Increased Urinary Albumin Excretion But Less Damaged Renal Tubular Structures in Mice with Genetically Decreased Elmo1 Post Ischemia/Reperfusion Injury

TL;DR

Mice with reduced Elmo1 expression show less kidney tubular damage after injury but have increased albumin in urine, suggesting a complex role for Elmo1 in kidney recovery.

Contribution

This study reveals a novel dual effect of decreased Elmo1 expression on kidney injury recovery, showing structural protection but increased albuminuria.

Findings

Mice with decreased Elmo1 had less severe tubular injury after ischemia/reperfusion compared to wild-type mice.

Elmo1L/L mice showed increased urinary albumin excretion despite preserved antioxidant marker expression.

Inflammatory marker expression was similar between Elmo1L/L and wild-type mice post-injury.

Abstract

Renal ischemia/reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI), a potentially fatal syndrome characterized by a rapid decline in kidney function. The major cause of AKI is IRI. Our prior studies have demonstrated that genetically increased Elmo1 expression in mice aggravated several kidney pathologies including diabetic nephropathy and transition of AKI to chronic kidney disease induced by IRI. However, the effects of decreased expression of Elmo1 on IRI is unclear. We compared the kidney structures and functions between wild type (WT) mice and mice with genetically decreased Elmo1 expression (Elmo1L/L) 5 days after unilateral renal IR surgery. The WT-IRI mice had typical tubular injuries including necrosis and shedding of proximal tubular cells, but these morphological changes were less severe in Elmo1L/L -IRI mice. In contrast, the urinary albumin excretion was elevated in Elmo1L/L -IRI mice compared with WT counterparts. While the expression of inflammatory markers (e.g., Il6, Tnfa, Cxcl1 and Tlr4) was comparable between WT and Elmo1L/L mice with IRI which significantly higher than control mice, the expression of antioxidant markers (e.g., Sod1 and Sod2) was preserved in Elmo1L/L -IRI mice which was significantly decreased in WT-IRI mice. We conclude that Elmo1L/L mice had preserved tubular structures but increased urinary albumin excretion after IRI, suggesting that the role of Elmo1 in IRI is complex and it merits future evaluation.

Figures

A](#F2)). The expression of markers of podocytes [*Wt1* encoding Wilms’ Tumor 1) and *Nphs1*(encoding Nephrin) was determined by qRT-PCR. While the expression of *Wt1* was not different between four groups of mice ([Figure 2B](#F2)), the expression of *Nphs1* tended to decrease in both WT and *Elmo*^*L/L*^ mice with IRI compared with control mice and there was no difference between WT-IRI and *Elmo*^*L/L*^-IRI mice ([Figure 2C](#F2)).

A](#F4)). *Elmo1*^*L/L*^ -IRI mice had a significant increase in cystatin C compared with *Elmo1*^*L/L*^ control mice, and higher than WT-IRI mice as well. WT-IRI mice had an increase in UACR compared with WT control mice as expected. *Elmo1*^*L/L*^ -IRI mice had higher UACR compared with WT counterparts ([Figure 4B](#F4)). The levels of cystatin C and UACR were not different between two groups of control mice ([Figure 4](#F4)).

Full text

INTRODUCTION

1

Acute kidney injury (AKI) is a clinical condition associated with high morbidity and mortality^[1,2]^. One of the leading causes of AKI is kidney ischemia/reperfusion injury (IRI), which occurs when the blood supply to the kidney is temporarily blocked then restored^[3,4]^. While reperfusion is essential to re-establish oxygen delivery, the sudden reintroduction of oxygen reacts with accumulated metabolic intermediates from the ischemic phase, causing a rapid increase in superoxide and other reactive oxygen species (ROS)^[5,6]^. This initial oxidative burst is further amplified by impaired mitochondrial electron transport and activation of NADPH oxidases (Nox), which sustains ROS generation during the injury and repair processes^[7,8]^. In AKI, excessive ROS damages renal proximal tubular cells mainly and contributes to both acute and chronic renal injury^[9,10]^.

