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Published in final edited form as: Am J Physiol Renal Physiol. 2025 Oct 14;329(5):F703–F711. doi: 10.1152/ajprenal.00231.2025

Renal tubular (pro)renin receptor deletion exacerbates kidney injury in db/db mice

Deborah Stuart 1, Caitlin S Peterson 1, Dhruvan Gopinath 1, Divya Sundar 1, Will Wheatley 1, Nirupama Ramkumar 1
PMCID: PMC12643321  NIHMSID: NIHMS2117925  PMID: 41087039

Abstract

The (pro)renin receptor (PRR) is a multifunctional protein implicated in blood pressure regulation and kidney fibrosis. Previous studies report enhanced PRR expression in non-diabetic and diabetic kidney disease. In this study, we investigated whether deletion of renal tubular PRR attenuates kidney injury in type 2 diabetes. Floxed PRR mice were bred with mice expressing Pax8 rtTA and LC1 transgenes and db/db mice (B6.BKS) to obtain renal tubular PRR knockout (KO)-db/db mice. Male, age matched non-diabetic floxed controls, db/db mice and PRRKO-db/db mice were studied at 16, 20, 26 and 30 weeks of age. PRR KO mice were only studied at 30 weeks of age. To induce PRR deletion, PRR KO and PRRKO-db/db mice were treated with 2 mg/ml doxycycline for 12 days at 8–10 weeks of age. Compared to controls, db/db mice and PRRKO-db/db mice had higher body weights throughout the study and elevated blood glucose levels at weeks 16 and 20. Compared to controls and db/db mice, PRRKO-db/db mice had higher urine volume, water intake and urinary albumin excretion. At 30 weeks, kidney histology showed minimal tubular or glomerular injury among all 4 groups. PRR KO mice had elevated expression of tubular injury markers compared to the other 3 groups. Plasma sPRR levels were almost two-fold higher in diabetic mice relative to controls with no difference between db/db mice and PRR KO-db/db mice. Renal tubular deletion of PRR does not protect against kidney injury in type 2 diabetes; rather the loss of PRR impairs baseline tubular function which is exacerbated by type 2 diabetes.

Keywords: (Pro)renin receptor, diabetes, kidney disease

New & Noteworthy

We investigated whether renal tubular deletion of the PRR would be protective in mice with type 2 diabetes. Longitudinal assessment up to 30 weeks of age demonstrated that diabetic mice with deletion of renal tubule PRR had higher albuminuria while gene expression of kidney injury markers was elevated in PRR KO mice at baseline compared to diabetic floxed controls and non-diabetic controls. Genetic deletion of PRR results in tubular cell dysfunction which is exacerbated in the setting of diabetes.

Graphical Abstract

graphic file with name nihms-2117925-f0006.jpg

Introduction

Diabetic kidney disease (DKD) is the leading cause of end stage renal disease in the United States and affects 20–30% of all diabetic patients (1). Activation of the renin angiotensin system (RAS) is an important contributor to the development and progression of DKD (2) and therapies that target the RAS are effective in slowing down the progression of DKD (1). However, these agents are not uniformly effective in all diabetic patients and a residual risk of progression to advanced kidney disease exists, despite treatment with RAS inhibitors, highlighting the need for development of new therapeutic agents that target other aspects of the RAS.

The (pro)renin receptor (PRR) is a unique component of the RAS (3) that is essential to nephron development (4) and plays an important role in renal tubular function including water transport (5, 6), sodium reabsorption (7–10) and acid excretion (11). We and others have demonstrated that the renal tubular PRR modulates blood pressure and kidney function through angiotensin-II dependent and independent pathways (7–10). Further, enhanced PRR expression has been described in experimental and clinical models of non-diabetic kidney disease (12–16).

Still, the role of the PRR in DKD is conflicting. While some studies report increased PRR expression in whole kidney extracts in diabetic rodents (17–19) and cultured renal cell lines exposed to high glucose (19–21), others found no difference in whole kidney PRR expression in diabetes (22). Early studies using the ‘handle region peptide’ which blocks prorenin from binding to the PRR, appeared to be promising in preventing the development of DKD in Type 1 and Type 2 diabetes (22–25); however, ensuing studies demonstrated that handle region peptide increased inflammatory markers and cardiac fibrosis in diabetes (26–28), questioning the specificity of handle region peptide in inhibiting PRR function. Based on the above studies, we hypothesized that the renal tubular PRR may contribute to the development of kidney injury and fibrosis in type 2 diabetes and renal tubule specific deletion of the PRR would be protective against tubular injury and interstitial fibrosis in DKD.

