Spencer Fox Eccles School of Medicine

43 Soluble (Pro)Renin Receptor in Aristolochic Acid Induced Model of Chronic Kidney Disease

Emma McManus and Nirupama Ramkumar

Faculty Mentor: Nirupama Ramkumar (Internal Medicine, University of Utah)

Abstract

The (pro)renin receptor (PRR) is a transmembrane protein that plays an important role in how the kidney nephrons develop and function. When the extracellular portion of PRR is cleaved, the soluble (pro)renin receptor (sPRR) is formed. Patients with chronic kidney disease have high plasma sPRR levels. Here we investigated whether loss of sPRR is protective in aristolochic acid (AA) induced kidney disease. A mutant sPRR mouse was developed using CRISPR-Cas 9, by mutating the cleavage site of PRR. Male mutant sPRR mice (n=6) and their littermate controls (n=6) were given intraperitoneal injections of AA adjusted to their body weight for a dosage of 1.5 mg/kg, given every day, for a total of 4 consecutive days. Male littermate controls (n=5) were given a vehicle volume (DMSO) instead of AA. Metabolic cage studies were done on days 8-9 and GFR measured on days 10-14. Urine assays were done to detect the kidney injury molecule (KIM-1) and albumin. The kidneys were harvested on day 16 and were examined for tubular damage (KIM-1) and fibrosis (Collagen-I, Collagen-III, Fibronectin-I) using qRT-PCR. Plasma assays were done to assess sPRR and BUN. One-way ANOVA was used to determine differences between the groups. Mutant sPRR mice had a lower body weight compared to both vehicle and drug controls (p<0.0001). AA injections resulted in an increase in KW/BW in both the AA control and the AA mutant sPRR (p<0.005, p<0.05) suggesting that the loss of sPRR did not result in improvement. Urine albumin excretion and urine KIM-1 were elevated in both AA treated groups (p<0.005, p<0.005) compared to the vehicle control. There was not a significant reduction in these markers between control and mutant sPRR mice. There was no significant difference in GFR between the 3 groups. The gene expression of kidney injury and fibrosis markers was slightly elevated in both AA groups, but there was no statistical significance. The absence of the sPRR did not significantly improve the markers of kidney injury in aristolochic acid- induced chronic kidney disease. There are some trends seen, but a larger cohort may be needed to account for variability.

Introduction

Chronic kidney disease (CKD) occurs when the kidneys are not functioning properly for an extended period (greater than 3 months). To be diagnosed with CKD, a patient typically has to have an eGFR of less than 60 mL/min per 1·73 m2. Other markers like albuminuria or urine abnormalities progressing for longer than 3 months can also be indicative of kidney damage and CKD. Often, CKD is asymptomatic until it is in the more advanced stages. End stage kidney disease occurs once a patient’s eGFR is below 15 mL/min per 1·73 m2.[1][2] There are a variety of different causes of CKD. Many other body systems and diseases can interact with the kidneys, causing CKD. Prevalent causes of CKD are diabetes, hypertension, and cardiovascular disease. Other factors such as old age, obesity and autoimmune diseases can also cause CKD (2). Many times, treatment of CKD involves treating underlying conditions that cause CKD such as hypertension, diabetes, or cardiovascular disease.

Decreasing medication that negatively affects the kidneys can also treat CKD. Once a patient progresses to end stage kidney disease, dialysis or a kidney transplant is needed.[3]

One common treatment for CKD is the use of renin-angiotensin system (RAS) inhibitors. The RAS system has a series of steps. First, renin cleaves Angiotensinogen to form Angiotensin I which is further converted into Angiotensin II (Ang II) through Angiotensin-converting enzyme (ACE). All of these are found within the kidney.[4] The (pro)renin receptor (PRR) also plays a role in regulating the RAS system because prorenin and renin can bind to the PRR.[5] PRR is found throughout many organs in the body such as the kidney, heart, brain, and many more tissues. The PRR when cleaved results in a soluble form called the soluble (pro)renin receptor (sPRR). It has been difficult to study the PRR since global or specific cell deletion of PRR often results in cell or animal death. However, the removal of sPRR does not produce animal death and can be a successful way to examine the role of sPRR in CKD.[6] In addition, previous studies have found elevated levels of plasma sPRR in CKD patients.[7] This is why the loss of sPRR is of significant interest.

