluate a potential route to prevention or treatment.

Heart-induced kidney ischemia causes distinct AKI from time-equivalent kidney-only ischemia

To test the hypothesis that acute cardiac injury contributes uniquely to kidney injury beyond ischemia alone, we developed time- and injury-equivalent models of CA/CPR and bilateral renal ischemia-reperfusion. To determine a bilateral kidney-only ischemia time which resulted in the same amount of kidney dysfunction 24h later as CA/CPR (i.e., injury-equivalent), we quantified GFR 24h after CA/CPR (8 min ischemia) and a selection of bilateral IRI clamp times. (Fig. S1A). 27 minutes of IRI (27min-IRI) resulted in 24h GFR and BUN not different from that of CA/CPR (Fig. S1B, C). Thereafter, we designated 27min IRI and CA/CPR as “GFR equivalent” and 8min IRI and CA/CPR as “time equivalent” and characterized renal injury 24h after these models (Fig. S2A). CA/CPR, 8min-IRI, and 27min-IRI demonstrated AKI characterized by reduced 24h GFR (Fig. S2B). This was accompanied by elevated BUN after CA/CPR and GFR-equivalent IRI but not after time-equivalent IRI (Fig. S2C). Additionally, immunofluorescence images of KIM-1 demonstrated that CA/CPR and GFR-equivalent IRI resulted in higher KIM-1 expression than time-equivalent IRI at 24h (Fig. S2D). These data establish the GFR-equivalence and time-equivalence models and indicate that the severity of AKI caused by CA/CPR was GFR-equivalent to 27min-IRI, and different from that caused by ischemia time-equivalent 8min-IRI.

Heart-induced kidney ischemia causes distinct CKD from GFR-equivalent kidney-only ischemia

Next, using the previously-determined outcome time of 49 days ¹⁵, we characterized development of CKD in each model (Fig. 1A). All three models (CA/CPR, time-equivalent IRI, and GFR-equivalent IRI) demonstrated reduced GFR compared with sham. 49 day GFR of CA/CPR and 27min-IRI were not different but both were reduced compared with 8min-IRI (GFR 732.3 ± 31.03 µL/min/100g in CA/CPR, vs. 933.3 ± 52.55 µL/min/100g in 27min-IRI (not significant). GFR 636.2 ± 20.82 µL/min/100g in 8min-IRI, p < 0.05 with respect to CA/CPR and p < 0.01 with respect to 27min-IRI, n = 7–8/group) (Fig. 1B). 49-day survival was significantly reduced in 27min-IRI animals (Fig. 1C), suggesting that CA/CPR imposes additional kidney injury over that of renal-only ischemia-reperfusion. Accordingly, BUN and cystatin-C (Cys-C) were elevated by CA/CPR and 27min-IRI but not by 8min-IRI, suggesting that 8min-IRI-induced CKD was milder than that induced by CA/CPR and 27min-IRI (Fig. S3A, B). Therefore, we investigated albuminuria, chronic KIM-1 expression, and fibrosis, critical pathologic and pathophysiologic correlates of CKD in each model.

CA/CPR caused higher KIM-1 expression than either 8min- or 27min-IRI (Fig. 1D) and more interstitial fibrosis as demonstrated by αSMA deposition (Fig. 1E). Histologic sections demonstrated that CA/CPR and 27min-IRI caused more atubular glomeruli and atrophic tubules than 8min-IRI (Fig. 1F). 8min-IRI and 27min-IRI caused albuminuria at 49 days, but CA/CPR did not (Fig. 1G). Histopathologic review by a blinded observer (N.A.) suggested a glomerulosclerosis pattern associated with CA/CPR which was not present in IRI-exposed animals with either time- or injury-equivalent ischemia. Therefore, we proceeded to assess arcuate artery morphometry (chosen because arcuate arteries are multiple and consistently identifiable on kidney sections) and systolic blood pressure. CA/CPR resulted in increased vascular wall thickness, characterized by smooth muscle hyperplasia in arcuate arteries at 49 days, but 8min- and 27min-IRI did not (Fig. 1H). Furthermore, CA/CPR resulted in elevated blood pressures at 49 days but 8min- and 27min-IRI did not (Fig. 1I). Together, CA/CPR-augmented functional and histologic injury, coupled with CA/CPR-augmented vascular intimal hyperplasia and hypertension suggested that there were different or additional mechanisms in AKI-CKD transition due to heart-induced kidney ischemia (CA/CPR) compared with those caused by kidney-only ischemia (IRI). We concluded that these findings specifically implicate acute cardiorenal syndrome in development of severe CKD with tubulointerstitial fibrosis, renovascular pathology and hypertension. Females did not experience severe GFR loss at 49 days (Fig. S3C); therefore further experiments were conducted in males only, except where noted.

CSRP3 directs CKD development after kidney-only ischemia-induced kidney injury

We previously identified CSRP3 as a putative endocrine mediator of cardiorenal syndrome ² Therefore we quantified plasma CSRP3 in the aftermath of CA/CPR (Fig. 2A). Using this data, we modeled CSRP3 plasma exposure using injection of recombinant human CSRP3 to mice to test whether CSRP3 mediates ischemia induced AKI-CKD transition. Quantification of the area under the curve for plasma CSRP3 after 1 µg injection demonstrated that a 5 repeated injection model would generate similar exposure to that after injury (Fig. 2B). We injected recombinant CSRP3 (rCSRP3, 1µg) or phosphate-buffered saline (PBS) into the retroorbital venous plexus daily for 5 days to mice starting after time-equivalent IRI (8 -min IRI) (Fig. 2C). IRI with CSRP3 administration (CSRP3-IRI) resulted in reduced GFR and increased BUN compared with PBS administration (Fig. 2D, E); this did not occur in IRI-exposed mice given a CSRP3-equimolar dose of myoglobin, a muscle protein with nearly identical molecular weight to that of CSRP3 (Fig. 2F). More severe tubulointerstitial fibrosis (Fig. 2G) and vascular wall hyperplasia (Fig. 2H) were observed in CSRP3-IRI than in PBS-treated IRI. Overall, CSRP3 injection augmented ischemia-equivalent IRI-induced renal functional injury and fibrosis, and imposed vascular pathology previously seen only after CA/CPR. Therefore, addition of CSRP3 to ischemia-equivalent IRI induced additional renal injury, such that CSRP3-IRI functional and pathologic injury was similar to that of CA/CPR. These data suggest that CSRP3 is a specific mediator of the distinct chronic kidney injury caused by heart-induced kidney ischemia.