Methods

Animals

All studies were conducted with the approval of the University of Utah Animal Care and Use Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Details on generation of renal tubular PRR KO mice have been published (5). Floxed PRR mice were bred with mice expressing Pax8-rtTA and LC-1 transgenes and db/db mice (B6.BKS(D)- Leprdb/J homozygotes) purchased from the Jackson laboratory (Bar Harbor, ME). PRRKO-db/db mice were homozygous for the floxed PRR and db genes and hemizygous for the Pax8-rtTA and LC-1 transgenes. Floxed PRR mice without the Pax8-rtTA and LC-1 transgenes and PRR KO mice without db gene were used as non-diabetic controls. Due to differences in susceptibility to diabetic kidney injury between males and females, only male mice were studied. To induce PRR deletion, PRRKO db/db mice and PRR KO mice were treated with 2mg/ml doxycycline in 2% sucrose drinking water for 12 days at 8–10 weeks after birth.

Experimental design

Age matched floxed PRR non-diabetic controls, db/db mice and PRRKO-db/db mice were studied at 16 weeks, 20 weeks, 26 weeks and 30 weeks of age. PRR KO mice were only studied at 30 weeks of age. Mice were placed in metabolic cages for measurement of food and water intake, body weight, and 24-h urine collection at each of the study points for 48 hours. At 30 weeks of age, mice were sacrificed and plasma and kidneys harvested for further analyses. All metabolic cage studies and non-fasting blood glucose measurements were performed at the same time of the day (noon).

Blood and urine assays

Non-fasting blood glucose levels were measured using a glucometer (Accu-chek, Roche, Basel, Switzerland) and urine albumin with the Albuwell M ELISA kit (Ethos Biosciences, Newton Square, PA). Blood urea nitrogen (BUN) and urine creatinine were measured using Quantichrom colorimetric assays (Bioassay System, Hayward, CA). Specific EIA kits were used to measure plasma and urine total prorenin/renin content which is the total amount of immunoreactive prorenin and renin (Innovative Research, Novi, MI, SKU: IMSPRENKTT) and plasma sPRR (IBL America, Minneapolis, MN, SKU: IB59131).

Histology

One kidney was fixed overnight in 10% formaldehyde and embedded in paraffin; 4-μm sections were obtained. Sections were rehydrated with xylene and ethanol. Kidney sections were stained with periodic acid Schiff (PAS) stain per protocol by the ARUP Research Histology lab at the University of Utah or Picrosirius Red Stain. Segmental and global glomerulosclerosis and tubular injury and fibrosis were examined in a blinded fashion.

Kidney mRNA expression by RT-PCR

RNA was isolated from 1/2 of the kidney containing cortex and medulla and reverse transcription performed on 0.5 μg of total RNA with oligo(dt) and Superscript III reverse transcriptase according to manufacturer’s protocol (Invitrogen, Grand Island, NY). The resulting cDNA was assayed for relative expression of kidney injury molecule-1 (KIM-1, Mm00506686_m1), neutrophil gelatinase-associated lipocalin (NGAL, Mm01324470_m1), collagen-I (Mm00801666_g1), renin (Mm02342887_m1), PRR (Mm00510396_m1) and GAPDH (Mm03302249) using TaqMan Gene Expression Assay (Applied Biosystems, Carlsbad, CA) and Nephrin using SYBRgreen assay.

