Podocyte-Specific Expression of the Stress Response Protein REDD1 Is Necessary for Diabetes-Induced Podocytopenia

Podocyte injury and death are major contributing factors in the pathogenesis of diabetic nephropathy (DN). Podocyte loss is a reliable early indicator of renal injury, as it is observed in patients with early and late stages of DN (1,2) and in animal models of type 1 and type 2 diabetes (3,4). Podocytes are terminally differentiated epithelial cells with specialized cell junctions known as the slit diaphragm (SD). The SD connects interdigitating podocyte foot processes and establishes a size-selective barrier to protein loss (5). The SD is a zipper-like structure made by molecular cross-linking via proteins that include podocin and nephrin. The SD not only serves as molecular sieve but also acts as a complex signaling platform that regulates podocyte function (5,6). In diseased states like diabetes, the expression of SD proteins is reduced, resulting in compromised barrier function and albuminuria (7,8). Thus, an improved understanding of the specific molecular events that cause changes in the SD may identify new therapeutic targets for addressing DN pathogenesis.

Another key component of the SD that does not participate directly in cell junction is the cation channel transient receptor potential canonical channel 6 (TRPC6). TRPC6 is essential for maintaining calcium homeostasis in podocytes (9). TRPC6 activation enhances the influx of calcium ions in podocytes to influence cellular processes, including cytoskeletal organization and apoptosis (10). TRPC6 activation is linked to hereditary and acquired forms of proteinuric kidney diseases (11). Indeed, TRPC6 expression and activity are upregulated in DN (12); however, the specific signaling events responsible for diabetes-induced TRPC6 upregulation remain to be identified. One possibility is that activation of the transcription factor nuclear factor-κB (NF-κB) in response to diabetes influences TRPC6 in podocytes, given that prior reports provide evidence that NF-κB acts as a regulatory effector upstream of TRPC6 in other contexts (13,14).

The stress response protein regulated in development and DNA damage response 1 (REDD1; also known as DDIT4 or RTP801) promotes NF-κB signaling in response to diabetes (15). REDD1 has been linked to the development of diabetes complications, including DN (3,16). REDD1 protein abundance is increased in the kidney of patients with diabetes (16), in the kidney of diabetic mice, and in podocyte cultures exposed to hyperglycemic conditions (3,17). Remarkably, whole-body REDD1 deletion prevents podocyte loss and albuminuria in diabetic mice (3). In response to hyperglycemic conditions, REDD1 contributes to oxidative stress and proinflammatory signaling in podocytes that are potentially responsible for cell death (3,17,18), raising the question of whether diabetes-induced REDD1 expression specifically in podocytes is sufficient to drive renal pathology. Herein, we investigated the role of podocyte-specific REDD1 in DN.

Please see the Supplementary Material for detailed methods.

All procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Penn State College of Medicine Institutional Animal Care and Use Committee. In male B6;129 mice that were homozygous for the REDD1 mutation (REDD1^−/−) (19) or in wild-type (REDD1^+/+) mice, diabetes was induced by administering 50 mg/kg streptozotocin (STZ) intraperitoneally for 5 consecutive days. Sodium citrate solution was administered as vehicle control. Diabetic phenotype was confirmed by fasting blood glucose concentrations >250 mg/dL. Male mice were used for all studies due to the resistance of female mice to developing hyperglycemia in response to low-dose STZ. To generate podocyte-specific REDD1 knockout (podKO) mice, floxed REDD1 mice (REDD1^fl/fl) (20) were crossed with mice expressing Cre recombinase in podocytes [B6.Cg-Tg(NPHS2-cre)295Lbh/J; The Jackson Laboratory, stock no. 008205]. At 16 weeks of diabetes, mice were euthanized, and kidneys were processed as described below. Protein excretion and urine albumin-to-creatinine concentrations were determined from spot urine samples, as previously described (3).