CSRP3 is heart-derived, plasma-borne, renally filtered, and taken up into proximal tubular epithelial cells

To determine the mechanism of delivery of CSRP3 to tubular epithelial cells, we evaluated expression and renal uptake of CSRP3 in vivo. CSRP3 was not detected in the kidney (Fig. 3A) at any time after sham or CA/CPR. At baseline, CSRP3 was expressed in myocardial tissue lysates in a sexually dimorphic fashion, with females demonstrating 55 ± 20% the CSRP3 expression of males (Fig. 3B, p < 0.05, n = 5/group). In male mice, CSRP3 was expressed in myocardial tissue lysate in all measured groups, and was dramatically increased 24h after CA/CPR, relative to sham procedure, 8min- or 27min-IRI (Fig. 3C). To evaluate renal trafficking of CSRP3, we injected FLAG-tagged recombinant CSRP3 followed by renal immunohistochemistry. CSRP3 was robustly detected at the renal brush border and apical intracellular punctae in the renal proximal tubule within minutes of injection (Fig. 3D). Taken together with data presented above, these findings indicate that CA/CPR specifically confers an increase of cardiac CSRP3 expression. Moreover, CSRP3, released into plasma after CA/CPR, enters the renal filtrate, where it is taken up by renal tubular epithelial cells in a process which concentrates CSRP3 into punctae, likely endocytic vesicles.

Heart-specific CSRP3 deletion ameliorates AKI-CKD transition after CA/CPR

Next, we tested whether cardiac CSRP3 deletion attenuates the AKI-CKD transition after CA/CPR. We generated inducible heart-specific CSRP3 deleted (CSRP3^fl/fl/Myh6^cre/Esr1*, iCSRP3KO) mice. After tamoxifen treatment, iCSRP3 KO mice did not express CSRP3 in the heart (Fig. 4A) and demonstrated unchanged blood pressure (Fig. S4A). Ejection fraction (EF) and cardiac output (CO) were reduced in iCSRP3KO mice (EF 50.0 ± 2.7 vs 62 ± 1.7%, p < 0.05, CO 13 ± 0.7 vs. 15.9 ± 0.4 mL/min, p < 0.05, n = 4–6/group Fig. S4B, C, (Representative images are shown in Fig. S5A, B)). Next, we performed CA/CPR in tamoxifen-treated iCSRP3KO mice and their littermate controls (Fig. 4B). Heart-specific CSRP3 deletion did not affect survival after CA/CPR (Fig. 4C). However, 49 days after CA/CPR, iCSRP3KO mice demonstrated reduced renal injury, as demonstrated by higher GFR compared with littermate controls (Fig. 4D) although BUN was not different (p = 0.07, Fig. 4E). iCSRP3KO mice demonstrated lower systolic blood pressure, attenuated renal fibrosis, renovascular hypertrophy, and compared with controls at 49 days after CA/CPR (Fig. 4F, G, H). Therefore, iCSRP3KO status conferred mild reduction in cardiac function 15 days after initiation of tamoxifen, yet nevertheless ameliorated 49-day loss of GFR, development of tubulointerstitial fibrosis, vascular wall hyperplasia, and systolic blood pressure elevation.

Renal CSRP3 uptake is megalin-dependent

We previously identified CSRP3 in the urine of mice with partial megalin deletion after CA/CPR ². Therefore to determine human-relevance and test the hypothesis that renal uptake of CSRP3 is megalin-dependent, we employed human proximal tubular epithelial cells (hPTEC). We validated cells as 95% pure culture of PTEC (Fig. S6A) Quantitative PCR and immunostaining for megalin revealed that hPTEC strongly expressed megalin (Lrp2, Fig. S6B C). We then treated hPTEC with Lrp2-directed siRNA (or scrambled control) and incubated cells for 6h with CSRP3 (Fig. 5A) (siRNA sequences in supplemental table 4). Lrp2 siRNA reduced Lrp2 expression by 60% (0.39 ± 0.19 in siRNA vs. 1.00 ± 0.09 in scramble, p < 0.005)) (Fig. S6D). Co-immunostaining for CSRP3 and megalin and evaluation of their intensity in each cell revealed that megalin interference inhibited CSRP3 uptake and the intensity of megalin signal and CSRP3 signal were strongly correlated to each other (Fig. 5B, C S6E). Next, we confirmed this result in vivo. iMegKO status dramatically inhibited rCSRP3 uptake in mouse proximal tubules (Fig. 5D). 24h after CA/CPR, megalin deletion caused > 2.5-fold increase in excreted CSRP3 (p < 0.05, n = 8–11/group) (Fig. 5E). Therefore, uptake of CSRP3 by proximal tubular epithelium is megalin-dependent, and in the absence of proximal tubular megalin, plasma-borne CSRP3 is excreted in the urine. This result is summarized in Fig. 5F.

