55 Loss of Soluble (Pro)Renin Receptor Attenuates Kidney Injury and Fibrosis in Unilateral Ureteral Obstruction

Fig. 3. CRISPR-Cas9 mutagenesis of the cleavage site of the PRR

We recently showed that sPRR attenuates Angiotensin-II mediated hypertension and kidney injury in male mice with loss of sPRR (Ramkumar et al., 2021). Additionally, preliminary studies have shown that sPRR is influential in attenuating kidney injury and inflammation. Thus, we hypothesized that compared to wild type, loss of sPRR will show decreased kidney injury, fibrosis, and inflammation.

The Unilateral Ureter Obstruction (UUO) is a widely used model of kidney injury in mice (Chevalier et al., 2009; Aranda-Rivera et al., 2021; Manucha, 2007; Rodríguez-Peña et al., 2008; Martinez-Klimova et al., 2019). A single ureter is tied off, obstructing the flow of urine and causing tubular injury to the obstructed kidney (Chevalier et al., 2009). The UUO model is most similar to chronic obstructive nephropathy, which, in the long term, leads to CKD. Typically, in the UUO, we are able to collect and analyze the kidneys post sacrifice of the mice. Messenger RNA (mRNA) is extracted to measure for 1) kidney injury markers, 2) markers of fibrosis or scarring, and 3) inflammation markers within the damaged kidneys. Markers of kidney injury include Kidney Injury Molecule 1 (KIM-1), which is not normally present unless the kidneys have been damaged (Timmeren et al., 2007; Bonventre, 2008). KIM-1 is a well-known marker of proximal renal tubular injury in laboratory settings (Han et al., 2002). Fibrosis markers include Collagen-I (COL-I), and Fibronectin (FN), and Transforming Growth Factor Beta (TGF-β). COL-I and FN are structural proteins seen in scar tissue (Buchtler et al., 2018; Dixon et al., 1980), while TGF-β is noted for its role in signaling collagen production (Roberts et al., 1986).  Inflammation markers include Interleukin 6 (IL6), and Tumor Necrosis Factor α (TNF-α). Both are proinflammatory cytokines  partly responsible for controlling immune response (Su et al., 2017; Ramseyer et al., 2013). Other inflammation markers include Monocyte Chemoattractant Protein-I (MCP-I), and Vascular Cell Adhesion Molecule-I (VCAM-I). Both are involved in the migration and action of immune cells in response to injury (Segerer et al., 2000; Wuthrich, 1992).

It is important to note that while we can measure mRNA levels within the kidneys, we are unable to analyze systemic markers such as blood urea nitrogen/creatinine or urinary markers ­­­ in the UUO model. This is because the tying of the ureter obstructs urine production from the damaged kidney, while the other kidney continues to function normally and does not change blood or urinary markers of injury, fibrosis, or inflammation.

Methods

Generation of Mutant sPRR Mice

Cleavage of the PRR gene is performed by furin and site-1 protease. Through use of CRISPR-Cas9, cleavage sites of both enzymes were mutated so that sPRR cannot be generated (Fig. 3). In short, guide RNAs introduced two point mutations (R276A and R279A) into exon 8 of the ATP6AP2 gene. Homology-directed repairs altered the furin and site-1 protease sites of the gene. Founder mice were developed through co-injection of Cas9 mRNA and gRNA with donor oligo and targeting vectors into mice embryos. Mice were later genotyped, and genome sequencing was performed.

Unilateral Ureteral Obstruction

The Unilateral Ureteral Obstruction (UUO) procedure is able to cause scarring, inflammation, and fibrosis within the obstructed kidney through forcing the backflow of urine. Male mutant sPRR (MUT) and littermate wild type (WT) mice between four to seven months of age underwent surgical procedures. All mice were on a C57BL/6J strain. UUO mice had their left ureter tied off at the lower pole of the left kidney, while Sham mice underwent the motions of the UUO without actual tying of the ureter. Incisions were sutured closed, and mice were closely monitored in the following days. All mice successfully survived the procedure and survived until the time of sacrifice. Each cohort was sacrificed at a period of 3 days or 7 days post operation. Due to limited number of mice, we were unable to perform sham surgery in mutant sPRR mice. Only male mice were examined in this study. Details on surgical protocol are included in the appendix.

Plasma and Kidney Collection

Mice were sacrificed at 3 days or 7 days post UUO/Sham operation. 500 μL of blood was collected by cardiac puncture under anesthesia to measure plasma sPRR concentration. Kidneys were weighed, flash frozen in liquid nitrogen, and stored at -80°C. UUO/Sham kidneys were sectioned into thirds. One third underwent fixation for histology, one third underwent mRNA extraction for qRT-PCR, and one third was stored for immunoblotting.