Formalin-fixed paraffin-embedded renal sections were processed for periodic acid Schiff (PAS) and hematoxylin and eosin (H&E) staining. Immunofluorescence labeling was done using the antibodies listed in Supplementary Table 1. Tissue sections were counterstained with 1 µmol/L Hoechst_33342 (Invitrogen), and photomicrographs were captured using a confocal laser microscope.

For transmission electron microscopy (TEM), kidneys were processed as described previously (3), and glutaraldehyde-fixed kidney cortical sections (65 nm) were mounted on a copper grid and photographed using a JEOL JEM1400 Transmission Electron Microscope located at the Penn State College of Medicine TEM Core Facility (RRID no.: SCR_021200).

All quantifications were performed in a masked manner. Mesangial matrix deposition and glomerular volume were evaluated as previously described (21). Glomerular tuft area (GA) was quantified using ImageJ and converted to glomerular volume (GV) by applying a spherical approximation formula (GV = 1.2545[GA]^1.5). Mesangial index was defined as the ratio of mesangial area to glomerular tuft area.

Mouse glomeruli were isolated from kidney cortex using a differential adhesion method, as previously described (24). Briefly, the kidney cortex was digested with collagenase IV (1 mg/mL) in Hanks' balanced salt solution at 37°C for 15 min. Digested tissue was filtered successively, and differential adhesion of tubules was performed. The filtrate was washed on a 40-μm cell strainer, and intact glomeruli were isolated.

Conditionally immortalized human podocytes (CIHP-1) were obtained from Dr. Moin Saleem (University of Bristol, Bristol, U.K.) and cultured as described (3). A stable CIHP-1 cell line deficient in REDD1 (REDD1 KO) was generated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing. To model hyperglycemia, cells were exposed to culture medium containing 30 mmol/L glucose (HG) or an osmotic control (OC; 11.1 mmol/L glucose plus 18.9 mmol/L mannitol). Cells were transfected using Lipofectamine 2000 (Life Technologies). Plasmids included pCMV5 vector, hemagglutinin (HA)-tagged pCMV-HA-REDD1, and pBabe-GFP-IKBalpha-mut (super repressor), which was a gift from William Hahn (Addgene plasmid no. 15264).

F-actin fibers in CIHP-1 cells were stained with rhodamine phalloidin (Actistain 535, Cytoskeleton), nuclei were counterstained with 1 µmol/L DAPI (Invitrogen), and images were captured using a Leica DMi8 microscope. Ten fields containing at least 200 cells were randomly evaluated by two independent investigators, and each podocyte was rated on a 3-point scale, as described previously (25). The mean cortical F-actin score was then computed.

Intracellular calcium (Ca²⁺) entry into the cytosol was measured by ratiometric imaging using fura-2, as described previously (26). In brief, podocytes were loaded with 2 µmol/L Fura-2/AM (no. F1221, Invitrogen), and fluorescence was measured using a Leica DMI 8 fluorescence microscope. Consecutive excitation at 340 nm (F340) and 380 nm (F380) was applied every 2 s, and emission fluorescence was collected at 505 nm for 3 min. To determine TRPC6-mediated Ca²⁺ entry, cells were treated with the TRPC6 selective agonist oleoyl-2-acetyl-sn-glycerol (OAG, 300 µmol/L; Cayman Chemicals). Intracellular Ca²⁺ flux was measured as F340-to-F380 ratios obtained from groups of >25 single cells from three independent experiments.

Total proteins were extracted from cells, renal cortical tissue, or isolated glomeruli using radioimmunoprecipitation assay lysis buffer. Western blot analysis was performed as previously described (3) with the appropriate antibodies (Supplementary Table 1).

Total RNA was extracted, reverse transcribed, and analyzed by real-time quantitative PCR (qPCR) (QuantStudio 12 K Flex Real-Time PCR System, RRID:SCR_021098) with primers listed in Supplementary Table 2. Mean cycle threshold values were determined. Fold change in mRNA expression relative to GAPDH mRNA was calculated.