Western blot analysis

The other half of the kidney was divided into cortex and medulla and homogenized in ice-cold isolation buffer (50 mM Tris; 5 mM EDTA, 1% Triton, 1 mM PMSF) and complete protease inhibitors (Roche, Pleasanton, CA). Protein content was determined using the modified Lowry assay, and samples were solubilized with Laemmli loading buffer containing 0.5% lithium dodecyl sulfate. Equal amounts of protein (20 μg/lane) were run on a denaturing NUPAGE 4–12% Bis-Tris minigel (Invitrogen) and were transferred to a polyvinylidene difluoride plus nylon membrane. Membranes were incubated with specific antibodies against prorenin/renin (1:1000; cat. no. ab109012, Abcam, Cambridge, MA), PRR (1:1000, cat. no. HPA003156, Sigma Aldrich, St. Louis, MO) and GAPDH (1:2000; cat. no. 2118S, Cell Signaling). Secondary horseradish peroxidase-conjugated antibodies (goat anti-rabbit, catalog no: ab6721 Abcam, Cambridge, MA) were used at a dilution of 1:2000. Immunoblots were visualized with the C-Digit blot scanner (LI-COR Biosciences, Lincoln, NE). Densitometry was performed with a Bio-Rad gel documentation system (Bio-Rad, Hercules, CA). Western blots were performed per published guidelines (29).

Statistical analysis

All mice in each group completed the study and were included in the analyses. GraphPad Prism 10 was used to perform all statistical analysis. A priori power calculation performed with alpha level of 0.05 and 80% power to detect a difference of 18% necessitated 5 mice/group. Normality was tested using Shapiro-Wilk test. All results are expressed as means ± SE. Mixed effects or one way ANOVA with Tukey’s multiple comparison test was used to examine differences between the groups. The criterion for significance was p ≤ 0.05.

Results

Diabetic parameters

Male db/db, PRR KO-db/db and nondiabetic control mice were studied from 16 weeks to 30 weeks of age. All mice in each group survived until the end of the study. Compared to controls, diabetic mice had higher body weight (p<0.001, Figure 1A) despite lower food intake (Figure 1B); PRR KO-db/db mice had relatively lower body weight compared to db/db mice at 26 and 30 weeks of age (p<0.01). No differences in water intake were observed between control and db/db mice throughout the study duration while PRR KO-db/db mice had higher water intake compared to controls and db/db mice (p<0.01, Figure 1C) starting at 16 weeks. Similarly, urine volume was higher in PRR KO-db/db mice compared to control and db/db mice with no differences noticeable between control and db/db mice (Figure 1D). Food intake, water intake and urine volume, unadjusted for body weight are shown in Supplementary Figure S1.

Figure 1.

Figure 1.

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Body weight (A), food intake adjusted for body weight (B), water intake adjusted for body weight (C) and urine volume adjusted for body weight (D) in male non-diabetic floxed control, db/db and PRR KO db/db mice over time (N=5–7/group). * p<0.0001 controls vs db/db, $ p<0.001 controls vs PRR KO db/db, # p<0.05 db/db vs PRR KO db/db using repeated measures ANOVA with Tukey post-hoc test.

Db/db mice had elevated non-fasting blood glucose level at 16 and 20 weeks of age although these levels seemed to be similar to controls and PRR KO db/db mice at 30 weeks (Figure 2A). PRR KO-db/db mice had elevated blood glucose levels at 20 weeks but not at 16 or 30 weeks of age. No differences in blood urea nitrogen were observed among the 4 groups at 30 weeks (Figure 2B). PRR KO mice were only studied at 30 weeks of age. Urinary albumin/creatinine excretion was markedly elevated in PRR KO-db/db mice relative to controls, db/db mice and PRR KO mice (p<0.001, Figure 2C). There were no differences in urine albumin/creatinine excretion among the 3 other groups (Figure 2C).

Figure 2.

Figure 2.

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Non-fasting blood glucose over time (A) blood urea nitrogen (B) and urine albumin/creatinine excretion (C) at 30 weeks in male non-diabetic control, db/db, PRR KO db/db and PRR KO mice (N=4–7/group). * p<0.0001 control vs db/db, $ p<0.001 controls vs PRR KO db/db, # p<0.05 db/db vs PRR KO db/db using repeated measures ANOVA (A) and **** p<0.0001 using one way ANOVA with Tukey post-hoc test (B, C).