Plasma sPRR Measurement

Plasma sPRR was measured using soluble (pro)renin receptor EIA assay kit (Immuno-Biological Laboratories Co., Gunma, Japan; 20898).  The kit is a solid phase ELISA that yields coloring that is proportional to the concentration of PRR. Colored samples were run in a UV-vis spectrophotometer against pre-prepared standards. Concentrations were calculated by comparing samples to a standard curve .

RNA Extraction and Verification

mRNA was isolated from UUO kidneys using PureLink RNA Mini Kit (Thermo Fisher Scientific, Carlsbad, CA, USA; 12183025). In short, one third of the UUO/Sham kidneys were homogenized with a rotor-stator homogenizer. Samples underwent multiple washes with Invitrogen’s wash buffers followed by centrifugation. Finally, mRNA was extracted with Invitrogen’s elution buffer.

Before analysis, RNA concentration was quantified and checked for purity via NanoDrop 2000 UV-vis Spectrophotometer. Both 260/280 and 260/230 ratios were compared to determine whether the mRNA was usable. Nucleic acids absorb at 260 nm, while common organic and inorganic contaminants absorb at 280 nm and 230 nm, respectively. For RNA, good purity at 260/280 are values between 2.0-2.3, while good values for 260/230 are between 1.6-1.8.  Once concentrations were calculated, the same amount of mRNA was used from each sample for reverse transcription.

Reverse Transcription

Isolated RNA (0.5-1 μg) was reverse transcribed to complimentary DNA (cDNA) using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Vilnius, Lithuania; 4368814). Samples were prepared in polymerase chain reaction (PCR) tubes and ran in Veriti 96-Well Thermocycler. Amount of RNA varied per cohort dependent on maximum RNA yield. Reverse transcription converts mRNA strands into cDNA preparing for rapid amplification during polymerase chain reaction.

Quantitative Reverse Transcription Polymerase Chain Reaction

mRNA levels were quantified through Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). qRT-PCR was performed using TaqMan Gene Expression Master Mix (Thermo Fisher Scientific, Waltham, MA, USA; 4369510) and SYBR Green PCR Master Mix (New England BioLabs, Ipswich, MA, USA; M3003) in a 96-well StepOne Real-time PCR System (Applied Biosystems, Foster City, CA, USA; 4376357). 2−ΔΔCT method was used to measure relative expression levels of mRNA. Markers were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH is 1) essential to cell survival and 2) expressed similarly in many tissue types (Sikand et al., 2012). TaqMan primer sequences (Table 1) (Thermo Fisher Scientific, Pleasanton, CA, USA; 4316034) were synthesized by Thermofisher (Thermo Fisher Scientific, Waltham, MA, USA ). SYBR Green primer sequences (Table 1) were synthesized at the University of Utah Primer Synthesis Core.

Table 1. List of TaqMan and SYBR Green primers for qRT-PCR. Top five rows are TaqMan, and the bottom three rows are SYBR Green. All primers were normalized to GAPDH.

Histology and Immunofluorescence

One third of UUO/Sham kidney  were fixed overnight in 10% formaldehyde and sent to the University of Utah Histology Core for paraffin fixation. Received slides were deparaffinized and Picro Sirius red staining was performed. To assess cross-sectional areas of inflammation and scarring, quantification was measured with ImageJ software. Picro Sirius red staining highlights structural tissue bright red, while other tissues are left as light yellow in color. ImageJ software allows a color threshold to be selected at which the percentage of red can be measured. Full protocols for staining and analysis are attached in the appendix.

Statistical Analysis

All mice were used for the analysis. 3-day UUO and 7-day UUO were analyzed separately. qRT-PCR data was compared through measuring the CT values. CT values are measured as the number of cycles required to amplify the cDNA to the preset threshold. 2−ΔΔCT values  were calculated and WT UUO/ mutant UUO were compared to the WT Sham. 1-way ANOVA with Tukey’s multiple comparisons test was used to compare fold values between each group. Statistical significance was marked at *P < 0.05.

Results

Male WT and mutant sPRR mice were used in this study for two reasons (1) ease of interpretation when comparing WT mice with mice with complete loss of sPRR in male mice as opposed to partial reduction of sPRR seen in female mutant sPRR mice, (2) female mice are less susceptible to kidney injury (Hodeify et al., 2013; Lima-Posada et al., 2017; Shepard, 2019).