The binding capacity of NF-κB p65 to the promoter region of the TRPC6 gene was confirmed using a chromatin-immunoprecipitation (ChIP) assay in conjunction with qPCR (ChIP-qPCR). The ChIP assay was performed using a Simple ChIP Plus Enzymatic ChIP Kit (Cell Signaling Technology), following the manufacturer’s instructions. In brief, proteins from CIHP-1 podocytes were cross-linked with chromatin, and subsequently, the protein-chromatin complex was broken using ultrasonication. The soluble chromatin was incubated with anti-p65 antibody or IgG (negative control) at 4°C overnight, and immunoprecipitation was done using protein G magnetic beads (Cell Signaling Technology). The recovered DNA was then analyzed by qPCR using primers listed in Supplementary Table 2. Results were tabulated as fold enrichment normalized to the IgG controls (34).

Data are expressed as mean ± SD. Statistical analysis of data with more than two groups was performed by two-way ANOVA, with the Tukey test for multiple comparisons used for pairwise analysis. The relationship between the urine albumin-to-creatinine ratio (ACR) and REDD1 protein abundance was tested by Spearman correlation analysis. Significance was defined as P < 0.05 for all analyses. Sample sizes for each experiment are listed in Supplementary Table 3, and exact P values for significantly different groups are listed in Supplementary Table 4.

Primary data, including unedited Western blot images and immunofluorescent images supporting the findings of this study, are reported within the supplementary information. Animation files for calcium imaging are available at https://doi.org/10.6084/m9.figshare.26790010.v1. Excel files with raw data supporting the findings will be made available upon request.

In the kidney of STZ-diabetic mice, REDD1 protein abundance was increased in coordination with elevated urinary albumin (Fig. 1A) (3). In support of prior reports (7), reduced Nphs2 (podocin) mRNA expression (Fig. 1B) and podocin protein abundance (Fig. 1C and D) were observed in the kidneys of STZ-diabetic wild-type mice. REDD1 deletion reduced the diabetes-induced attenuation in podocin mRNA and protein in the kidney. A similar REDD1-dependent reduction in NPHS2 mRNA expression (Fig. 1E) and podocin protein abundance (Fig. 1F and G) was also seen in human podocytes exposed to hyperglycemic culture conditions. To confirm the impact of REDD1 on podocin abundance in podocytes, REDD1 was restored in REDD1-deficient cells by expression of the HA-tagged REDD1 plasmid. Ectopic REDD1 expression reduced podocin levels in REDD1 KO cells when exposed to OC or hyperglycemic conditions (Fig. 1H). Thus, increased REDD1 protein content was sufficient to attenuate podocin protein abundance in podocytes.

Hemizygous podocin-cre mice were crossed with REDD1^fl/fl mice to achieve podocyte-specific recombination (NPHS2-cre⁺, REDD1^fl/fl; REDD1 podKO). PCR analysis of genomic DNA was used to confirm homozygous expression of the floxed REDD1 gene and NPHS2-Cre in REDD1 podKO mice (Supplementary Fig. 1A). Compared with REDD1^fl/fl mice, podocyte-specific REDD1 deletion nearly eliminated REDD1 mRNA expression in isolated glomeruli and partially reduced REDD1 mRNA expression in kidney homogenates from REDD1 podKO mice (Fig. 2A). REDD1 mRNA expression in liver, heart, and retina were unchanged between the two groups. To evaluate the role of podocyte-specific REDD1 expression on diabetes-induced renal injury, mice were administered STZ. Diabetic and nondiabetic REDD1 podKO mice exhibited similar fasting blood glucose concentrations as those observed in diabetic and nondiabetic REDD1^fl/fl mice, respectively (Fig. 2B). REDD1 protein abundance was increased within glomeruli of diabetic REDD1^fl/fl mice compared with nondiabetic controls (Fig. 2C and D). Podocyte-specific REDD1 deletion reduced glomerular REDD1 protein abundance and prevented an increase in REDD1 in response to diabetes. Diabetes reduced Nphs2 mRNA expression within glomeruli isolated from REDD1^fl/fl mice, and the suppressive effect was blunted in REDD1 podKO mice (Fig. 2E). Similarly, glomerular podocin protein levels were also reduced in diabetic REDD1^fl/fl mice but not in diabetic REDD1 podKO mice compared with their respective nondiabetic controls (Fig. 2F and G). The data support that podocyte-specific REDD1 contributes to diabetes-induced reduction in podocin.