Kidney injury

At 30 weeks, compared to control mice, db/db mice had similar KIM-1, NGAL and collagen-1 mRNA expression in kidney cortex and inner medulla (Figure 3A–C). PRR KO db/db mice had increased cortical NGAL expression compared to control and db/db mice (Figure 3B). No differences were observed in cortical or inner medullary KIM-1 and collagen-1 expression in in PRRKO-db/db mice compared to control and db/db mice (Figure 3A, 3C). In contrast, PRR KO mice increased cortical KIM-1 and NGAL and inner medullary collagen-I expression relative to controls, db/db and PRR KO db/db mice (p<0.001, Figure 3A–C). No differences were observed with nephrin expression among the 4 groups (Figure 3D).

Figure 3.

Figure 3.

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Kidney tubular injury and fibrosis markers in male non-diabetic control, db/db, PRR KO db/db and PRR KO mice. Results from RT-PCR results for kidney injury molecule-1 (A), neutrophil gelatinase-associated lipocalin (B), collagen-I (C) and nephrin (D), (N=4–7/group). ****p<0.0001, ***p<0.001 using two-way ANOVA with Tukey post-hoc test.

Kidney histology assessed with PAS stain showed minimal tubular injury, glomerulosclerosis or interstitial inflammation among the 4 groups (Figure 4A). Picrosirius staining showed enhanced staining for collagen-I (Figure 4B) in PRRKO-db/db mice relative to control, db/db and PRR KO db/db mice at 30 weeks of age.

Figure 4.

Figure 4.

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Kidney histology at 30 weeks in male non-diabetic control, db/db, PRR KO db/db and PRR KO mice (N=4/group, 5 sections per mouse). Representative images from periodic acid schiff stained kidney sections 40x magnification (A), picrosirius red stained kidney sections 10x magnification (B).

Renin angiotensin system markers

No observable differences in plasma total prorenin/renin concentration were noted between control and db/db mice (Figure 5A) while PRR KO mice had significantly lower plasma total prorenin/renin concentration compared to db/db or PRR KO db/db mice. Plasma sPRR was almost two-fold higher in db/db mice and PRR KO db/db mice relative to controls or PRR KO mice (p<0.001, Figure 5B). Plasma sPRR levels were comparable between db/db and PRR KO db/db mice. Similarly, plasma sPRR levels were similar between non-diabetic controls and PRR KO mice. Urinary prorenin/renin/creatinine ratio was significantly elevated in PRR KO db/db compared to controls, db/db and PRR KO mice (Figure 5C). Similar levels or prorenin/renin excretion were found between controls, db/db mice and PRR KO mice. Urinary sPRR was undetectable in control and PRR KO mice (<0.5 pg/ml) and detectable in three out of 7 db/db mice (7.5 ± 0.7 pg/ml) and modestly elevated in PRR KO-db/db mice (47.6 ± 22.3 pg/ml).

Figure 5.

Figure 5.

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Plasma prorenin/renin concentration (A), plasma sPRR levels (B) urine prorenin/renin/creatinine ratio at 30 weeks (C), kidney renin mRNA expression (D), kidney PRR mRNA expression by RT-PCR (E), kidney cortical protein expression by immunoblotting (F) and quantification (G) in male non-diabetic control, db/db, PRR KO db/db and PRR KO mice (N=5–7/group). **** p<0.0001, *** p<0.001, ** p<0.01 * p<0.05 using two-way ANOVA with Tukey post-hoc test.

No difference in prorenin/renin mRNA expression was detected between control and db/db or PRRKO-db/db mice in either kidney cortex or inner medulla (Figure 5D). PRR KO mice had significantly lower inner medullary expression of prorenin/renin compared to controls and PRR KO db/db mice (Figure 5D). While cortical PRR mRNA expression was similar among all 4 groups (Figure 5D), PRRKO-db/db mice had markedly reduced inner medullary PRR expression (Figure 5E) relative to the other 3 groups. Compared to controls, abundance of cortical kidney prorenin/renin was increased in PRRKO-db/db mice while db/db mice had no change in prorenin/renin abundance (Figure 5F–G, Supplementary Figure S3). PRR abundance tended to be lower in PRRKO-db/db mice and db/db mice relative to controls, although this was non-significant (Figure 5F–G). We were unable to obtain good quality Western blots of kidney inner medulla due to low protein yield.

