Progression of established non-diabetic chronic kidney disease is halted by metformin treatment in rats
Authors: Raphaëlle Corremans, Ellen Neven, Stuart Maudsley, Hanne Leysen, Marc E. De Broe, Patrick C. D’Haese, Benjamin A. Vervaet, Anja Verhulst
Journal: Kidney International (2022)

Current treatment strategies for chronic kidney disease (CKD) mainly focus on controlling risk factors. Metformin, a first-line drug for type 2 diabetes, exerts beneficial pleiotropic actions beyond its prescribed use and incipient data have revealed protective effects against the development of kidney impairment. This study evaluated the therapeutic efficacy of metformin and canagliflozin, a sodium-glucose cotransporter-2 (SGLT2) inhibitor recently approved by the United States Food and Drug Administration to treat diabetic nephropathy, in slowing the progression of established non-diabetic CKD. Rats with adenine-induced CKD were assigned to different treatment groups to receive either 200 mg/kg metformin, four or five weeks after the start of the adenine diet (established mild-moderate CKD), or 25 mg/kg canagliflozin four weeks after the start of the diet, by daily oral gavage administered during four weeks. Each treatment group was compared to a vehicle group. Chronic adenine dosing resulted in severe CKD in vehicle-treated rats as indicated by a marked rise in serum creatinine levels, a marked decrease in creatinine clearance, and a disturbed mineral metabolism. Metformin, but not canagliflozin, halted functional kidney decline. Additionally, kidneys of metformin-treated animals showed less interstitial area and inflammation as compared to the vehicle group. Proteomic analyses revealed that metformin's kidney-protective effect was associated with the activation of the Hippo signaling pathway, a highly conserved multiprotein kinase cascade that controls tissue development, organ size, cell proliferation, and apoptosis. Thus, metformin demonstrated therapeutic efficacy by halting the progression of established CKD in a rat model. Current treatment strategies for chronic kidney disease (CKD) mainly focus on controlling risk factors. Metformin, a first-line drug for type 2 diabetes, exerts beneficial pleiotropic actions beyond its prescribed use and incipient data have revealed protective effects against the development of kidney impairment. This study evaluated the therapeutic efficacy of metformin and canagliflozin, a sodium-glucose cotransporter-2 (SGLT2) inhibitor recently approved by the United States Food and Drug Administration to treat diabetic nephropathy, in slowing the progression of established non-diabetic CKD. Rats with adenine-induced CKD were assigned to different treatment groups to receive either 200 mg/kg metformin, four or five weeks after the start of the adenine diet (established mild-moderate CKD), or 25 mg/kg canagliflozin four weeks after the start of the diet, by daily oral gavage administered during four weeks. Each treatment group was compared to a vehicle group. Chronic adenine dosing resulted in severe CKD in vehicle-treated rats as indicated by a marked rise in serum creatinine levels, a marked decrease in creatinine clearance, and a disturbed mineral metabolism. Metformin, but not canagliflozin, halted functional kidney decline. Additionally, kidneys of metformin-treated animals showed less interstitial area and inflammation as compared to the vehicle group. Proteomic analyses revealed that metformin's kidney-protective effect was associated with the activation of the Hippo signaling pathway, a highly conserved multiprotein kinase cascade that controls tissue development, organ size, cell proliferation, and apoptosis. Thus, metformin demonstrated therapeutic efficacy by halting the progression of established CKD in a rat model. see commentary on page 874 see commentary on page 874 Translational StatementTreatment strategies for chronic kidney disease (CKD) should ideally (i) directly target the kidney and (ii) be able to halt further progression of already established CKD. Recent studies on antidiabetic drugs, such as metformin and canagliflozin, are pointing toward a putative beneficial effect on kidney function decline. This paper reports the results of a rat study investigating the