Compared with nondiabetic mice, diabetic mice had reduced body weights (Supplementary Fig. 1B) but increased kidney-to-body weight ratio (Supplementary Fig. 1C). No genotype differences were observed. Glomerular morphology and mesangial proliferation were examined by H&E and PAS staining, respectively (Fig. 3A). Histology revealed increased glomerular size, basement membrane thickening, and mesangial matrix deposition in diabetic REDD1^fl/fl mice compared with nondiabetic controls. Podocyte deletion of REDD1 prevented increased glomerular volume and reduced the Bowman space in the kidneys of diabetic mice (Fig. 3B). Increased mesangial proliferation was also attenuated in mice lacking podocyte REDD1 postdiabetes induction (Fig. 3C). Podocytopenia is a clinical hallmark of early DN in patients with type 1 or type 2 diabetes (1,2). Wilms tumor-1 (WT-1) immunolabeling was used to identify podocytes. Podocyte-specific ablation of REDD1 attenuated diabetes-induced podocyte loss and prevented reduced nephrin protein abundance compared with diabetic REDD1^fl/fl mice (Fig. 3D). Quantification of WT-1–positive nuclei within glomeruli revealed a reduction in podocytes within glomeruli of diabetic REDD1^fl/fl mice but not in diabetic REDD1 podKO mice (Fig. 3E). Podocyte apoptosis was also characterized by visualizing TUNEL-positive nuclei colocalized with WT-1 within the glomerulus (Fig. 3F). Whereas an increase in TUNEL-positive cells colocalized with WT-1 cell staining was present in glomeruli of diabetic REDD1^fl/fl mice, an increase in apoptotic cells was attenuated in diabetic REDD1 podKO mice (Fig. 3G). In summary, REDD1 deletion in podocytes was sufficient to prevent development of glomerular pathology and podocytopenia in DN.

Foot processes function and the preservation of SD integrity depend on focal podocyte adhesions and actin cytoskeletal structure (27). To determine whether REDD1 contributed to impairment of podocyte cytoskeletal architecture, we assessed podocyte F-actin arrangement. Hyperglycemic conditions lead to overt F-actin disorganization in wild-type podocytes, which was characterized by the loss of dispersed central stress fibers and the appearance of cortical F-actin (Fig. 4A and B). Unlike wild-type cells, REDD1-deficient cells maintained their actin cytoskeletal structure after exposure to hyperglycemic environments. Calcium (Ca²⁺) homeostasis is important in regulating podocyte cytoskeleton dynamics, and hyperglycemic conditions increase cytosolic Ca²⁺ entry in podocytes (12). Upon exposure to hyperglycemic conditions, basal Ca²⁺ levels were increased in wild-type podocytes but not in REDD1-deficient podocytes (Fig. 4C and D). In wild-type cells exposed to hyperglycemic conditions, TRPC6 protein abundance was increased (Fig. 4E). However, a similar increase in TRPC6 was not observed in REDD1-deficinet cells. Ca²⁺ entry in response to the TRPC6-selective agonist OAG was augmented in wild-type cells exposed to hyperglycemic conditions (Fig. 4F). REDD1 deletion reduced OAG-mediated Ca²⁺ entry and prevented an increase in the response under hyperglycemic conditions. To determine whether REDD1 had a similar effect in the kidney, renal sections were immunolabeled for TRPC6. Diabetes increased TRPC6 colocalization with nephrin within glomeruli of REDD1^fl/fl mice but not in REDD1 podKO mice (Fig. 4G). Western blot analysis of glomerular protein isolates from diabetic mice also showed a REDD1-dependent increase in TRPC6 protein compared with nondiabetic controls (Fig. 4H).