Fig. 4. Plasma sPRR concentrations of wild type and mutant mice for both 3-day and 7-day; Mean +/- SEM. *P < 0.05.

Stark differences in plasma sPRR levels can be seen between the mutant sPRR and WT groups. WT mice on average had a plasma sPRR concentration of 18,420 +/- 1,990 pg/mL while mutant sPRR mice on average had a plasma sPRR concentration of 258.2 +/- 86.6 pg/mL. This suggests that mutant mice on average showed a 99.6% decrease in plasma sPRR levels when compared to WT mice.

The length of time until the UUO mice are sacrificed is directly proportional to the extent of kidney damage, scarring, and inflammation. Preliminary studies were performed at 21 days following UUO. 21-Day mice showed far too severe structural damage and scarring to the kidney, thus, further studies were performed at 3 and 7 days post- UUO. Results of 3-day and 7-day qRT-PCRs are shown below.

Fig. 5. Gene expression of KIM-1 by RT-PCR at 3-day (left; n = 7-10/group  ) and 7-day UUO (right; n = 3-4/group); Mean +/- SEM of fold difference comparing GAPDH to KIM-1 is shown. All groups are compared to WT Sham. P-values determined by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05.

For our kidney injury marker, both WT UUO and MUT UUO groups had significantly greater levels of Kidney Injury Marker 1 (KIM-1) compared to WT Sham and MUT Sham for both   3-day and 7-day.  For the 3-day mice, WT UUO had an average fold increase of 332 +/- 113, while MUT UUO had an average fold of 159 +/- 76 compared to WT Sham with an average fold of 1.34 +/- 0.46. Meanwhile, for the 7-day mice, WT UUO had an average increase of 390 +/- 60, while MUT UUO had an average fold of 391 +/- 204. Unlike the 7-day mice, however, the 3-day mice had a trend toward attenuated KIM-1 presence for MUT UUO compared to WT UUO. Although the difference is not statistically significant, this trend may be of interest, suggesting that the MUT UUO shows less kidney injury compared to wild type.

Fig. 6. Gene expression of COL-I by RT-PCR at 3-day (left; n = 7-10/group) and 7-day UUO (right; n = 6-8/group); Mean +/- SEM of fold difference comparing GAPDH to COL-I is shown. All groups are compared to WT Sham. P-values determined by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05.

Markers of kidney scarring and fibrosis show similar results. Firstly, the UUO procedure causes dramatically increased levels of Collagen-I (COL-I) when compared to Sham as expected. COL-I levels are similar for 7-day mice with values of 20.3 +/- 4.0 and 15.0 +/- 4.1 for WT and MUT, respectively. COL-I levels for 3-day mice, however, are moderately attenuated for MUT  UUO compared to WT UUO despite lacking statistical significance (MUT: 24.4 +/- 9.3 and WT: 70.4 +/- 20.0).

Fig. 7. Gene expression of FN by RT-PCR at 3-day (left; n = 7-10/group) and 7-day UUO (right; n = 6-8/group); Mean +/- SEM of fold difference comparing GAPDH to FN is shown. All groups are compared to WT Sham. P-values determined by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05.

Similar to COL-I, Fibronectin (FN) was significantly attenuated in MUT UUO mice compared to WT UUO at the 3-day time-point. WT UUO and MUT UUO for 3-day show average folds of 29.4 +/- 6.8 and 11.5 +/- 3.7, respectively. Unlike COL-I, the difference between WT UUO and MUT UUO is statistically significant with a p-value of 0.033. Meanwhile, WT UUO and MUT UUO for 7-day show average increases of 16.7 +/- 2.2 and 11.4 +/- 2.2, respectively, which were no longer statistically different.

Fig. 8. Gene expression of TNF-α by RT-PCR at 3-day (left; n = 2-3/group) and 7-day UUO (right; n = 6-8/group); Mean +/- SEM of fold difference comparing GAPDH to TNF-α is shown. All groups are compared to WT Sham. P-values determined by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05.

Unlike the kidney injury and fibrosis markers, there was no difference in TNF-α expression in the MUT UUO mice compared to WT UUO for either the 3-day or the 7-day mice. WT UUO and MUT UUO for 3-day show average increases of 25.7 +/- 6.8 and 30.3 +/- 6.1, respectively. WT UUO and MUT UUO for 7-day show average folds of 7.10 +/- 1.06 and 6.20 +/- 1.11, respectively relative to WT Sham.