therapeutic efficacy of metformin and canagliflozin in a setting of already established, nondiabetic CKD. Our data show that metformin has renoprotective potential and holds perspective for being used as a therapeutic to retard or even halt progression of CKD. Treatment strategies for chronic kidney disease (CKD) should ideally (i) directly target the kidney and (ii) be able to halt further progression of already established CKD. Recent studies on antidiabetic drugs, such as metformin and canagliflozin, are pointing toward a putative beneficial effect on kidney function decline. This paper reports the results of a rat study investigating the therapeutic efficacy of metformin and canagliflozin in a setting of already established, nondiabetic CKD. Our data show that metformin has renoprotective potential and holds perspective for being used as a therapeutic to retard or even halt progression of CKD. Chronic kidney disease (CKD) is characterized by a decreased kidney function lasting more than 3 months. It is currently recognized as a worldwide public health problem with an estimated global prevalence of 3% to 16%.1Jha V. Garcia-Garcia G. Iseki K. et al.Chronic kidney disease: global dimension and perspectives.Lancet. 2013; 382: 260-272Google Scholar, 2Benghanem Gharbi M. Elseviers M. Zamd M. et al.Chronic kidney disease, hypertension, diabetes, and obesity in the adult population of Morocco: how to avoid "over"- and "under"-diagnosis of CKD.Kidney Int. 2016; 89: 1363-1371Google Scholar, 3National Kidney FoundationK/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification.Am J Kidney Dis. 2002; 39: S1-S266Google Scholar The leading causes of CKD in developed countries are diabetes and hypertension.1Jha V. Garcia-Garcia G. Iseki K. et al.Chronic kidney disease: global dimension and perspectives.Lancet. 2013; 382: 260-272Google Scholar Additionally, CKD also contributes to the global burden of cardiovascular diseases.1Jha V. Garcia-Garcia G. Iseki K. et al.Chronic kidney disease: global dimension and perspectives.Lancet. 2013; 382: 260-272Google Scholar,4Luyckx V.A. Tonelli M. Stanifer J.W. The global burden of kidney disease and the sustainable development goals.Bull World Health Organ. 2018; 96: 414-422DGoogle Scholar Renal impairment can further progress to kidney failure, which inevitably requires dialysis or kidney transplantation, and consequently significantly reduces quality of life.5Zoccali C. Kramer A. Jager K.J. Chronic kidney disease and end-stage renal disease—a review produced to contribute to the report "The Status of Health in the European Union: Towards a Healthier Europe.".NDT Plus. 2010; 3: 213-224Google Scholar Current treatment strategies for CKD mainly focus on controlling risk factors and relieving symptoms. Metformin, a biguanide drug, is the first-line oral antidiabetic agent used by 200 million patients suffering from type 2 diabetes mellitus.6Schernthaner G. Schernthaner G.H. The right place for metformin today.Diabetes Res Clin Pract. 2019; 159: 107946Google Scholar,7Bailey C.J. Metformin: historical overview.Diabetologia. 2017; 60: 1566-1576Google Scholar Although metformin has already been used for 6 decades, the molecular pathways by which metformin lowers blood glucose are still not fully elucidated.8Rena G. Pearson E.R. Sakamoto K. Molecular mechanism of action of metformin: old or new insights?.Diabetologia. 2013; 56: 1898-1906Google Scholar,9Marshall S.M. 60 years of metformin use: a glance at the past and a look to the future.Diabetologia. 2017; 60: 1561-1565Google Scholar Data from various research groups however have shown that metformin exerts many benign pleiotropic effects beyond its initial prescribed use. Independent of a patient's diabetic status, the use of metformin has shown promise for the treatment of cardiovascular diseases10Rena G. Lang C.C. Repurposing metformin for cardiovascular disease.Circulation. 