Engulfment and cell motility protein 1 (Elmo1) was first discovered in Caenorhabditis elegans as the CED-12 protein for its role on cell corpse engulfment which helps internalize apoptotic cells. It was first identified as part of the CED-2/CED-5/CED-12 signaling pathway in which cytoskeletal remodeling occurred during phagocytosis and cell migration^[11]^. Later, researchers found that it was a cytoplasmic adaptor protein involved in regulating cytoskeletal dynamics, phagocytosis, and cell survival signaling. Genome-wide association studies have identified multiple single-nucleotide polymorphisms in the ELMO1 gene that are strongly associated with kidney disease including diabetic nephropathy^[12,13]^ and nephrotic syndrome^[14]^. Elmo1 has also been implicated in modulating oxidative stress pathways. Previous work has demonstrated that increase in Elmo1 expression (200% of normal) linked to increased ROS generation, which aggravated diabetic complications, while decreased Elmo1 expression (30%) had protective effects in diabetic mice^[15,16]^.

Our previous studies also showed that Elmo1 overexpression exacerbated the severity of IRI-induced kidney disease^[17]^. However, the effects of reduced Elmo1 expression on IRI is unknown. Because (1) Elmo1 regulates autophagy induction^[18]^ and excessive or insufficient autophagy is observed during kidney disease progression^[19,20]^, (2) Elmo1 deficiency causes cell cycle arrest and deceased proliferation^[21]^ and cell proliferation is critical for kidney repair after IRI^[3]^, it is important to investigate the role of Elmo1 deficiency in IRI.

In the current study, we applied renal unilateral IR surgery to mice with decreased expression of Elmo1 and found that haploinsufficiency for Elmo1 led to increased urinary albumin excretion but less damaged tubular structures in acute phase of IRI (5 days after surgery) in mice.

MATERIALS AND METHODS

2

Mice

2.1

Male littermates of WT and Elmo1^L/L^ (C57BL/6J background) at ages 12–15 weeks were used^[15]^. Mice were maintained under standard housing conditions with a 12-hour light/dark cycle and free access to food and water. All animal procedures complied with the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill (Protocol #: 25–190). WT or Elmo1^L/L^ Mice were randomly enrolled into different groups.

Renal IRI Procedure

2.2

The surgery was performed on mice using a dorsal approach. Mice were anesthetized with 1.5% isoflurane inhalation, and body temperature was maintained at 37 °C throughout the procedure. Following anesthesia, the surgical site was disinfected, a small incision was made through the muscle fascia to expose the left kidney. The left renal pedicle was occluded using a microaneurysm clamp for 30 minutes to induce ischemia (successful ischemia was confirmed by the kidney turning dark). After clamp removal, reperfusion was initiated and the incision was closed. The muscle fascia was closed with two interrupted absorbable sutures, and the skin was closed using wound clips^[22]^. Intact WT and Elmo1^L/L^ mice were included as respective controls.

Biochemical Analysis

2.3

Spot urines were collected 5 days after surgery, prior to euthanasia. Following euthanasia, blood and kidneys were collected. Plasma cystatin C and urinary albumin were measured by ELISA kits (MSCTC0, R&D system; Albuwell M kit #1011, Ethos Biosciences). Mouse urine creatinine was measured by an enzymatic assay kit (80350, Crystal Chem)^[22]^. The experiments were done by investigators who were blinded to the experimental groups

Histological Examination

2.4

Kidney tissues (both affected and contralateral) were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5μm), and stained using the Periodic acid–Schiff (PAS). Images were captured with cellSENS Microscope Imaging Software (version 4.3, Evident Olympus)^[17]^. Semi-quantification of tubular injury was scored using a scale of 0 to 4: 0, no injury; 1. <25%; 2. 25–50%; 3. 50–75%; 4. >75%. Scoring was done by an investigator who was blinded to the experimental groups^[22]^.

Quantitative RT-PCR

2.5

Total RNA from tissues was extracted using Trizol (Life Technologies) following the manufacturer’s instructions. NanoDrop spectrophotometer method and gel electrophoresis were used to check quantity and quality of RNA. mRNA was quantified with QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific) by using one-step RT-PCR Kit (Bio Rad) with Actb as reference gene^[22]^. All used qRT-PCR primers and probes were shown in Table 1. The experiments were conducted by investigators who were blinded to the experimental groups.

Statistical Analysis

2.6

The sample size was determined by power calculation based on our previous study^[17,22]^. Data are shown as mean±SEM. The two-way ANOVA was performed using JMP version 17.2.0 (SAS Institute Inc.). Two-tailed Tukey–Kramer HSD test was used for post hoc comparison. All Statistical significance was set at P<0.05.