Trpc6 mRNA expression in glomerular isolates from REDD1^fl/fl mice was significantly increased with diabetes, and podocyte-specific deletion of REDD1 prevented the effect (Fig. 5A). A similar REDD1-dependent increase was also observed in podocyte cultures exposed to hyperglycemic conditions (Fig. 5B). NF-κB promotes transcription of TRPC6 by increased binding of the p65 subunit at the TRPC6 promoter (14). To determine the impact of NF-κB activity on TRPC6 transcription, wild-type cells expressing an empty vector or an I-κB superrepressor mutant were exposed to hyperglycemic conditions. Suppression of NF-κB activity prevented an increase in TRPC6 mRNA expression upon exposure to hyperglycemic conditions (Fig. 5C). To assess the impact of REDD1 on p65 NF-κB binding to the TRPC6 promoter, a ChIP PCR assay was performed. Increased binding of p65 at the TRPC6 promoter was observed in wild-type cells exposed to hyperglycemic conditions compared with OCs (Fig. 5D). REDD1 deletion suppressed p65 binding to the TRPC6 promoter and prevented an increase in TRPC6 enrichment in the p65-ChIP in cells exposed to hyperglycemic conditions.

TEM was used to determine changes in glomerular ultrastructure. Compared with nondiabetic mice, we observed diffused thickening of the GBM and extensive fusion of foot processes within glomeruli of diabetic REDD1^fl/fl mice (Fig. 6A). Morphometric analysis of TEM photomicrographs showed a significant increase in GBM thickness that was reduced in glomeruli of diabetic REDD1 podKO mice (Fig. 6B). We also observed increased podocyte foot processes width within the glomeruli of diabetic REDD1^fl/fl mice compared with nondiabetic controls (Fig. 6C). Podocyte REDD1 deletion prevented foot process effacement in diabetic mice. Microalbuminuria is an important early indicator of DN progression and is often caused by the dysfunction of the glomerular filtration barrier (28). Urinalysis of diabetic REDD1^fl/fl mice revealed an increased urinary protein band at 60 kDa that was reduced in the urine from diabetic REDD1 podKO mice (Fig. 6D). Quantification of urine albumin and creatinine levels showed a marked increase in urine ACR in diabetic REDD1^fl/fl mice that was attenuated in diabetic REDD1 podKO mice (Fig. 6E). These data support that REDD1 deletion from podocytes is sufficient to attenuate the glomerular structural changes and filtration function decline that is observed in diabetic mice.

Podocyte injury, podocytopenia, and, consequentially, microalbuminuria are hallmarks of early DN. Using a combination of in vivo and in vitro loss-of-function approaches, we demonstrated herein that podocyte-specific expression of the stress response protein REDD1 was required for podocyte loss, deterioration of the glomerular filtration barrier, and the subsequent decline in filtration function in a well-characterized preclinical model of type 1 diabetes. The studies here also provide evidence that REDD1 in podocytes acts to augment TRPC6-mediated intracellular Ca²⁺ influx, disorganization of the actin cytoskeleton, and reduced SD integrity. Overall, the findings support a model wherein diabetes-induced podocyte-specific REDD1 expression disrupts calcium homeostasis, resulting in glomerular injury and filtration function deficits (Fig. 7).