2018; 137: 422-424Google Scholar and polycystic ovary syndrome11Lord J.M. Flight I.H.K. Norman R.J. Metformin in polycystic ovary syndrome: systematic review and meta-analysis.BMJ. 2003; 327: 951-953Google Scholar and has demonstrated substantial efficacy in different types of cancer (prostate12Zaidi S. Gandhi J. Joshi G. et al.The anticancer potential of metformin on prostate cancer.Prostate Cancer Prostatic Dis. 2019; 22: 351-361Google Scholar and breast13Dowling R.J.O. Zakikhani M. Fantus I.G. et al.Metformin inhibits mammalian target of rapamycin–dependent translation initiation in breast cancer cells.Cancer Res. 2007; 67: 10804-10812Google Scholar). Additionally, recent data from preclinical studies in various kidney disease models have also pointed toward a putative protective effect of metformin on the kidney.14Neven E. Vervaet B. Brand K. et al.Metformin prevents the development of severe chronic kidney disease and its associated mineral and bone disorder.Kidney Int. 2018; 94: 102-113Google Scholar, 15Cavaglieri R.C. Day R.T. Feliers D. et al.Metformin prevents renal interstitial fibrosis in mice with unilateral ureteral obstruction.Mol Cell Endocrinol. 2015; 412: 116-122Google Scholar, 16Christensen M. Norgard M.O. Jensen M.S. et al.Metformin modulates immune cell infiltration into the kidney during unilateral ureteral obstruction in mice.Physiol Rep. 2019; 7: e14141Google Scholar, 17Morales A.I. Detaille D. Prieto M. et al.Metformin prevents experimental gentamicin-induced nephropathy by a mitochondria-dependent pathway.Kidney Int. 2010; 77: 861-869Google Scholar, 18De Broe M.E. Jouret F. Does metformin do more benefit or harm in chronic kidney disease patients?.Kidney Int. 2020; 98: 1098-1101Google Scholar, 19Pastor-Soler N.M. Li H. Pham J. et al.Metformin improves relevant disease parameters in an autosomal dominant polycystic kidney disease mouse model.Am J Physiol Renal Physiol. 2022; 332: F27-F41Google Scholar In a previous experimental study, we have shown that preventive metformin treatment was able to strongly attenuate the development of severe CKD.14Neven E. Vervaet B. Brand K. et al.Metformin prevents the development of severe chronic kidney disease and its associated mineral and bone disorder.Kidney Int. 2018; 94: 102-113Google Scholar In this scenario we found that metformin preserved kidney function, reduced renal inflammation, and prevented excessive fibrosis.14Neven E. Vervaet B. Brand K. et al.Metformin prevents the development of severe chronic kidney disease and its associated mineral and bone disorder.Kidney Int. 2018; 94: 102-113Google Scholar Unfortunately, most patients with CKD are diagnosed when GFR is already reduced (GFR category G2–G3a or even G3b–G4), therefore therapeutic treatment of already established CKD is a more clinically relevant paradigm to study. The potential exploration of the clinical use of metformin during CKD has been hampered however by the historical fear for the development of lactic acidosis.20Lalau J.D. Arnouts P. Sharif A. De Broe M.E. Metformin and other antidiabetic agents in renal failure patients.Kidney Int. 2015; 87: 308-322Google Scholar,21DeFronzo R. Fleming G.A. Chen K. Bicsak T.A. Metformin-associated lactic acidosis: current perspectives on causes and risk.Metabolism. 2016; 65: 20-29Google Scholar Revised guidelines though state that metformin can now be used in patients with mild to moderate renal insufficiency.22Crowley M.J. Diamantidis C.J. McDuffie J.R. et al.Metformin Use in Patients with Historical Contraindications or Precautions. US Department of Veterans Affairs, 2016Google Scholar Furthermore, a recent clinical study of Lalau et al.23Lalau J.D. Kajbaf F. Bennis Y. et al.Metformin treatment in patients with type 2 diabetes and chronic kidney disease stages 3a, 3b, or 4.Diabetes Care. 2018; 41: 547-553Google Scholar indicated that metformin treatment also appeared to be safe and still pharmacologically efficacious in moderate to severe CKD (GFR category G3a–b to G4), provided that the dose is adjusted for the degree of kidney failure. Sodium-glucose co-transporter-2 (SGLT2) inhibitors have been shown to reduce the risk of renal impairment in patients with diabetes and have therefore been approved to treat diabetic nephropathy.24Perkovic V. Jardine M.J. Neal B. et al.Canagliflozin and renal outcomes in type 2 diabetes and nephropathy.N Engl J Med. 2019; 380: 2295-2306Google Scholar SGLT2 inhibitors are a novel class of oral antidiabetic drugs that target renal tubular glucose reabsorption.25Cuypers J. Mathieu C. Benhalima K. SGLT2-inhibitors: a novel class for the