RESULTS

3

The Kidney Tubular Structure Was Improved in Elmo1L/L Mice 5 Days Post IRI

3.1

IRI causes structural and functional losses in acute and chronic kidney injury^[17,22]^. To evaluate how reduced Elmo1 expression influences the structures of kidneys after IRI acutely, we first examined the renal tubular structures of affected kidneys. As expected, WT-IRI mice showed protein casts and shedding of proximal tubular cells^[22]^. Surprisingly, Elmo1^L/L^-IRI showed only minimal renal injury compared with WT counterparts. The kidney structures were not different between WT-control and Elmo^L/L^ -control mice (Figure 1).

The structures of the glomeruli from mice with IRI did not show obvious abnormalities examined by a light microscope (Figure 2A). The expression of markers of podocytes [Wt1 encoding Wilms’ Tumor 1) and Nphs1(encoding Nephrin) was determined by qRT-PCR. While the expression of Wt1 was not different between four groups of mice (Figure 2B), the expression of Nphs1 tended to decrease in both WT and Elmo^L/L^ mice with IRI compared with control mice and there was no difference between WT-IRI and Elmo^L/L^-IRI mice (Figure 2C).

The right kidney weight (unaffected)/body weight ratio was lower in Elmo^L/L^ mice with IRI compared with WT counterparts (Table 2), however, the structures of contralateral kidneys examined by a light microscope did not reveal obvious difference between four groups of mice (Figure 3).

Elmo1L/L Mice with IRI Had Higher Urinary Albumin Excretion than WT Counterparts 5 Days Post IRI

3.2

Since IRI caused a decline in kidney function^[22]^, we measured two markers of kidney function: plasma cystatin C and urinary albumin-to-creatinine ratio (UACR) ^[23–25]^. In WT mice, plasma cystatin C slightly increased after IRI compared with WT control mice (Figure 4A). Elmo1^L/L^ -IRI mice had a significant increase in cystatin C compared with Elmo1^L/L^ control mice, and higher than WT-IRI mice as well. WT-IRI mice had an increase in UACR compared with WT control mice as expected. Elmo1^L/L^ -IRI mice had higher UACR compared with WT counterparts (Figure 4B). The levels of cystatin C and UACR were not different between two groups of control mice (Figure 4).

The expression of inflammatory markers was not different between WT-IRI and Elmo1L/L-IRI mice

3.3

Inflammation response plays pivotal roles in the IRI process^[26,27]^. To determine whether low Elmo1 alters inflammatory responses to IRI, we evaluated the mRNA levels of Il6, Tnfα, Cxcl1 and Tlr4 in the affected kidneys. Compared with their respective control groups, both WT and Elmo1^L/L^ mice showed significant increases in the mRNA levels of these markers of inflammation following IRI. There was no difference between WT and Elmo1^L/L^ mice with IRI (Figure 5).

The Expression of Antioxidant Enzymes was Preserved in Elmo1L/L -IRI Mice

3.4

Because prior studies show antioxidant enzymes decreased in kidneys with IRI^[22,28]^, we examined the expression of antioxidant enzymes: superoxide dismutase 1/2/3 (Sod1, Sod2 and Sod3) in affected kidneys. In WT mice, the expression of Sod1, Sod2 and Sod3 was reduced after IRI as expected. However, this decrease of Sod1 and Sod2 was not observed in Elmo1^L/L^ mice with IRI, only the expression of Sod3 (extracellular SOD) was decreased in Elmo1^L/L^ mice with IRI. Rac1-GTP dependent NADPH oxidase (Nox2) mRNA level was increased in WT mice with IRI consistent with previous reporting^[5,29]^. Interestingly, the expression of Nox2 increased in Elmo1^L/L^ mice with IRI as well, and there was no difference between WT and Elmo1^L/L^ mice with IRI (Figure 6).

Because proximal tubule protein uptake is mediated by 2 receptors, megalin and cubulin^[30,31]^, we determined the expression of Lrp2 (encoding megalin) and Cubn (encoding cubulin) and found the expression of them was not different in affected kidneys from four groups of mice (Figure 7).