Fig. 9. Gene expression of TGF-β by RT-PCR at 3-day (left; n = 2-3/group) and 7-day UUO (right; n = 6-8/group); Mean +/- SEM of fold difference comparing GAPDH to TGF-β is shown. All groups are compared to WT Sham. P-values determined by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05.

Transforming Growth Factor β (TGF-β) expression was also similar between MUT UUO mice compared to WT UUO for either the 7-day or the 3-day mice. WT UUO and MUT UUO for 3-day show average folds of 23.9 +/-7.9 and 27.0 +/- 2.8, respectively compared to WT Sham. WT UUO and MUT UUO for 7-day show average folds of 6.24 +/- 1.35 and 4.75 +/- 1.04, respectively relative to WT Sham.

Fig. 10. Gene expression of IL6 by RT-PCR at 3-day (left; n = 7-10/group) and 7-day UUO (right; n = 6-8/group); Mean +/- SEM of fold difference comparing GAPDH to IL6 is shown. All groups are compared to WT Sham. P-values determined by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05.

Similar to TNF-α and TGF-β, WT UUO and MUT UUO mice had similar Interleukin 6 (IL6) concentrations for both 3-day or 7-day time-points. WT UUO and MUT UUO for 3-day show average folds of 2.08 +/- 0.47 and 1.89 +/-0.69, respectively compared to WT Sham. WT UUO and MUT UUO for 7-day show average folds of 3.37 +/- 0.49 and 2.71 +/- 0.53, respectively relative to WT Sham.

Fig. 11. Gene expression of MCP-I by RT-PCR at 3-day (left; n = 9/group) and 7-day UUO (right; n = 6-8/group); Mean +/- SEM of fold difference comparing GAPDH to MCP-I is shown. All groups are compared to WT Sham. P-values determined by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05.

At 3 days, there is a statistically significant difference between WT UUO and MUT UUO mice in Monocyte Chemoattractant Protein-I (MCP-I) concentrations with a p-value of 0.011. MUT UUO MCP-I concentrations appear to be much less than the WT UUO concentrations, almost similar to the Sham concentrations. Meanwhile, 7-day mice had similar expression of MCP-I between WT and MUT UUO. WT UUO and MUT UUO for 3-day show average folds of 36.0 +/- 10.0 and 9.13 +/- 3.86, respectively. WT UUO and MUT UUO for 7-day show average folds of 23.9 +/-2.3 and 18.4 +/- 4.2, respectively.

Fig. 12. Gene expression of VCAM-I by RT-PCR at 3-day (left; n = 6-10/group) and 7-day UUO (right; n = 6-8/group); Mean +/- SEM of fold difference comparing GAPDH to VCAM-I is shown. All groups are compared to WT Sham. P-values determined by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05.

At 3 days, there was a trend toward lower Vascular Cell Adhesion Molecule-I (VCAM-I) expression in the MUT UUO mice compared to WT UUO. This difference, however, was not statistically significant. No difference in VCAM-I expression was seen at the 7-day time-point. For the 3-day mice, WT UUO and MUT UUO for 3-day show average increase of 31.9 +/- 13.3 and 12.0 +/- 6.3, respectively compared to WT Sham. WT UUO and MUT UUO for 7-day show average folds of 26.6 +/- 3.6 and 21.8 +/- 4.7, respectively relative to WT Sham.

Fig. 13. Sirius Red staining for Sham, WT UUO, and MUT UUO at 3 days following UUO. Strong pink coloring indicates fibrous tissue.

In the above slides, Picro Sirius Red stains normal tissue a light yellow in color, while Collagen-I found in fibrous and scar tissue stains bright pink as a. Vascular tissue (i.e. arteries and veins) and structural tissue contain Collagen-I, and thus, it is important to recognize these areas to discern them from scarring due to UUO. It is interesting to note that UUO causes tubular dilatation and injury in comparison to the Sham tissue due to the obstruction.

At 3 days, the Sham kidneys can clearly be seen to have the least scarring. Structural tissue can be seen closer to the center of the kidney at the renal pelvis, while normal blood vessels can be seen on occasion throughout the tissue. Comparatively, the WT UUO appears to have the most scarring as the greatest pockets of interstitial fibrosis are visible all throughout the sample. The MUT UUO tissue appears to have attenuated scarring in the WT UUO. Unfortunately, due to time constraints, staining is yet to be performed for the 7-day mice, and quantitative analysis for both groups is ongoing.