treatment of type 2 diabetes introduction of SGLT2-inhibitors in clinical practice.Acta Clinica Belg. 2013; 68: 287-293Google Scholar,26Kalra S. Sodium glucose co-transporter-2 (SGLT2) inhibitors: a review of their basic and clinical pharmacology.Diabetes Ther. 2014; 5: 355-366Google Scholar Furthermore, in a recent randomized controlled trial (Dapagliflozin and Prevention of Adverse Outcomes in Chronic Kidney Disease [DAPA-CKD] trial) the SGLT2 inhibitor, dapagliflozin, was shown to slow the progression of CKD independent of the presence of type 2 diabetes.27Heerspink H.J.L. Stefánsson B.V. Correa-Rotter R. et al.Dapagliflozin in patients with chronic kidney disease.N Engl J Med. 2020; 383: 1436-1446Google Scholar For that reason the US Food and Drug Administration very recently approved the use of dapagliflozin for patients with CKD irrespective of diabetes status.28Jafar T.H. FDA approval of dapagliflozin for chronic kidney disease: a remarkable achievement?.Lancet. 2021; 398: 283-284Google Scholar,29US Food and Drug AdministrationFDA approves treatment for chronic kidney disease. April 30, 2021.https://www.fda.gov/news-events/press-announcements/fda-approves-treatment-chronic-kidney-diseaseDate accessed: December 9, 2021Google Scholar To help clarify these data with respect to interventions for established CKD, our present study aimed to evaluate the therapeutic efficacy of metformin and canagliflozin on the progression of established CKD in the nondiabetic rat model with adenine-induced CKD. Metformin (Merck KGaA) and canagliflozin (Janssen) were dissolved in 1% carboxymethylcellulose at a concentration of 20 and 2.5 mg/ml, respectively, before the daily oral gavage. A dose volume of 10 ml/kg was used. Ninety-four (94) male Wistar rats (Iffa, Credo) were housed 2 per cage, exposed to a 12-12 hour light-dark cycle, with ad libitum access to water and their allotted diet. CKD was induced by administration of an adenine-enriched diet (with low vitamin K content, 0.25% adenine, 0.2 mg/kg K, 1% Ca, 1% P, 1 IU/g vitamin D, and 6% protein; SSNIFF Spezialdiäten), as described elsewhere,30Neven E. Bashir-Dar R. Dams G. et al.Disturbances in bone largely predict aortic calcification in an alternative rat model developed to study both vascular and bone pathology in chronic kidney disease.J Bone Miner Res. 2015; 30: 2313-2324Google Scholar during the entire study periods of 8 and 9 weeks. CKD rats were randomly assigned to 6 different groups to receive a 4-week treatment of 200 mg/kg metformin, 25 mg/kg canagliflozin, or vehicle (1% carboxymethylcellulose) that started either 4 or 5 weeks into adenine-induced CKD regimen. The 4- and 5-week treatment starting points were included to evaluate whether therapeutic efficacy was dependent on the initial severity of CKD. To assess renal histology and function at these respective starting points, 2 additional groups were administered the 0.25% adenine diet for 4 and 5 weeks, respectively, after which they were sacrificed. As a study reference, a group of control rats with normal kidney function was also included (Figure 1). Animals were housed in metabolic cages for 24 hours to collect urine samples followed by blood sampling through the tail vein in a restrained, conscious condition. Guidelines recommend a metformin dose reduction in patients with impaired kidney function to avoid lactic acidosis.23Lalau J.D. Kajbaf F. Bennis Y. et al.Metformin treatment in patients with type 2 diabetes and chronic kidney disease stages 3a, 3b, or 4.Diabetes Care. 2018; 41: 547-553Google Scholar,31Guideline Development GroupClinical practice guideline on management of patients with diabetes and chronic kidney disease stage 3b or higher (eGFR <45 mL/min).Nephrol Dial Transplant. 2015; 30: ii1-ii142Google Scholar,32Duong J.K. Kumar S.S. Kirkpatrick C.M. et al.Population pharmacokinetics of metformin in healthy subjects and patients with type 2 diabetes mellitus: simulation of doses according to renal function.Clin Pharmacokinet. 