Deletion of megalin has sexually dimorphic effect on chronic renal injury

Next, to test the hypothesis that that renal megalin mediates long-term renal effects of CSRP3, we performed CA/CPR on iMegKO mice and littermate controls (Fig. 6A). Tamoxifen injection of LRP2^fl/fl;Ndrg1^CreERT2 + mice induced proximal tubule-specific megalin deletion (iMegKO) (Fig. S7A) and albuminuria, demonstrating reduced uptake of this cognate megalin ligand (Fig. S7B). Megalin deletion did not mediate CRS1; GFR 24h after CA/CPR was mediated by sex but not iMegKO status (Fig. S7C). However, 49 days after CA/CPR, iMegKO male mice demonstrated higher GFR than littermate controls with similar BUN (Fig. 6B, C). Survival was not different between iMegKO and littermate control after CA/CPR (Fig. S7D). iMegKO status ameliorated CA/CPR-induced interstitial fibrosis and renovascular hypertrophy (Fig. 6D, E). Interestingly, iMegKO did not result in amelioration of GFR loss in females (male vs. female 49day GFR; p = 0.031 in iMegKO, p = 0.27 in control, n = 8–11/group Fig. S7E). Therefore, although megalin deletion had no effect on acute cardiorenal syndrome, iMegKO status attenuated development of CA/CPR-induced chronic renal injury –only in males. Taken together these results suggest that AKI-CKD transition after CA/CPR is mediated by sex, and by megalin only in males.

CSRP3 provokes transcriptional change associated with repair in hPTEC

To gain insight to the role of CSRP3 in the kidney tubular epithelial cells, we performed bulk RNA sequencing (bulk RNA seq) in CSRP3-treated hPTEC and control cells, followed by cell subtype deconvolution analysis. CSRP3 induced differential expression of 37 transcripts (Figs. S8A, B and 7A). Gene set enrichment analysis (GSEA) demonstrated activation of RNA binding and cell-matrix adhesion, cell-substrate junction organization processes, with suppression of negative regulatory controls on transcription and biosynthesis (Fig. 7B). A protein-protein interaction search seeded with CSRP3 and gene set driving genes from each highly regulated ontology demonstrated networks centered on extracellular matrix control and RNA processing and synthesis (Fig. 7C). Second-order interactions of CSRP3 controlled both of these networks through a convergence on Smad3 (Fig. 7D). Because the identified specific ontologic and interaction protein sets are frequently identified in repairing and failed repair PT cells, we next performed cell subtype deconvolution. CSRP3 nearly-exclusively induced the repairing PT subtype (Fig. 7E, F), although the failed repair subtype was not regulated. Since repair cells have fibrosis- and functional regulation roles in the injured kidney, GSEA was performed on this subpopulation, indicating suppression of cell differentiation, developmental processes, and response to stimulus (Fig. 7G). Taken as a whole, these data indicate that CSRP3 mediates transcription in human PT cells, changing their phenotypic distribution and perhaps shifting repairing PT cells toward a more stable, less responsive state.

Pharmacologic inhibition of megalin ameliorates CSRP3-augmented AKI-CKD transition

We found that CSRP3 administration augmented chronic renal injury induced by IRI and entered tubular epithelial cells in a megalin-dependent fashion. CSRP3 is transiently present in the plasma after acute cardiac injury such as CA/CPR, but then returns to baseline. Therefore, transient pharmacotherapy in accordance with the plasma release kinetics of CSRP3, which interfered with renal uptake of CSRP3, could ameliorate chronic renal injury. Therefore, we tested translational use of cilastatin sodium, a megalin inhibitor, as pharmacotherapy for CSRP3 excess²². First, we determined that as observed in iMegKO mice, CSRP3 uptake in the proximal tubules was inhibited by cilastatin (Fig. 8A). Next, we performed 8min-IRI in mice. After IRI, mice received CSRP3 and either cilastatin sodium or vehicle control. (Fig. 8B). Urine CSRP3 was increased in cilastatin-treated CSRP3-IRI mice at day 5, suggesting that pharmacological megalin inhibition by cilastatin prevented CSRP3 uptake (Fig. 8C). 49 days after IRI, cilastatin-treated CSRP3-IRI mice demonstrated reduced renal functional injury, as demonstrated by higher GFR (1028.0 ± 153.9 in Cilastatin vs. 875.3 ± 136.3 in vehicle, µL/min100g, p < 0.05) and lower BUN compared with vehicle-treated CSRP3-IRI mice (18.88 ± 3.31mg/dL in Cilastatin vs. 25.05 ± 5.97mg/dL in vehicle, p < 0.05) (Fig. 8D, Fig. S9A) Furthermore, cilastatin ameliorated renal fibrosis (0.0082 ± 0.0012 in Cilastatin vs. 0.0102 ± 0.0026 in vehicle, V_αSMA/V_kidney, p < 0.05), renovascular hypertrophy (0.585 ± 0.033 in Cilastatin vs. 0.666 ± 0.028 in vehicle, p < 0.01), and elevation of systolic blood pressure (130.5 ± 4.9 mmHg in Cilastatin vs. 143.1 ± 11.5 mmHg in vehicle, p < 0.01) compared with vehicle treated CSRP3-IRI mice (Fig. 8E, F, G). Therefore translationally-relevant megalin inhibition prevented CSRP3-augmented interstitial fibrosis and renal function loss, resulting in amelioration of AKI-CKD transition as summarized in Fig. 8H.

The most important finding of this investigation is that the heart-derived soluble factor CSRP3 directs change in kidney function caused by acute cardiorenal syndrome. Acute heart disease such as myocardial infarction, cardiac arrest, and cardiac surgery are widely recognized to worsen kidney function, in turn worsening outcomes of the most common cause of death, cardiovascular disease^{23, 24}. Therefore, understanding the mechanisms by which heart disease drives kidney disease is significant. Clinical observations of linkage between heart and kidney dysfunction has led multiple groups to suspect the role of non-flow mediators such as endocrine factors, so called “cardiorenal connectors”^{6, 25}. Linkage between cardiovascular disease and kidney disease has best been characterized in the kidney-to-heart direction, as in cardiorenal syndromes type 3 and 4^{26, 27}. However translational study of CRS1 has been hampered by a paucity of models with transient cardiac dysfunction^{28, 29}, which is precisely a feature of the CRS model used in this work³⁰.