We developed a mouse model that prohibits the cleavage of the PRR into sPRR using CRISPR-Cas9. The sPRR is cleaved via the furin and site-1 protease cleavage site found in the ATP6AP2 gene (Figure 1). This gene, however, is found on the X chromosome, and the male mice harboring the mutated sPRR are infertile. As a result, male mice are hemizygous, while female mice are heterozygous.[8][9][10] Our lab has done previous studies with sPRR mutant mice. A recent study showed the loss of sPRR reduces the effects of Angiotensin-II hypertension and kidney injury. We are in the process of a study examining the role of sPRR in CKD with a model of unilateral urethral obstruction, in which mutant sPRR mice had acute protective effects for kidney injury.[11]

Figure 1: Structure of the (pro)renin receptor and result of CRISPR-Cas9 mutagenesis of the cleavage site.
Figure 1. Structure of the (pro)renin receptor and result of CRISPR-Cas9 mutagenesis of the cleavage site. R – Arginine, X- any amino acid, K- Lysine, L – Leucine, I – Isoleucine, V – Valine, Z – Leucine or Threonine. The mutation induced in the Furin and S1P cleavage site, results in the inability to form sPRR in mice.

This study examines another model of inducing chronic kidney disease with the use of aristolochic acid. AA was originally found to cause renal fibrosis and kidney disease from old herbal remedies. AA causes severe tubular and epithelial cell injury within the kidneys. Initially the glomeruli are not as affected, but as injury within the tubules progresses, it can lead to damage in the glomeruli.[12] We hypothesize that the loss of sPRR in male mutant mice will provide protective effects against AA induced CKD.

Methods

Figure 2: Protocol for AA or vehicle injection
Figure 2. Protocol for AA or vehicle injection

Generation of mutant sPRR Mice

The generation of the mutant sPRR mice was done using CRISPR-Cas9 (Cyagen Inc, Santa Clara, CA). The sPRR is cleaved through furin and site-1 protease (S1P) (8-10). Two-point mutations on exon 8 of the ATP6AP2 gene alter the furin and S1P site prohibiting the cleavage of sPRR (Figure 1). Founder mice were developed by co-injection of Cas9 mRNA and gRNA. Once the line was established, pups were genotyped via PCR and sequencing. Confirmation of sPRR absence is done using enzyme immunoassay.

Aristolochic Acid Injections

Male sPRR mice (n=6) and littermate controls (n=6) were given intraperitoneal injections of AA adjusted to their body weight for a dosage of 1.5 mg/kg. This was given for a total of 4 days consecutively. Male controls (n=5) were given a vehicle volume (DMSO) instead of AA.

Urine Assays and ELISA

Urine was collected via 24-hour metabolic cages on days 8-9. Urine was centrifuged to remove impurities, then stored at -80 ℃ for further analysis. Urine kidney injury molecule-1 (KIM-1) and Albumin levels were completed using ELISA (Abcam Mouse TIM 1 ELIASA (KIM-1) ab213477 and Ethos Biosciences Albuwell M Kit, Ethos).

Glomerular Filtration Rate (GFR)

GFR was measured based on the change in fluorescence of FITC-sinistrin though a fluorometer (MediBecon, St. Louis, MO). FITC was given via intraorbital injection at a dosage of 0.15 mg/g bodyweight. Calculations were done via MediBecon software to determine the filtration; body weight was considered. GFR was measured on days 11, 14, 15.

Plasma and Kidney Collection

Mice were sacrificed on day 16. Blood was collected via cardiac puncture under anesthesia. Blood was centrifuged and plasma was collected and stored at -80℃ for further analysis of blood urea nitrogen and plasma sPRR. Each kidney was weighed and flash frozen in liquid nitrogen. Three full kidneys from each group were fixed for histology, with the other kidney sectioned into halves for mRNA extraction for qRT-PCR. The remaining kidneys were saved in full for future analyses.

Quantitative Reverse Transcription Polymerase Chain Reaction

The total RNA was first isolated from homogenized half sections of left kidney tissue and purified using the PureLink RNA Mini Kit (Thermo Fisher Scientific, Cat. No 12183018A). RNA concentration and purity was found via NanoDrop 2000 UV-vis, from this equal amount of mRNA was reverse transcribed to complimentary DNA (cDNA) using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Cat. No 4368814). The relative expression of kidney inflammation and injury markers (Collagen I, Collagen III Fibronectin I, Kidney Injury Marker-1 normalized to glyceraldehyde 3-phosphate dehydrogenase) were measured using TaqMan gene expression master mix (Thermo Fischer Scientific, Cat. No 4369016).

Histology and Immunofluorescence

Three kidneys from each group were fixed overnight in 10% formaldehyde and sent to the University of Utah Histology Core for paraffin fixation and staining in PAS and Trichrome.