The expression of Edn1 was not different between WT-IRI and Elmo1L/L -IRI mice

3.5

Endothelin-1 (ET-1) is involved in ischemic renal damage^[32,33]^, and Elom1 is associated with Edn1 (encoding pre-pro-ET-1) expression (GSE299038^[21]^). Therefore, we determined the expression of Edn1 in the affected kidneys and found that the expression of Edn1 increased in both WT and Elmo1^L/L^ mice with IRI compared with their respective controls, and there was no difference between WT and Elmo1^L/L^ mice with IRI (Figure 8).

DISCUSSION

4

In this study, we characterized the effects of genetically reduced Elmo1 expression on renal outcomes following IRI, assessing structural injury, functional impairment, and the expression of markers of inflammation and antioxidant. Improved tubular structure was observed in Elmo1^L/L^ mice following IRI. The kidney function of Elmo1^L/L^ -IRI mice was worse compared with WT counterparts, as evidenced by higher urinary albumin excretion. The expression of markers of inflammation (e.g., Il6 and Tnfα) was increased similarly in WT and Elmo1^L/L^ mice with IRI. The expression of antioxidants (e.g., Sod1 and Sod2) decreased in WT-IRI mice but not in Elmo1^L/L^ -IRI mice.

In our prior studies, we found that the damaged kidney structure was associated with damaged kidney function^[17]^. Surprisingly, here we found that the damaged tubular structure from Elmo1^L/L^ mice with IRI was not as severe as WT mice with IRI; however, these mice had higher urinary albumin excretion compared with WT counterparts. How the macromolecules present in urine is still an unanswered fundamental question for the renal field. Our prior study suggests that both glomerular filtration barrier and tubular reabsorption are two main determinants of presence of albumin in urine^[34]^. If filtered albumin exceeds the capacity of proximal tubular reabsorption, albumin appears in urine. In the current study, the inconsistency between relative preserved tubular structure and increased urinary albumin could be due to 1) the more damaged glomeruli especially podocytes in Elmo1^L/L^ mice with IRI. Although the main target of renal IRI is the proximal tubular cell, podocytes are also affected by IRI^[35]^. It is reported that excess Rac activation disturbs actin remodeling in podocyte foot processes causing foot process effacement and proteinuria^[36]^ and Elmo1 influences actin through Rac^[37]^. While the effect of low Elmo1 on actin is unknown, it is possible that low Elmo1 may decrease actin leading to poor podocyte foot processes. Taken together, we hypothesize that (1) Elmo1 deficiency could aggravate the podocyte injury induced by IRI and subsequent proteinuria. In the current study, our methods could not detect the pathological changes of podocytes. We will utilize electron microscope (EM) to examine glomeruli especially podocytes in our future study. (2) Although we did not observe severe tubular injury in Elmo1^L/L^ mice with IRI than WT counterparts, and the expression of megalin/cubulin was comparable between WT-IRI and Elmo1^L/L^-IRI mice, the tubular function could be declined more in these mice due to aggravated insufficiency of Elmo1/Rac signaling. Currently, we are testing this hypothesis using cultured mouse proximal tubular cells (BU.MPT cell line^[18]^).

Prior studies demonstrated that antioxidant enzymes Sod1 and Sod2 are reduced in the kidneys of WT mice following IRI^[22]^. We confirmed this in our study. In contrast, Elmo1^L/L^-IRI mice had the same levels of Sod1 and Sod2 as control groups. The expression of inflammatory markers was not different between WT and Elmo1^L/L^ mice with IRI, which significantly increased compared with control mice. Taken together, the preserved antioxidative response could contribute to the less tubular injury observed in Elmo1^L/L^-IRI mice.

A number of important limitations of the present study need to be considered. The morphological examination was performed by using a light microscope, which can not detect the changes of ultrastructures of glomeruli and tubules, especially podocytes. Further studies using EM are warranted to elucidate the alteration of ultrastructures of kidneys with IRI. Although we did not find obvious abnormality of the tubular structures of contralateral kidneys in WT and Elmo1^L/L^ mice with IRI, the functional analysis of these kidneys is needed. We acknowledge as a limitation of the study that we only measured the mRNA levels of megalin and cucblin.

CONCLUSION

5

In summary, mice with decreased expression of Elmo1 had less tubular injury but more severe kidney dysfunction 5 days post IRI. The data prompt us to hypothesize that low Elmo1 may have different effects on different types of cells in the kidneys. Future comprehensive stereological and/or single cell RNA-seq analysis may provide insight into the complex effects of Elmo1 on AKI.