We found that CRS1 modeled by CA/CPR caused a phenotypically distinct AKI-CKD transition compared with AKI induced by injury-equivalent kidney-only ischemia/reperfusion. In injury-equivalent models, CRS1-induced AKI-CKD was distinguished by prolonged injury to tubular epithelial cells, increased interstitial fibrosis, renovascular intimal hyperplasia, and elevated blood pressure. These findings, with histologic atubular glomeruli and relatively low urine albumin suggest a cardiovascular etiology, as human renal pathologic samples with these findings are typically associated with hypertension over other causes of CKD³¹. The development of intimal hyperplasia and systolic blood pressure elevation were unique to CRS1; they did not occur in ischemia-equivalent IRI or GFR-equivalent IRI. Blood pressure elevation after renal IRI has been investigated by other groups; hypertension eventually develops after 6 month recovery^{27, 32} but development of systolic pressure elevation within 2 months of IRI requires a second insult – either high-salt diet or angiotensin II infusion^{33, 34}. Therefore, we conclude that equivalent renal ischemia which includes CA/CPR causes distinct AKI-CKD transition, likely because CA/CPR entails additional signaling beyond that within the kidney itself. This finding suggests a directing component which originated in the heart, and prompted investigation of CSRP3.

CSRP3, a 21 kD LIM-only protein, was identified in 1994 as a myogenesis and muscle cell differentiation factor, highly expressed in cardiac muscle³⁵. It is not expressed in the kidney³⁶, and expressed at very low level in skeletal muscle³⁷ in adults. One prior study found CSRP3 is released by injury in an isolated rabbit heart preparation and identified it in 6 of 8 human plasma samples obtained after myocardial infarction¹⁷. We previously² found that CA/CPR specifically upregulates heart-derived proteins in the renal filtrate and urine, and identified CSRP3 as a target of interest. Our investigation confirmed that CSRP3 is specifically delivered to the renal proximal tubular epithelium from the plasma, and determined that CSRP3 given to mice exposed to CA/CPR time-equivalent IRI recapitulated the blood pressure elevation, vascular hyperplasia, fibrosis, and GFR loss pattern which were specific to CRS1. Conditional deletion of CSRP3 from the heart ameliorated the AKI-CKD transition induced by CA/CPR in control animals. Therefore, CSRP3 is both necessary and sufficient to drive ischemia-induced renal injury toward the nephrosclerotic phenotype caused by CRS1.

We found that short-term exposure to nanomolar CSRP3 modulates transcription of cell-matrix adhesion and RNA transcriptional control in human PTEC. CSRP3 has well-described role in cardiomyocyte differentiation, where it activates myogenic transcription factors myoblast determination protein 1 (MyoD), myogenic factor 6, and myogenin²¹, and drives expression of α-smooth muscle actin (αSMA) ³⁸. Interestingly, CSRP3 and closely-related proteins comprised of repetitions of the LIM (Lin-11, Islet-3, Mec-3) domain (“LIM-only proteins”) are broadly involved in mesenchymal developmental, proliferative, and structural regulation^{39, 40, 41, 42}.

Therefore, CSRP3 mediation of cell matrix and transcriptional control in hPTEC is only surprising because it takes place in renal epithelial cells, which do not express CSRP3. A driving factor may be CSRP3 interaction with SMAD3 through ANKRD1⁴³, potentially connecting CSRP3 to cell-matrix adhesion and muscle structure development pathways in vitro. CSRP3 and ANKRD2 coimmunoprecipitate and share spatiotemporal expression patterns and functional roles – both serve as myocyte differentiation factors by transcription factors, and both transit from nucleus to cytoplasm at the completion of cell differentiation^{44, 45}. That CSRP3 drove lasting tubulointerstitial fibrosis in vivo and cell development and adhesion pathways in hPTEC suggests that CSRP3 modulates cell plasticity. Cell plasticity and developmental change such as epithelial-mesenchymal transition (EMT) enables proximal tubule epithelial cells to acquire mesenchymal phenotypes including motility, leading/trailing polarity, and extracellular matrix deposition⁴⁶ and plays a critical but incompletely understood role in AKI-CKD transition⁴⁷. The mechanism by which tubular injury drives extracellular matrix accumulation is of great interest. Proximal tubular cells undergo plastic change to several phenotypes after injury, yielding cells which participate in repair, and failed repair cells, which express profibrotic, proinflammatory transcripts including NF κ B and Relb and are thought to drive fibrosis ⁴⁸. Our finding that CSRP3 increased fibrosis in vivo, but did not increase failed repair cells, increasing the number and apparent stability of repairing PT cells in vitro instead, adds to the complexity of this mechanism. We speculate that CSRP3-altered repairing PT cells may modulate fibrotic change, perhaps supporting more stable phenotypes. A variant of EMT, termed EMyT, has been described, in which EMT invokes myogenesis. This program has complex regulation by TGFβ1 signaling, in which SMAD3 acts as a repressor/terminator which, when removed, allows myogenesis to proceed ^{42, 49}. Thus, EMT induction may be facilitated by removal of stability factors which on return may consolidate lasting phenotypic change. We speculate that CSRP3-induced downregulation of repair cell stimulus response and developmental processes of may prolong the plastic state of other cells, perhaps immune cells, supporting the observed fibrosis and vascular hyperplasia. Recently, the proinflammatory microenvironment surrounding injured proximal tubules was observed to support development of tertiary lymphoid tissue, which provides an interactive space for CXCR3 + T-cells and fibroblasts ⁵⁰. Consistent with this report, our recent study demonstrated that natural killer T cells, which express CXCR3, played a role in renal interstitial fibrosis after CA/CPR ⁵¹. In this context, there is a possibility that CSRP3-caused proinflammatory or profibrotic transcriptional change in proximal tubular cells mediates the interaction between TLTs and immune cells resulting in chronic kidney injury characterized by fibrosis and myogenesis.