Statistical Analysis

GraphPad Prism 9 was used to perform all statistical analysis using one-way ANOVA with Tukey post-hoc analyses. Findings were considered significant when p was less than or equal to 0.05.

Results

All mice survived the entirety of the study. One sPRR mutant mouse was excluded from urine studies due to the lack of urine output during the collection phase. Otherwise, all mice were included in the study. There was a total of 5 mice in vehicle controls, 6 mice in AA controls, and 6 mice in AA mutants. Mutant mice had a lower body weight compared to both control groups with a mean 22.6 ±1.8 g while the AA and vehicle control had a mean of 31.2 ±2.7 g and 34.4 ± 2.0 g, respectively (Figure 3B). There were differences seen in KW/BW ratio. Vehicle controls had a mean KW/BW ratio of 0.0058 ± 0.0004 g, the AA control and AA mutant sPRR mice had elevated means of 0.008 ± 0.0011 g and 0.0075 g ± 0.0009 respectively (Figure 3A). There was no statistical difference between the AA control and AA mutants, but there is a significant difference between vehicle control and both the AA control and AA mutants (p<0.005 and p<0.05 respectively). There was no difference in food, water or urine volume during metabolic cage studies, therefore the difference in body weight is not contributed to the amount of food or water consumed (Figure 4).

Figure 3. Mass Measurements A: Body weight to kidney weight and B: Body weight of mice before GFR in vehicle controls.
Figure 3. Mass Measurements A: Body weight to kidney weight ratio (g) in vehicle (n=5) and in AA (n=6) B: Body weight of mice before GFR in vehicle controls (n=5) and in AA (n=6). (*p<0.05, **p<0.005, ***p<0.001****p<0.0001, by ANOVA).

Figure 4: Metabolic characteristics of mice on day 8-9 in vehicle control (n=5) and AA controls and mutants(n=6).
Figure 4. Metabolic characteristics of mice on day 8-9 in vehicle control (n=5) and AA controls and mutants
(n=6).A: Food consumed(g), B: Water consumed (mL), C: Urine excreted (mL) D: Change in BW (g) (ns by ANOVA.

Plasma sPRR and BUN

Mutant sPRR mice had relatively undetectable amounts of plasma sPRR with a mean of 0.4 ±0.1 ng/mL compared to littermate controls of both vehicle and AA (16.3 ±2.6 ng/mL and 14.6 ±2.6 ng/mL respectively). An increase in plasma sPRR was not seen in control AA mice. Blood urea nitrogen showed no difference among the 3 groups. Both AA treated control and mutant mice tended to have higher end of BUN levels (27.1 ±9.4 mg/dL and 28.4 ±19.2 mg/dL respectively) compared with the control (20.5 ±5.2 mg/dL). There was high variability between the groups (Figure 5A, 5B).

 

Figure 5. Plasma sPRR, BUN, and GFR of in vehicle control (n=5) and AA controls and mutants (n=6).
Figure 5. Plasma sPRR, BUN, and GFR of in vehicle control (n=5) and AA controls and mutants (n=6).) A: sPRR(ng/mL) B: BUN (mg/dL) C: GFR (mL/min/100g BW) day 10-14 (****p<0.0001 by ANOVA).

Glomerular Filtration Rate

There was no significant difference in GFR between any of the groups (Figure 5C). They were relatively similar across each group with vehicle control mean of 791.0 ±58.5 uL/min/100g BW, AA control of 738.3 ±166.7 uL/min/100g BW, and AA sPRR mutant of 851.6 ±194.1 uL/min/100g BW. Since AA targets the tubule function of the kidneys, the damage might not have had enough time to affect the glomerulus. During preliminary studies, there was a change in GFR seen in 21 days, however it caused the kidneys to be very fibrotic leading to unsuccessful RNA extraction.

Urine Albumin and KIM-1

Elevated levels of urine albumin excretion were seen in both AA control and AA mutant sPRR (929.5 ±396.4 ug/day, and 546.1 ±339.9 ug/day respectively) compared with vehicle control (66.6 ±29.5). There was a significant difference between the AA control and vehicle control (p<0.005). There was no difference between the AA mutant sPRR mice and the vehicle control, indicating that there is some protective effect, but not a significant amount when compared to the AA drug control (Figure 6). Urine KIM-1 showed very similar trends. AA control and AA mutant sPRR mice both had elevated levels (89.6 ±43.3 pg/day and 55.1 ±21.4 pg/day respectively) compared to the vehicle control (7.0 ±3.7 pg/day). There was a significant difference between the AA control and vehicle control (p<0.005) but no other difference was significant. These urine ELISAs indicate a trend toward lower kidney injury markers in the sPRR mutants, but there is high variability, leading to insignificant findings.