2013; 52: 373-384Google Scholar Therefore, dose adjustment was based on evaluation of serum lactate levels and pH as indicators of lactic acidosis (Supplementary Table S1). In the group receiving metformin following 4 weeks of adenine-induced CKD, a dose reduction to 150 mg/kg was implemented at week 7 until the end of the study. When treatment started after 5 weeks of adenine-induced CKD, the metformin dose was reduced to 150 mg/kg at the start of week 7 and further to 100 mg/kg at the end of week 8. After 4 weeks of metformin or canagliflozin treatment (experiencing 8 and 9 weeks of adenine-induced CKD), animals were killed by exsanguination through the retro-orbital plexus after anesthesia through i.p. administration of 80 mg/kg ketamine (Ketalar; Pfizer) and 16 mg/kg xylazine (Rompun; Bayer). Experimental procedures were conducted according to the National Institutes of Health Guide for the care and Use of Laboratory Animals 85-23 (1996) and approved by the University of Antwerp Ethics Committee (permit number 2017-56). To assess kidney function, creatinine was measured in serum and urine samples according to the Jaffe principle. Serum levels of phosphorus were determined with Ecoline S Phosphate kit (Diasys), calcium with flame atomic absorption spectrometry (Perkin-Elmer), blood glucose (GlucoMen Lx Plus+, A. Menarini Diagnostics), lactate, pH, and bicarbonate (i-STAT Point of Care, Abbott). Urinary glucose concentration (Atellica CH 930 Analyzer, Siemens 11067000) was also measured. After isolation and decapsulation of the left kidney, a transverse slice was fixed (4 hours) in neutral buffered formalin, rinsed with isopropanol, and embedded in paraffin (52 °C; Kendall). Tissue blocks were sectioned, after which 4-μm sections were deparaffinized and processed for staining. Periodic acid–Schiff–stained renal sections were used for general morphological evaluation and quantification of the interstitial area. Four random pictures (original magnification ×200) were taken from the cortex of each periodic acid–Schiff–stained renal section. Using Fiji image analyses software (US National Institutes of Health), the area that contains tubules and glomeruli was measured and subtracted from the total analyzed area (μm2). The ratio of the remaining area and the total analyzed area, resulted in the area percentage taken by the interstitium. Deparaffinized tissue sections were stained for evaluation of inflammation (CD45, pan leukocyte marker) using the avidin-biotin complex method. After blocking with goat serum and overnight incubation with primary antibody rabbit polyclonal anti-CD45 (1:1000, ab10558, Abcam), subsequent incubation with biotinylated goat anti-rabbit IgG antibody (PK-4001, Vector Laboratories), followed by a complex of avidin-biotin peroxidase (AB-complex, Vector Laboratories) allowed visual detection of a dark brown color on addition of diaminobenzidine. CD45 positivity (area percentage) was quantified in a blinded way (original magnification ×100) using Axiovision image analysis software (Carl Zeiss). Total mRNA of a snap-frozen kidney section was extracted using the PureLink RNA Mini Kit (Invitrogen) and reverse transcribed to cDNA by the High-Capacity cDNA archive kit (Applied Biosystems). Quantitative real-time polymerase chain reaction was performed using QuantStudio 3 (Thermo Fisher Scientific). TaqMan probe and primers were purchased from Thermo Fisher Scientific (Supplementary Methods). Expression level of each tested gene was analyzed in triplicate for each sample and normalized to the expression of the housekeeping reference gene Gapdh. The left kidney from CKD rats treated with metformin or vehicle after 5 weeks of adenine diet was collected and prepared for mass spectrometry and quantitative proteomics as described in the Supplementary Methods. The significantly altered proteins of the acquired datasets were functionally annotated using Kyoto Encyclopedia of Genes and Genomes (KEGG) and WikiPathways pathway analysis using the Enrichr suite (https://maayanlab.cloud/Enrichr/).33Chadwick W. Zhou Y. Park S.