Renal uptake and actions of CSRP3 are megalin-dependent. The kidney’s megalin-mediated endocytic system serves a sensor/adaptor role for numerous filtered, plasma-borne signaling molecules, including peptides, proteins, steroid hormones, and drugs, facilitating endosomal uptake and sorting^{52, 53}. Inhibition or deletion of megalin abrogates tubular uptake of megalin ligands, altering tubular cell response and increasing ligand excretion in urine^{18, 54}. In our study, proximal tubule-specific megalin deletion increased CSRP3 excretion and ameliorated CKD development due to CRS1 in males. Co-administration of the megalin inhibiting drug cilastatin sodium and CSRP3 similarly increased urinary CSRP3 excretion and ameliorated systolic pressure elevation, vascular hyperplasia, and GFR loss induced by CSRP3 with renal ischemia. Therefore, renal uptake of CSRP3 and functional renal megalin are necessary for development of CSRP3-directed CKD. These effects of CSRP3 were shared with the megalin ligand myoglobin: when delivered in equimolar dose to CSRP3 (~ 0.3 mg/kg), myoglobin did not mediate development of CKD even though myoglobin is renotoxic in rodents at 35 g/kg⁵⁵. There are several possible explanations for the finding that megalin deletion did not ameliorate CRS1-induced CKD in females. Wild type and control females did not develop CKD as severe as that of males, so it is possible that experiments were underpowered for this outcome in females. Megalin mediates renal uptake of 17β-estradiol⁵⁶, which attenuates development of CKD through a SMAD3-dependent mechanism. Reduced kidney exposure to protective 17β-estradiol in females may have obscured a protective effect of increased CSRP3 excretion. A third possibility is that because females express less cardiac CSRP3, reducing already-low renal exposure to CSRP3 had insignificant effect. These findings add important context to sex difference in cardiorenal disease and will guide our further investigation. Lastly, in addition to delineating a novel cardiorenal signaling mechanism, megalin-dependence of CSRP3 action in the kidney has important translational implications. Cilastatin sodium is currently FDA-approved (at lower dose, in combination with another medication). A phase I trial of cilastatin (NCT03595189) at the dose range used in our experiments was recently completed. Were the mechanisms demonstrated here confirmed in clinical studies, repurposing of cilastatin could offer the tantalizing potential of secondary prophylaxis for CRS1-induced CKD.

Our study has limitations. First, our data were obtained in mouse models and human relevance was confirmed in primary culture of human cells. Although we previously showed that humans with cardiac injury elevate plasma CSRP3, additional study in humans will be required to determine the translational importance of our findings. Second, though we developed inducible, cardiac-specific CSRP3 deleted mice specifically to for this study, iCSRP3KO mice demonstrated 12% reduced body weight and 16% reduced ejection fraction, in comparison to littermate controls, prior to CA/CPR. Humans with CSRP3 mutations develop dilated cardiomyopathy⁵⁷; mice with constitutive CSRP3 deletion or overexpression develop heart failure ⁵⁸. The observed reduced cardiac function could be due to CSRP3 deletion, or it could be a result of cre-recombinase expression in the heart, which transiently reduces myocardial function⁵⁹. However, since reduced cardiac function worsens CKD, the finding of improved renal function in iCSRP3KO mice strongly suggests that CSRP3 itself has significant renally injurious function. Moreover, that CSRP3 administration in wild-type mice with no CSRP3 elevation recapitulated CRS1-directed CKD confirms the specific role of CSRP3 without confounding from reduced ejection fraction. Lastly, the mechanism by which CSRP3 induces vascular change, and its extent, is unknown; this is the focus of further, extensive investigation.

In conclusion, we demonstrate that the heart-derived protein CSRP3 directs a distinct AKI-CKD transition after acute cardiorenal syndrome, a previously unsuspected role for the myocyte development factor CSRP3 in epithelial cells, outside the heart. We describe an elegant and novel mechanism of cardiorenal interaction which depends on injury-induced release of CSRP3 from the heart and renal uptake through the megalin-mediated endocytic system. Important questions remain, particularly the importance of CSRP3 upregulation after CA/CPR and the mechanism of release from the heart, and specific mechanisms by which CSRP3’s renal uptake induces renovascular change and blood pressure elevation. These compelling findings may support further translational investigation of treatment or secondary prophylaxis for CKD induced by acute cardiorenal syndrome, perhaps involving repurposing of cilastatin sodium.

In all experiments, samples were randomly allocated, and analysis was blinded. All animal procedures were approved by the Oregon Health & Science University Institutional Animal Care and Use Committee or the VA Portland Health Care System Institutional Animal Care and Use Committee. Detailed methods are available in Supplemental Methods.