Figure 6. Urine injury markers in vehicle control (n=5), AA controls (n=6) and AA mutant sPRR (n=5).
Figure 6. Urine injury markers in vehicle control (n=5), AA controls (n=6) and AA mutant sPRR (n=5). A: Urine albumin excretion (ug/day) B: Urine KIM-1 excretion (pg/day) (**p<0.005 by ANOVA).

Gene Expression of Kidney Injury and Fibrosis

There was no difference between any of the groups for all the kidney fibrosis and injury markers (Collagen I, Collagen III, Fibronectin 1, Kidney Injury Marker-1). However, for all markers both AA control and AA mutant sPRR had a higher mean compared to vehicle control. For markers Collagen I, Collagen III, and Kidney Injury Marker-1 an elevated value was seen in AA mutant SPRR compared to AA control, however this was statistically insignificant (Figure 7).

Figure 7: Gene expression of kidney injury and fibrosis markers using RT-PCR in vehicle control (n=5) and AA (n=6).
Figure 7. Gene expression of kidney injury and fibrosis markers using RT-PCR in vehicle control (n=5) and AA (n=6). A: KIM-1, B: Collagen I, C: Collagen III, D: Fibronectin 1 (ns via ANOVA).

Histology

Histology staining for this cohort is currently pending. Figure 8 represents the damage seen in vehicle control compared to AA control mice within a previous cohort that did not contain mutant sPRR mice. Within the AA control group, the PAS staining shows cast within the lumen as well as dilation on the tubules. The trichrome staining shows the AA control group contains more collagens built up in the tubules, an indicator of fibrosis and scaring. These comparisons show that the AA injections do indeed cause damage within the kidney.

 

Figure 8: Kidney histology by trichrome and PAS staining.
Figure 8. Kidney histology by trichrome and PAS staining (past cohort shown as current cohort staining is being processed, n=3 in vehicle controls, n=5 in =AA control).

Discussion

This study reports the findings that 1) AA injections caused kidney injury and fibrosis in both control and mutant sPRR mice; 2) No changes in GFR, urine albumin or urine KIM-1 excretion was seen in AA treated mice compared to vehicle control; 3) No difference in gene expression markers in kidney injury and fibrosis. Overall, the absence of the sPRR did not significantly improve the markers of kidney injury in aristolochic acid-induced chronic kidney disease.

There are some trends seen, but a larger cohort may be needed to account for variability. For instance, in urine albumin and KIM-1, lower excretion levels were seen in mutant sPRR mice, but there is such large variability between both AA groups making it difficult to see if a trend is present. GFR was relatively consistent between both the vehicle and AA groups. Since AA targets the tubule function of the kidneys, the damage might not have had enough time to affect the glomerulus. During preliminary studies, there was a change in GFR seen in 21 days; however, it caused the kidneys to be very fibrotic, leading to unsuccessful RNA extraction. Similar results were seen in gene expressions of kidney injury and fibrosis. There are trends in sPRR mutants having higher expressions in genetic markers of kidney injury, but values vary between groups. It is important to mention that the urine was collected on day 8-9 of the study, where the kidneys were not as damaged. The data shows a trend of lower albumin and KIM-1 in mutants. However, on day 16 when kidneys were harvested, there was a slightly higher mean of injury and inflammation markers in the kidney. This could mean that sPRR mutants may initially have acute protective effects, but their protective role diminishes over time as CKD progresses. This correlates to some of the results we are currently working on in a UUO model of AKI. In the future, it might be necessary to look at the effects of mutant sPRR mice in an AA model inducing AKI instead of CKD. It is, however, difficult to do, since AA produces damage quite quickly. Expanding the number of mice in each group may help reduce variability.

Acknowledgements

I am extremely grateful for my wonderful lab mentors and colleagues. My work would not have been possible without the help and support of my principal investigator, Dr. Nirupama Ramkumar. I am forever grateful for her teaching and mentorship during this project and allowing me to work in her lab. I am grateful to Deborah Stuart for teaching me all there is to know about laboratory work and for her assistance and expertise in this project. I also greatly appreciate Divya Sundar and Sherie Agcaoili for their help with my experiments, friendship, and growth in college.

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About the authors

name: Emma McManus

name: Nirupama Ramkumar

institution: University of Utah

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RANGE: Undergraduate Research Journal (2025) Copyright © 2025 by University of Utah is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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