-S. et al.Minimal peroxide exposure of neuronal cells induces multifaceted adaptive responses.PLoS One. 2010; 5e14352Google Scholar34Martens M. Ammar A. Riutta A. et al.WikiPathways: connecting communities.Nucleic Acids Res. 2021; 49: D613-D621Google Scholar To provide a functional interpretation of these proteins, the Database for Annotation, Visualization and Integrated Discovery (DAVID Bioinformatic Recourses 6.8: https://david.ncifcrf.gov/) was used. Details on the further bioinformatic analyses are outlined in the Supplementary Methods. Results are expressed as means ± SEM. Nonparametric statistical analyses were performed using Prism (GraphPad Software). P < 0.05 was considered statistically significant. Details on body weight, food intake, and urinary output of all study groups are presented in Supplementary Figures S1 and S2. Blood glucose levels in this nondiabetic CKD model were, compared to vehicle treatment, not altered by metformin, nor canagliflozin (Table 1).Table 1Blood glucose in the nondiabetic rat model of adenine-induced CKD treated with metformin, canagliflozin, or vehicle, 4 or 5 weeks after induction of CKD for a period of 4 weeksGlucose (mg/dl)Treatment start week 4Week 0Week 2Week 4Week 6Week 8Metformin129.6 ± 3.5112.4 ± 2.2aP < 0.05 versus week 0.122.9 ± 2.5127.4 ± 3.6195.6 ± 12.3aP < 0.05 versus week 0.Vehicle126.1 ± 4.7112.8 ± 3.1aP < 0.05 versus week 0.123.2 ± 11.2125.3 ± 2.5193.1 ± 8.0aP < 0.05 versus week 0.Treatment start week 5Week 0Week 2Week 5Week 7Week 9Metformin141.5 ± 4.4118.6 ± 2.9aP < 0.05 versus week 0.126.4 ± 4.9aP < 0.05 versus week 0.119.4 ± 4.7aP < 0.05 versus week 0.194.9 ± 15.4aP < 0.05 versus week 0.Vehicle134.6 ± 5.2116.0 ± 3.0aP < 0.05 versus week 0.114.4 ± 9.1aP < 0.05 versus week 0.119.0 ± 4.1aP < 0.05 versus week 0.162.3 ± 8.7aP < 0.05 versus week 0.Treatment start week 4Week 0Week 2Week 4Week 6Week 8Canagliflozin141.6 ± 3.0128.3 ± 3.5133.4 ± 3.8129.1 ± 4.2126.9 ± 4.7Vehicle136.1 ± 3.4127.8 ± 2.8aP < 0.05 versus week 0.136.8 ± 3.6127.2 ± 4.3aP < 0.05 versus week 0.126.2 ± 9.0aP < 0.05 versus week 0.CKD, chronic kidney disease.Values are mean ± SEM.a P < 0.05 versus week 0. Open table in a new tab CKD, chronic kidney disease. Values are mean ± SEM. Chronic adenine dosing resulted in severe CKD in vehicle-treated rats as indicated by the rise in serum creatinine levels (Figure 2a and b). This increase was significantly different after 4 and 5 weeks of adenine-induced CKD, indicating 2 treatment starting points with different CKD severity (P < 0.0001; Supplementary Figure S3). The progressive decline in kidney function was almost completely halted by metformin administration from weeks 4 and 5 on as indicated by stabilization of serum creatinine levels at 8 and 9 weeks (P < 0.0001 vs. vehicle), respectively (Figure 2a and b). Creatinine clearance, as a measure for glomerular filtration rate, revealed a decrease over time in the vehicle-treated CKD rats, while metformin dosing attenuated this decrease (P < 0.05 vs. vehicle; Figure 2c and d). Also here, the difference in CKD severity at the 2 starting points was demonstrated by a significantly reduced creatinine clearance after 5 weeks compared to 4 weeks of CKD induction (P < 0.001; Supplementary Figure S3). In line with kidney dysfunction, a disturbed mineral metabolism became apparent in vehicle-treated CKD rats. Serum phosphorus levels increased significantly from weeks 6 and 7 onward, whereas calcium decreased, particularly at the later time points. Metformin treatment also abrogated these disturbances (Table 2).Table 2Serum parameters of mineral metabolism in the nondiabetic rat model of adenine-induced CKD treated with metformin or vehicle, 4 or 5 weeks after induction of CKD for a period of 4 weeksTreatment start week 4Week 0Week 2Week 4Week 6Week 8Calcium (mg/dl)Metformin14.3 ± 0.114.8 ± 0.1bP < 0.05 versus week 0.14.9 ± 0.1bP < 0.05 versus week 0.15.1 ± 0.2aP < 0.05 versus vehicle.,bP < 0.05 versus week 0.15.9 ± 0.3aP < 0.05 versus vehicle.,bP < 0.05 versus week 0.Vehicle14.6 ± 0.114.8 ± 0.114.8 ± 0.114.5 ± 0.411.8 ± 0.5bP < 0.05 versus week 0.Phosphorus (mg/dl)Metformin13.3 ± 0.98.0 ± 0.3bP < 0.05 versus week 0.9.7 ± 0.3bP < 0.05 versus week 0.10.5 ± 0.4aP < 0.05 versus vehicle.,bP < 0.05 versus week 0.12.8 ± 0.5aP < 0.05 versus vehicle.Vehicle11.6 ± 0.58.4 ± 0.3bP < 0.05 versus week 0.10.8 ± 0.414.3 ± 1.1bP < 0.05 versus week 0.19.0 ± 0.7bP < 0.05 versus week 0.Treatment start week 5Week 0Week 2Week 5Week 7Week 9Calcium (mg/dl)Metformin14.6 ± 0.115.1 ± 0.1bP < 0.05 versus week 0.15.2 ± 0.2bP < 0.05 versus week 0.15.7 ± 0.2aP < 0.05 versus vehicle.,bP < 0.05 versus week 0.14.3 ± 0.5aP < 0.05 versus vehicle.Vehicle14.4 ± 0.115.1 ± 0.1bP < 0.05 versus week 0.14.8 ± 0.2bP < 0.05 versus week 0.12.1 ± 0.7bP < 0.05 versus week 0.10.0 ± 0.7bP < 0.05 versus week 0.Phosphorus (mg/dl)Metformin10.6 ± 0.58.1 ± 0.3bP < 0.05 versus week 0.10.8 ± 0.412.5 ± 0.7aP < 0.05 versus vehicle.,bP < 0.05 versus week 0.12.9 ± 0.9aP < 0.05 versus vehicle.Vehicle10.1 ± 0.48.6 ± 0.2bP < 0.05 versus week 0.12.5 ± 0.918.0 ± 1.0bP < 0.05 versus week 0.15.2 ± 0.9bP < 0.05 versus week 0.CKD, chronic kidney disease.Values are mean ± SEM.a P < 0.05 versus vehicle.b P < 0.05 versus week 0. Open table in a new tab CKD, chronic kidney disease. Values are mean ± SEM. Adenine-fed CKD rats presented tubular brush border loss, basement membrane thickening, and tubular dilatation, which were all attenuated on metformin treatment (Figure 3a). As compared to control animals with normal kidney function, the interstitial area percentage increased 2- and 2.3-fold at weeks 4 and 5, respectively, after induction of CKD (Figure 3b and 3c). In accordance with these findings, mRNA expression of the fibrotic marker Col1a1 was markedly increased at the latter time points (Figure 3d and e). In contrast to vehicle treatment, there was no significant increase in interstitial area percentage between the start of metformin treatment (week 4 and week 5, respectively) and the end of the study (week 8 and week 9, respectively), providing evidence for the capacity of metformin to attenuate further interstitial enlargement and histopathological decay. These findings were reinforced by measuring the expression of the key profibrotic cytokine, TGFβ, which was significantly lower in the metformin-treated animals as compared to the vehicle-treated animals (Figure 3f and g). With respect to inflammation, 4 weeks of vehicle treatment resulted in a significantly increased Cd45 expression at the end of the study (i.e., weeks 8 and 9) as compared to control rats with normal kidney function (Figure 4a). This Cd45 expression increment was significantly (P < 0.01) halted when metformin was administered after 5 weeks of adenine-induced CKD (Figure 4c). Likewise, metformin treatment, initiated after 5 weeks of adenine-induced CKD, led to a significantly lower expression of the inflammatory cytokine Tnfα at the end of the study compared with the vehicle-treated animals at the same time point (P < 0.01; Figure 4d and e). Similar results were seen with the expression of Cd68, which suggested the suppression of macrophage infiltration (Figure 4f and g). Even though metformin treatment showed the ability to slow down the progression of already established CKD, it must be noted that a higher mortality was found in the metformin-treated animals (Figure 5). Nonetheless, to combat and mitigate the risk of lactic acidosis, that is, increased lactate levels, metformin dose adjustment was performed (see Methods), which resulted in a decrement of the values at the end of the study (Figure 6).Figure 6Effect of metformin on blood lactate levels. Blood lactate levels of chronic kidney disease (CKD) rats treated with metformin (a,b), canagliflozin (c), or vehicle starting 4 (a,c) or 5 weeks (b) after induction of chronic kidney disease. Data are presented as mean ±SEM. The lactate levels at week 8 were not measured, because blood, obtained through the retro-orbital plexus instead of the tail vein, was used at killing, leading to distorted results. ∗P < 0.05 versus vehicle; #P < 0.05 versus week 0.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Differential proteomic analysis of metformin-treated CKD animals versus vehicle-treated CKD animals revealed the significant (P < 0.05) alteration of 195 renal proteins (83 upregulated and 112 downregulated) at week 9 (Supplementary Table S2). To identify signaling pathways that are regulated by metformin, a minimal protein-protein interaction network was generated (https://www.networkanalyst.ca/) and interrogated using KEGG signaling pathway database. This resulted in a series of pathways linked to both developmental (e.g., Hippo signaling) and aging (e.g., FoxO signaling) pathways as well as metabolic activities (e.g., pyruvate metabolism, adenosine monophosphate kinase [AMPK] signaling). With respect to enrichment scores and probabilities, the strongest