Cardiac arrest and cardiopulmonary resuscitation model (CA/CPR)

Eight- to twelve-week-old male and female C57BL/6 mice (obtained from Jackson Laboratories) and megalin- and CSRP3 gene-deleted mice (described below) underwent normothermic, potassium chloride-induced cardiac arrest followed by cardiopulmonary resuscitation with chest compressions and intravenous epinephrine (CA/CPR) as previously described ⁶⁰. Briefly, under 5% isoflurane in a 2:1 air:oxygen mixture, followed by 2.5% isoflurane for maintenance. Mice were placed in a supine position on a heated pad, and temperatures were maintained between 36.5 and 37.5°C monitoring with a rectal temperature probe. Tracheal intubation was performed with a 22-ga Teflon catheter (InSyte-W, BD, Franklin, NJ), and mice were mechanically ventilated with 150 uL tidal volume, 150 breaths per minute. Subcutaneous electrocardiography (EKG) leads were placed. After a PE-10 catheter tube incretion into the right jugular vein, cardiac arrest (CA) was induced by the intravenous infusion of 20 µEq (40 µl) of 0.5 M potassium chloride. CA was confirmed by isoelectric EKG and absence of cardiac contraction by visually checking chest wall movement. Upon confirmed CA, oxygen, air, and isoflurane were turned off. At the same time, mechanical ventilation was discontinued and disconnected from the endotracheal tube. Seven minutes and 30 seconds after CA, the endotracheal tube was reconnected with mechanical ventilation using 100% oxygen with 180 breath per minute. Eight minutes after CA, mechanical chest compressions were initiated at a rate of 500 beats per minute. Up to 1.0 ml (16 µg) of epinephrine diluted in 0.9% sodium chloride solution, was administrated via the catheter. Return of spontaneous circulation (ROSC) was confirmed by EKG and visible cardiac contractions on the chest wall. After the achievement of > 60 breaths/min spontaneous respiration, the trachea was extubated, and the jugular catheter was removed. Animals were placed in a recovery cage, which was placed on a warming pad controlled at 37°C. For sham-treated mice, general anesthesia was performed as described for CA/ CPR without endotracheal intubation and catheter placement.

Bilateral ischemia-reperfusion injury model (IRI)

Eight- to twelve-week-old male C57BL/6 mice underwent normothermic bilateral renal IRI (transient occlusion of the renal pedicle) as previously described ⁶¹. Briefly, under the same isoflurane general anesthesia as CA/CPR, the mouse was placed on the homothermic heat pad by monitoring the body temperature in the range of 36.5–37.5°C through a rectal probe. Flank hair was removed with clippers and depilatory. Then the surgical area was sterilized with povidone iodine. After the skin preparation, the skin and muscle on the left flank side were cut open to expose the left kidney. The kidney was then pushed out from the cut and dissected of the pedicle tissue to identify the renal blood vessels. The right kidney was exposed by a similar procedure to the left kidney, but the incision was lower than the left side. Renal ischemia was started by clamping the right kidney vessels with a micro clip, then the left kidney vessels. The ischemia time was measured from the time point of clamping each side separately so that both kidneys received the same durations of ischemia. Complete ischemia was confirmed by the turning of the kidney color from red to dark purple immediately. After verification of the successful ischemia, the kidney was covered with a warm wet gauze. After the ischemia, the micro clips were released at the indicated times for each kidney to start the reperfusion. Reperfusion was indicated by the change of kidney color returned to red. After confirming hemostasis, the muscle layer of the incision and the skin wound were closed with 5 − 0 silk.

Generation and breeding of proximal tubule–specific inducible megalin deleted (iMegKO) mice

Ndrg1^CreERT2 mice were proximal tubule specific inducible Cre mice generated as described ⁶². Using these mice, iMegKO mice,were generated and bred as previously described ¹⁸. Deletion of megalin in the proximal tubule was induced by intraperitoneally injection of tamoxifen (150 mg/ kg body weight, 5 continuous days). Fifteen days after the first tamoxifen injection, mice were subjected to CA/CPR or sham surgery. Cre-negative littermates served as controls, and received tamoxifen identically to experimental mice.

Generation and breeding of heart specific inducible CSRP3 deleted mice (iCSRP3KO) mice

The csrp3 gene floxed mouse (CSRP3 ^flox/wt) was generated by GemPharmatech (China) by using CRISPR/Cas9 technology. Homozygosity of CSRP3 flox (CSRP3 ^flox/flox) was confirmed after F1 breeding by polymerase chain reaction (primer sequences in Supplemental table 2), CSRP3^flox/flox mice were bred to mice expressing tamoxifen-dependent Cre recombinase in cells that expressed the mouse cardiac-specific alpha-myosin heavy chain (αMHC; Myh6) promoter (Myh6^cre/Esr1*). Mice were genotyped using quantitative PCR by Transnetyx (Cordova, TN). Then, experimental mice (CSRP3^fl/fl/Myh6^cre/Esr1*+, iCSRP3KO) were subjected to tamoxifen injection (150 mg/kg body wt, intraperitoneally, 5 continuous days) to induce heart-specific CSRP3 deletion. Urine was collected and blood pressure was measured at 15 days after the first tamoxifen injection, then mice were subjected to CA/CPR or sham surgery at age 8–12 weeks. Cre-negative littermates received tamoxifen and served as control to CSRP3 gene-deleted mice.

CSRP3 injection model

1µg of filter-sterilized rCSRP3 (1µg/150µL, Origene) was retroorbitally injected after 8min-IRI (day 0) and following 4 days in a row (day1-4). In experiments in which cilastatin sodium was administered also rCSRP3 was as above following retroorbital injections of cilastatin sodium (4mg/day* 5 days, Sigma). Vehicle treatment consisted of an identical volume of PBS.

Blood pressure measurement

Blood pressures were measured by tail-cuff plethysmography using the multi-channel MC4000 Blood Pressure Analysis System (Hatteras Instruments). All measurements were performed by the same investigator between 10:00AM-1:00PM, 3 days in a row after training the mice for 3 days. Mice were placed on the 37˚C pre-heated experimental platform, and tails were put into the cuff and gently fixed by taping. Following 5 preliminary measurement cycles, blood pressures were recorded as an average of 10 experimental measurement cycles.

Echocardiography

Echocardiography was performed in the OHSU Small Animal Research Imaging Core facility under isoflurane anesthesia. Ejection fraction and cardiac output were measured in multiple views obtained using a 38 Mhz probe Vevo 2100 (FUJIFILM VisualSonics).