Discussion

We expected to find that: 1) Loss of sPRR would attenuate kidney injury, fibrosis, and kidney inflammation when compared to wild type; 2) similar results would be found at both 3-day and 7-day time-points; 3) Sirius red staining would show attenuated fibrosis in the mutant sPRR mice compared to WT.

Our results show that at 3 days following UUO, Collagen-I (COL-I), Fibronectin (FN), Monocyte Chemoattractant Protein-I (MCP-I), and perhaps Vascular Cellular Adhesion Molecule-I (VCAM-I) are all reduced in the mutant sPRR mice compared to WT UUO mice. Loss of sPRR appears to attenuate, but not entirely reduce expression of kidney injury and fibrosis markers, and some, but not all kidney inflammation markers in the setting of UUO. This suggests that other factors than sPRR may influence kidney injury, fibrosis, and kidney inflammation. Furthermore, out of all markers, only FN and MCP-I show statistically significant differences while a strong trend towards attenuated presence in MUT UUO can still be seen in the other markers (KIM-1, COL-I, VCAM-I). It is likely that this is due to variation between individual mice; further testing increasing the sample size may potentially show significance. In addition, inconsistencies during mice surgeries, RNA sample purity, and analysis may have led to variances in the data. Regardless, sPRR does appear to play a role in kidney injury and fibrosis in the setting of UUO.

It is interesting to note that the 7-day results are quite different from the 3-day time-point. Differences between the MUT UUO and WT UUO at seven days do not show support towards a trend. This may presumably be due to the length of progression of the kidney damage. 7-day mice may have sustained more damage than protection afforded by loss of sPRR. In other words, loss of sPRR may have an acute protective effect which is no longer present with ongoing obstruction at the 7-day time-point. This is not entirely unexpected as UUO models tend to increase in severity with ongoing obstruction and several pathways of injury, inflammation, and fibrosis are likely to be activated by then. Additional models not involving UUO may be helpful in determining the temporal trends of how sPRR protects against kidney injury and fibrosis.

In our 3-day qRT-PCR results, we found that both our fibrosis markers, COL-I and FN were reduced in MUT sPRR mice compared to wild type. Upon visible inspection, the Sirius red staining reflects this wherein interstitial fibrosis for the MUT UUO mice appears to be greatly reduced in comparison to the WT UUO mice although not quite to the same degree as the WT Sham mice. While the slides are straightforward to compare visually, due to time constraints, quantitative analyses are still ongoing and will be completed in the next month. We anticipate that the staining results would be in accordance with RT-PCR results with loss of sPRR reducing kidney fibrosis at 3-day time-point and not as much at the 7-day time-point.

In addition to performing quantitative analysis on the staining, immunoblotting of kidney injury, inflammation, and fibrosis is planned as RNA and protein expression may be quite different. Moreover, considering alternative mouse models of CKD such as treatment with adenine or folic acid, may provide valuable insights into the role of sPRR in kidney disease. These models induce fibrosis, inflammation, and injury differently from the UUO model, and may be able to further clarify the role of sPRR in kidney disease. Another potential consideration is whether female mutant sPRR mice are protected against kidney disease. While mutant sPRR males have near complete reduction of sPRR, mutant females retain close to 50%, potentially resulting in different outcomes when subjected to UUO or other kidney disease models. Further investigations of these aspects could potentially enhance the understanding of the sPRR.

The results of this study are humbly contributive to the nature of the soluble (pro)renin receptor. The work presented here does not, by any means, represent a comprehensive explanation of the sPRR. Our aim is to describe sPRR in an instance of CKD such as the UUO. Although sPRR might be a promising target for medical treatments, further study of the sPRR is required to help understand the RAAS pathway, and the implications of sPRR on CKD, CVD, and hypertension.

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Acknowledgements

My work here and my thesis would not have been possible without the guidance of my mentor and principal investigator, Dr. Nirupama Ramkumar.  Dr. Ramkumar has been instrumental in my understanding of this project, her research, and the work that I have completed. She is an amazing mentor for not only my research aspirations, but also for my path to medical school. I will be eternally grateful for the two years that I have had the opportunity to spend in her laboratory.

I am further grateful for Deborah Stuart, BS and Will Wheatley, MS, in the laboratory, not only for their assistance and contributions to this project, but also for their roles as mentors and leaders in my scientific journey. They have taught me much over the years and I am grateful for their willingness to help me learn and grow as a student in my college career.

Lastly, I am grateful to my parents for continuing to support me through all the ups and downs. It has not been the smoothest journey, but in the end, I am grateful for their love and support.

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