A growing body of evidence suggests that REDD1 expression in the kidney contributes to the development of DN pathophysiology (3,16,17). Single-cell sequencing provides evidence that REDD1 expression is not restricted to a specific type of kidney cell (29). We previously reported that REDD1 expression was increased within the renal cortex of STZ-diabetic mice and that germline deletion of REDD1 prevented diabetes-induced podocyte loss (3). Moreover, REDD1 deletion in cultured podocytes prevented podocyte apoptosis in response to hyperglycemic conditions (3). Because podocyte loss is a clinical hallmark of early DN pathogenesis, we used Cre-lox recombination to KO REDD1 specifically in podocytes. REDD1 deletion in podocytes nearly eliminated REDD1 expression within glomerular isolates and attenuated REDD1 levels in renal cortical homogenates. In further support of podocyte specificity, REDD1 expression in other tissues, including the liver, retina, and heart, remained unchanged. Although the NPHS2-cre is a well-established tool to modify gene function precisely in podocytes (30), it has been reported that mice expressing the NPHS2-cre demonstrate modest GBM thickening and altered foot process morphology (31). We did not observe a change in these end points in nondiabetic REDD1 podKO mice compared with nondiabetic REDD1^fl/fl mice. Rather, podocyte-specific deletion of REDD1 in mice expressing NPHS2-cre reduced the diabetes-induced changes in GBM and foot process morphology. Importantly, REDD1 deletion in podocytes was sufficient to prevent early renal pathology and attenuate podocyte loss with STZ-induced diabetes. Notably, single-nucleus and single-cell transcriptomic analyses of human kidneys have indicated REDD1 expression in other cell types, including endothelial cells and immune cell populations (32). However, REDD1 deletion in podocytes was sufficient to reduce REDD1 levels in both glomerular isolates and renal cortical homogenates from the kidneys of diabetic mice. This supports that REDD1-dependent processes in podocytes are potentially necessary for REDD1 upregulation in other renal cells in the context of diabetes.

Podocyte injury is a key event in the development of glomerular filtration defects. In the current study, we used a well-established model of type 1 diabetes to examine this phenomenon. A limitation of this study is that compared with other inbred mouse strains (e.g., DBA/2J, KK-Ay), C57BL/6 mice with STZ-induced hypoinsulinemia and secondary hyperglycemia have modest changes in glomerular pathology and mild-to-moderate increases in urine albumin excretion (33,34). Whereas DBA/2 mice develop albuminuria within 5 weeks of diabetes induction, the onset of albuminuria with STZ-diabetes is delayed in C57BL/6 mice (33). Importantly, low-dose STZ has no direct toxic effects on podocytes after 2 weeks of administration (35). Consistent with our prior report (3), we observed a close association between renal REDD1 levels and increased albumin excretion. Remarkably, podocyte-specific REDD1 deletion was sufficient to maintain the structural and functional integrity of the glomerular filtration barrier and prevented albuminuria in STZ-diabetic mice. Together, the observations support a key role of podocyte REDD1 expression in the failed adaptive response of the kidney to diabetes.

In diseased states like diabetes, expression of SD proteins is reduced, resulting in compromised barrier function and albuminuria. In support of prior observations (10), SD proteins, including podocin and nephrin, were significantly attenuated by diabetes or exposure to hyperglycemic conditions. We also observed that the reduction in SD protein content was concomitant with compromised glomerular ultrastructure. REDD1 deletion in podocytes preserved SD protein levels and prevented the deterioration of podocyte architecture in response to diabetogenic stimuli. Podocyte-specific REDD1 deletion also prevented characteristic signs of early DN pathology that included increased volume of the glomerular tuft, GBM thickening, foot process effacement, and podocyte apoptosis (7).