Transdermal glomerular filtration rate (GFR) measurement

Transdermal GFR measurement was performed using FITC-fluorimetry (MediBeacon, St. Louis, MO) as previously described ⁶³. Under isoflurane anesthesia, a transdermal FITC-fluorimeter device was attached on pre-shaved mouse skin, then FITC-sinistrin (MediBeacon, FTC-FS001, 35mg/mL, 150 µL) was injected retroorbitally. The fluorescence intensity of FITC-sinistrin was recorded for 2h. GFR was evaluated as the clearance of FITC sinistrin using a single-compartment model.

Primary culture of human kidney proximal tubular epithelial cells (hPTEC)

Human Kidneys for primary culture of proximal tubular epithelial cells (hPTEC) were provided from Pacific Northwest Transplant Bank (PNTB). Kidneys were recovered from donors with intention for donation that were deemed unsuitable for transplant into a recipient and were subject to inclusion/exclusion criteria (Supplemental table 5). After receiving kidneys, they were dissected and digested as previously described ⁶⁴. Briefly, on the cold tray, fat and the renal capsule were removed, then papilla was exposed. The cortex, about the depth of 8-10mm from the surface, was dissected and rinsed with cold phosphate buffered saline (PBS). The dissected tissue was minced until it got homozygous. Debris in minced tissue was removed by washing with Dulbecco's Modified Eagle's Medium (DMEM)/F12 solution (See supplemental table 6) several times. Then, the tissue was digested by 0.26U/mL liberase in DMEM/F12 solution and incubated for 2h at 37C. Digestion was stopped by adding 10% FBS in DMEM/F12 solution supplemented with 1mL/L Insulin, transferrin, selenium supplement (ITS, Corning, Corning NY). Cells were filtered with the 100um and 70um cell strainer respectively. Then cells were placed on collagen coated flask, and incubated overnight. The next day, following rinsing with PBS, culture medium was replaced to DMEM/F12 solution with ITS and 5% FBS. Cells were incubated until full confluence and split until passage 2–3. Next, cells were trypsinized to single cell suspension, and stained with CD10 antibody, CD13 antibody, and DAPI for 30min. Then cells were subjected to fluorescence activated cell sorting (FACS). CD10+/CD13 + live cells were selected and recultured, then cryostored or used for experimentation. All experiments were performed on cells at passage 4 or less. Solutions and reagents used for cell culture are described in Supplemental table 6.

Treatment of human kidney proximal tubular epithelial cells (hPTEC)

To assess the role of megalin for CSRP3 uptake in vitro, hPTECs were treated with 60pg/mL Lrp2-directed siRNA or scrambled siRNA for 24h. After siRNA treatment, cells were treated with 150ng/mL (7 nM) rCSRP3 for 20 min. Then cells were rinsed with ice-cold PBS and stained with antibodies to megalin and CSRP3. For bulk RNA sequencing, hPTECs were treated with 10 nM rCSRP3 for 6h following 24h serum starvation. After rCSRP3 treatment, cells were harvested, then total RNA was extracted.

Bulk RNA Sequencing of hPTEC and Downstream Analysis

hPTEC were treated with either PBS or CSRP3 and then subjected to RNA isolation and bulk RNA sequencing. Differential expression analysis was performed using DNASeq2⁶⁵ with independent hypothesis weighting⁶⁶ prior to downstream analysis including gene set enrichment analysis ⁶⁷, and protein interaction analysis⁶⁸. Cell type deconvolution, and differential gene expression inference was achieved using CIBERSORTx ⁶⁹.

Quantitative polymerase chain reactions (qPCR)

TaqMan gene expression assay probes were used for qPCR. Cells were homogenized using QIAshredder (QIAGEN, Netherland) then extracted RNA. After reverse transcribing RNA, target genes were amplified by real-time PCR with a Step One Plus Real-Time PCR System (Applied Biosystems, Carlsbad, CA) with duplicating all samples. Expression of target genes were quantitatively evaluated performing the ΔΔCT method. mRNA expression was normalized to gapdh expression. Used kits and probes are listed in Supplemental Tables 1 and 3

Western blotting

Protein was extracted from kidney or heart homogenate in iced radioimmunoassay precipitation buffer with protease inhibitor. The concentration of lysate was measured by bicinchoninic acid assay. Ten-µg of protein was loaded to 4–12% gradient precast gels (Invitrogen, Carlsbad, CA), electrophoresed, and transferred to polyvinyl difluoride membranes. After blocking with 5% milk, membranes were incubated overnight in primary anti-CSRP3 at 4C, then incubated for 2h in HRP-conjugated secondary antibody at room temperature (see Supplementary Table 1). Blots were imaged using ChemiDoc MP Bioimaging System (Bio-Rad, Hercules, CA). For quantification, total protein was semi-quantified using the Ponceau stain image of the same membrane, and the specific protein quantity expressed as ratio of the specific band density to total Ponceau density. Primary and secondary antibodies and dilutions are shown in Supplemental Table 1. (Full length blots are shown in Fig. S10.)

Blood and urine biochemical analysis

Blood plasma was collected at the time of euthanasia by the puncture of cardiac left ventricular apex. Urine was collected for 24h before, at 24h, and at 49 days after surgery. Blood urea nitrogen (BUN), urine albumin, and urine CSRP3 were quantified using commercially available kits listed in Supplemental Table 1.