Discussion

DKD is the leading cause of end stage kidney disease in the United States and encompasses a progressive spectrum of clinical and histological changes (1). Although glomerular injury was thought to be the classical presentation of DKD, it is now well recognized that tubulointerstitial damage is equally important and may potentially precede the development of glomerular damage in some patients (30). Moreover, the extent of tubulointerstitial injury has prognostic implications in DKD and can predict decline in kidney function (30). While the precise mechanisms for tubulointerstitial dysfunction in DKD is not fully understood, tubular cell dysfunction, inflammation and oxidative stress in the setting of hyperglycemia are thought to play a role.

The PRR is a multifunctional protein that is widely expressed within the kidney including the proximal tubule, distal tubule, collecting duct, mesangial cells and podocytes (31). The PRR is involved in maintenance of renal tubule function including sodium and water transport (7–10) acid excretion (11) as well as a component of the Wnt/beta catenin signaling pathway (13, 32). Since constitutive deletion of the PRR during kidney development leads to abnormal development and early lethality (4, 33–35), inducible models of PRR deletion have been used to examine the role of PRR in blood pressure regulation (8–10), non-diabetic kidney disease (12, 13) and lysosomal function (11). In this study, we examined whether inducible, renal tubular deletion of the PRR is protective in DKD in a mouse model of type 2 diabetes.

Contrary to our hypothesis, deletion of the PRR in the renal tubules exacerbated albuminuria, tubular injury and kidney fibrosis with minimal glomerular damage. Since the c-terminal domain of the PRR serves as an accessory protein to the vacuolar H+ATPase (36), deletion of the PRR appears to have altered tubule cell lysosomal function which worsened in the setting of diabetes. Although we did not observe any differences in kidney function or histology under basal conditions (7–10), Trepiccione et al, using a similar inducible renal tubular PRR deletion mouse model, noted accumulation of autophagosomes within the epithelial cells of the collecting duct and thick ascending limb by electron microscopy along with enhanced expression of lysosomal markers such as LAMP2 and p62 (11). Additionally, tubule cells in the medulla demonstrated high levels of lipid droplets suggestive of impaired lipophagy suggesting a dysfunctional autophagy-lysosome system, particularly in the distal nephron segments (11). Indeed, we found increased expression of tubular injury and fibrosis markers in PRR KO mice compared to all 3 groups at 30 weeks of age. A more recent study described that mice with inducible renal tubular deletion of the PRR had albuminuria and low molecular weight proteinuria indicative of proximal tubule dysfunction resulting from abnormal receptor mediated endocytosis (37). Similar results were observed in transgenic rats with knockdown of the PRR (38). Of note, abundance of megalin, cubilin or other proximal tubule sodium transporters were unchanged in this study (38). While both studies were conducted in non-diabetic, basal conditions, Culver et al found that deletion of the renal tubule PRR altered endocytosis by reducing expression of megalin and increasing mTOR activity in mice treated with high fat diet for 6 months (37). These studies suggest that the PRR in both the proximal tubule and distal tubule are essential to maintaining normal tubular function. Hence, the deletion of the PRR in the setting of diabetes likely aggravated tubular injury and fibrosis in our model, resultant of impaired autophagy and receptor mediated endocytosis through both megalin dependent and independent mechansims.

Another interesting finding from our study is that PRR KO-db/db mice had modest reduction in body weight compared to db/db mice at 26 and 30 weeks of age. This is consistent with a previous study where mice with renal tubular deletion of the PRR, fed a high fat diet for 6 months, had lower body weight and fat mass compared to high fat diet fed floxed controls (37), presumably from high levels of glucosuria despite similar blood glucose levels between the two groups. We found no differences in non-fasting blood glucose levels between the diabetic mice, although PRR KO-db/db mice in our study took longer to develop hyperglycemia (around 20 weeks) compared to db/db mice (16 weeks). PRR KO-db/db mice also had increased urine volume and water intake from 16 weeks of age which coincided with the onset of albuminuria. Therefore, it is conceivable that the polyuria in the PRRKO-db/db mice might have lowered blood glucose levels in the early stages of diabetes, akin to SGLT2 inhibition, and contributed to reduced weight gain during the study duration. Another possibility is altered vasopressin signaling as previously noted in our prior studies (5, 6). Despite persistent and high-level proteinuria, however, PRR KO-db/db had no differences in BUN levels, mesangial expansion in the glomeruli or changes in nephrin expression. Notably, markers of tubular injury and fibrosis were several-fold higher in the PRR KO db/db compared to db/db mice highlighting the importance of the tubulointerstitial compartment in DKD.