TRPC6 is a stress-induced cation channel that associates with the SD and is required for proper renal function (36). Clinical observations and gain-of-function mutation studies demonstrate that increased TRPC6 expression correlates with aberrant calcium homeostasis and proteinuric kidney disease (11). In metabolic diseases like diabetes, TRPC6 expression is associated with reduced SD protein expression and disorganized F-actin assembly that leads to foot process effacement, podocyte loss, and eventually, the development of proteinuria (10,37,38). Hyperglycemia has previously been implicated as a major driver of TRPC6 expression and activity (10,38). Herein, REDD1 was required for increased TRPC6 expression and an increase in cytoplasmic Ca²⁺ in podocytes exposed to hyperglycemic conditions, suggesting a potential mechanism by which REDD1 regulates podocyte dysfunction. It is worth noting that the data herein do not exclude the possibility that REDD1 also acts to influences TRPC6 localization or activity.

Although some reports suggest that NF-κB mediates suppression of canonical TRPC6 expression (13), others have shown that the p65 subunit of NF-κB binds to the promoter region of TRPC6 to increase its transcription (14). An abundance of literature in recent years has supported a role for REDD1 in mediating NF-κB–mediated inflammatory response in diabetes complications (39–43). In response to hyperglycemic conditions, NF-κB suppression prevented increased TRPC6 mRNA expression. Notably, ChIP PCR analysis indicated that REDD1 deletion in podocytes reduced NF-κB binding to the TRPC6 promoter and prevented an increase in response to hyperglycemic conditions. It is worth noting that other transcription factors, including AP-1 and SP-1, share consensus-binding motifs in the TRPC6 promoter region and might also influence the expression of the TRPC6 gene through NF-κB–independent mechanisms (13). Moreover, studies in multiple cell types, including podocytes (37,44–46) and vascular myocytes (47), attribute a role for increased oxidative stress in the activation and expression of the TRPC6 channel. However, studies have also shown that reactive oxygen species–dependent activation of protein kinase C in mesangial cells can reduce TRPC6 expression (13).

Overall, the data herein are consistent with a model wherein enhanced REDD1 expression in podocytes leads to their failure to properly adapt to the diabetic metabolic environment, resulting in podocytopenia and microalbuminuria. The results provide new insight into mechanisms of podocyte injury and support targeting REDD1 as a therapeutic approach in the context of DN. Inhibiting REDD1 could also have additional benefits on account of its known inhibitory action on GSK3β activity (3,40,48). Although targeting REDD1 using siRNA therapeutics has previously been used in a clinical setting to target retinal complications such as macular degeneration (49) and diabetic macular edema (50), the efficacy and safety of inhibiting REDD1 as a potential strategy for DN remains to be established.

Acknowledgments. The authors thank Elena Feinstein (Quark Pharmaceuticals) for permission to use the REDD1 KO mice and David Williamson (Penn State Harrisburg) for the use of the REDD1^fl/fl mice. The authors are very grateful to Ellen Mullady and Gretchen Snavely, of the Department of Comparative Medicine at Penn State College of Medicine, for their preparation of histology sections.

Funding. This research was supported by grants from the American Diabetes Association (grant no. 11-23-PDF-84), Children’s Miracle Network Trainee Research Award, the Judy S. Finkelstein Memorial Student Research Award, and the Penn State College of Medicine’s Comprehensive Health Studies Program (to S.S.), as well as National Institutes of Health National Eye Institute grant R01 EY032879, and an Innovative Award 1-INO-2024-1538-A-N from JDRF (to M.D.D.).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. S.S., E.I.Y., A.L.T., H.C., and Y.Z. contributed to the investigation. S.S., A.L.T., H.C., Y.Z., and M.D.D. contributed to methodology. S.S., D.L.G., S.R.K., and M.D.D. conceptualized the study. S.S., D.L.G., S.R.K., and M.D.D. reviewed and edited the manuscript. S.S. and M.D.D. curated the data and contributed to formal analysis, funding acquisition, visualization, and writing the manuscript. S.R.K. and M.D.D. provided resources. S.R.K. and M.D.D. supervised the study. M.D.D. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and accuracy of the data analysis.