Histological evaluation and unbiased stereology

Kidneys were perfused and fixed with 4% paraformaldehyde, paraffin embedded, and sectioned into 6 µm thickness, 3 sections per animal. After periodic acid-Schiff (PAS) staining slides were scanned using a Nikon Axioscan scanner. Vascular wall thickness of arcuate arteries and the number of atubular glomeruli were evaluated by a treatment-blind renal pathologist. Vascular wall thickness was then quantified as vascular wall thickness index. To quantify vascular wall thickness index, ImageJ software (NIH, Bethesda, MD) was used to draw region of interest (ROI) lines on each identified renal arcuate artery cut in circular cross section (9–12 arcuate arteries per animal). First, one ROI (A) was drawn around the outer perimeter of the vascular wall and then a second ROI (B) was drawn around the outer perimeter of the vascular lumen. Vascular wall thickness index was then quantified as (A-B)/A: the area of the vascular wall, divided by the total area of the vessel including lumen. Vascular wall thickness index did not correlate with vessel area (R² = 0.06, Fig S2E).

For the evaluation of the extracellular matrix component α-smooth muscle actin (αSMA), and kidney injury molecule-1 (KIM-1), immunofluorescence staining was performed with the reagent and antibodies listed in Supplemental Table 1. After staining, sections were scanned and quantitatively evaluated using semi-automated unbiased stereology as previously described ⁷⁰. Briefly, random superimposed grids on cut sections were used to create a ratio of αSMA or KIM-1 positive grids to area, which translates to the volume of positive tissue with reference to overall kidney volume, expressed as the volume faction of αSMA orKIM-1 positive tissue volume per total kidney volume (VαSMA /VKidney, VKIM-1/VKidney).

Detection of injected recombinant CSRP3

Renal uptake of CSRP3 was evaluated by immunohistochemistry conducted on perfusion-fixed kidneys. Briefly, 20ug of DDK FLAG-tagged recombinant human CSRP3 (rCSRP3, TP325217, Origene) was retroorbitally injected to non-treated wild type mouse, cilastatin treated wild type mouse, or iMegKO mouse. Mice were dissected and perfused with cold saline and 4% PFA at 6min after CSRP3 injection. After 24h incubation at 4C, kidneys were placed in 15% sucrose in PBS until they sank, then transferred to 30% sucrose in PBS until they sank, and followed by cryo-embedding in Optimum Cutting Temperature compound (OCT, Tissue-Tek, Sakura FineTek, Torrance CA). Kidneys were sectioned into 5 or 7µm thickness then proceeded to immunohistochemistry. Reagents and antibodies used in this experiment are listed in Supplemental table 1.

Statistical Analysis:

Statistical analyses were primarily performed with GraphPad Prism software 9.0 (GraphPad Software). Mouse survival was assessed using Kaplan-Meier survival curves followed by log-rank (Mantel-Cox) test. Student’s t-test was used for comparison between 2 groups. One-way ANOVA followed by the Tukey post hoc test was used for the comparison of 3 or more groups. Correlation was analyzed using Pearson’s test. RNAseq statistical and analysis pipelines are in Supplementary information. Values are reported in text and figures as mean ± SEM except where noted.

Acknowledgements:

The authors gratefully acknowledge the assistance of William Packwood, B.S. of the OHSU Small Animal Research Imaging Core, and the engineering/technological support of Takahisa Funahashi, MEng. Graphical representations of mechanisms were created using Biorender.

Funding:

Merit Award from United States Department of Veterans Affairs I01BX004288 (MPH)

Technology Development grant from the United States Department of Defense W81XWH2010196 (MPH)

Author contributions:

Conceptualization: YF, MPH

Methodology: YF, AM, KB, JH, MN, EN, MPH

Investigation: YF, AM, KB, JH, MN, MPH

Visualization: YF, AM, MPH

Funding acquisition: YF, TG, MPH

Project administration: TG, MPH

Supervision: NA, EN, MY, SG

Writing – original draft: YF, MPH

Writing – review & editing: YF, AM, TG, EN, MY, SG, MPH

Competing interests:

Authors declare that they have no competing interests.

Data and materials availability:

All data are available in the main text or the supplementary materials.

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A heart derived-soluble factor which controls kidney and cardiovascular function after acute cardiorenal syndrome

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Abstract

Figures

INTRODUCTION

RESULTS

Heart-induced kidney ischemia causes distinct AKI from time-equivalent kidney-only ischemia

Heart-induced kidney ischemia causes distinct CKD from GFR-equivalent kidney-only ischemia

CSRP3 directs CKD development after kidney-only ischemia-induced kidney injury

CSRP3 is heart-derived, plasma-borne, renally filtered, and taken up into proximal tubular epithelial cells

Heart-specific CSRP3 deletion ameliorates AKI-CKD transition after CA/CPR

Renal CSRP3 uptake is megalin-dependent

Deletion of megalin has sexually dimorphic effect on chronic renal injury

CSRP3 provokes transcriptional change associated with repair in hPTEC

Pharmacologic inhibition of megalin ameliorates CSRP3-augmented AKI-CKD transition

DISCUSSION

MATERIALS AND METHODS

Cardiac arrest and cardiopulmonary resuscitation model (CA/CPR)

Bilateral ischemia-reperfusion injury model (IRI)

Generation and breeding of proximal tubule–specific inducible megalin deleted (iMegKO) mice

Generation and breeding of heart specific inducible CSRP3 deleted mice (iCSRP3KO) mice

CSRP3 injection model

Blood pressure measurement

Echocardiography

Transdermal glomerular filtration rate (GFR) measurement

Primary culture of human kidney proximal tubular epithelial cells (hPTEC)

Treatment of human kidney proximal tubular epithelial cells (hPTEC)

Bulk RNA Sequencing of hPTEC and Downstream Analysis

Quantitative polymerase chain reactions (qPCR)

Western blotting

Blood and urine biochemical analysis

Histological evaluation and unbiased stereology

Detection of injected recombinant CSRP3

Statistical Analysis:

Declarations

Acknowledgements:

Funding:

Author contributions:

Competing interests:

Data and materials availability:

References

Additional Declarations

Supplementary Files

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