As expected, plasma prorenin levels tended to be higher in diabetic mice at the end of the study with no differences between db/db mice and PRR KO-db/db mice. Conversely, urine prorenin/renin excretion was markedly elevated in PRR KO-db/db mice relative to controls and db/db mice with db/db mice having higher levels of urinary prorenin/renin levels compared to non-diabetic controls. This is in line with recent findings of elevated levels of urinary renin in patients and mice with diabetes (39) attributed to a combination of increased filtration from systemic circulation and impaired proximal tubule reabsorption. Prorenin abundance by western blot was also notably higher in the PRR KO-db/db mice compared to the other two groups despite no differences in renin gene expression within the cortex or inner medulla. While it is plausible that deletion of the renal tubular PRR might have altered tubule cell function leading to reduced reabsorption of prorenin from the tubule lumen, increased synthesis of prorenin/renin from the collecting duct, particularly during early stages of diabetes, cannot be completely ruled out (40). Further studies using the newer PRR pharmacological inhibitor, PRO20 (which differs significantly from the aforementioned handle region peptide) could help clarify the role of prorenin-PRR interaction in the setting of diabetes.

We did not detect any differences in PRR gene expression or protein abundance in db/db mice compared to controls. This contrasts with prior work where enhanced PRR expression have been described in humans, mice and rats with diabetes (17, 18) as well as in cultured cell lines exposed to high glucose (20, 21). The main reasons for this discrepancy may be attributed to the model used (type 1 vs type 2 diabetic model, high fat diet treatment vs genetic model of diabetes) and the time course of the study (early versus late stages of hyperglycemia). The PRR KO-db/db mice had reduced PRR gene expression in the inner medulla with a modest reduction in both gene expression and protein abundance in the cortex as noted in our prior studies (5, 10). Curiously, plasma and urinary soluble PRR (sPRR) levels were increased in the diabetic mice compared to controls indicating that plasma sPRR levels were independent of the kidney and most likely originated from adipose tissue in the diabetic mice. Whether elevated plasma and/or urinary sPRR levels can predict kidney damage in diabetes remains to be determined.

Few limitations exist in our study – firstly, the B6.BKS strain of the db/db mice is less susceptible to kidney damage compared to the KS/J strain and may have contributed to reduced severity of kidney damage observed in our study. We also did not employ uninephrectomy to hasten the severity of kidney damage as reported by others to allow examination of a more ‘natural’ progression of DKD. It is also possible that PRR deletion may have altered blood pressure in PRR KO-db/db mice which was not measured in our study. Finally, dose dependent effect of doxycycline to induce tubular PRR deletion have been reported (38) and lower doses of doxycycline could have limited tubular autophagy. Although it should be noted that doxycycline is considered safe for use in kidney disease with experimental data in rodents showing protective effects of doxycycline in kidney injury induced by ischemia/reperfusion, hemorrhagic shock or cisplatin treatment (41–43). We did not observe any evidence of tubular injury in wild type mice treated with doxycycline (Supplementary Figure S2). It is also unclear whether inhibition of the prorenin-PRR interaction, with the novel PRO20 (44), might help in attenuating DKD. Nonetheless, our results demonstrate definitively the importance of the tubulointerstitial injury in DKD (45) with the loss of tubular PRR exacerbating diabetes induced tubular cell damage and inflammation well before the onset of glomerular injury.

Supplementary Material

Supplementary figures S1-S3 available https://doi.org/10.6084/m9.figshare.30128887.v1

Acknowledgements

This research is supported by the American Diabetes Association Junior Faculty Development Award, National Institutes of Health (NIH) DK133271 and HL155345. We are also grateful to the Office of Undergraduate Research for their support through the Undergraduate Research Opportunity Program (to DG and DS).

Footnotes

Disclosures

The authors declare no conflict of interest.

Data Availability

All the data and materials supporting the findings of this study are available within the article. Further enquiries can be directed upon email to the corresponding author.

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Data Availability Statement

All the data and materials supporting the findings of this study are available within the article. Further enquiries can be directed upon email to the corresponding author.

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