Randomized Trial of Dapagliflozin in Patients With Nondiabetic Stage 4 CKD

Abstract

Introduction

Sodium-glucose cotransporter-2 (SGLT2) inhibitors are nephroprotective in patients with chronic kidney disease (CKD) and mild-to-moderate renal insufficiency.

Methods

This prospective, randomized, cross-over, placebo-controlled, double-blind study compared the effects of 6-week dapagliflozin (10 mg/d) with placebo treatment in 31 consenting nondiabetic Caucasian adults with stage 4 CKD and proteinuria > 0.5 g/24 h. Participants were identified at the Nephrology Unit of Papa Giovanni XXIII Hospital and treated at Mario Negri Institute (Bergamo, Italy) between December 2021 and December 2023. Normalized glomerular filtration rate (GFR) (using iohexol plasma clearance) and 24-hour proteinuria (median of 3 urinary measurements) were co–primary outcomes. Analyses were by modified intention-to-treat.

Results

At 6 weeks, dapagliflozin significantly decreased GFR by 1.88 ± 5.00 ml/min per 1.73 m² (P = 0.022) and proteinuria by 0.50 (−0.10 to 0.80) g/24 h (P = 0.026) versus placebo. The dapagliflozin-induced GFR (P < 0.001) and proteinuria (P = 0.003) reduction was already significant at 1 week. At 6 weeks, dapagliflozin reduced absolute GFR (P = 0.026), the CKD-Epidemiology Collaboration (CKD-Epi) equation–based estimated GFR (eGFR) (P = 0.003), the Modification of Diet in Renal Disease (MDRD) equation–based eGFR (P = 0.002), 24-hour albuminuria (P = 0.001), total protein (P = 0.057) and albumin (P = 0.009) fractional clearances, and fasting blood glucose (P < 0.001); and increased serum albumin (P = 0.001), renin activity (P = 0.020), glucosuria (P < 0.001), and glucose fractional clearance (P < 0.001) versus placebo. All changes reversed completely after treatment withdrawal. GFR changes correlated inversely with changes in renal plasma flow (RPF) (P = 0.010) and positively with changes in postglomerular resistance (P < 0.001) but did not correlate with changes in preglomerular resistance. There were no serious adverse events.

Conclusion

Dapagliflozin safely ameliorates (compensatory) glomerular hyperfiltration and proteinuria and is glycosuric in nondiabetic patients with preterminal CKD. GFR reduction is likely because of postglomerular vasodilation rather than preglomerular vasoconstriction.

Graphical abstract

CKD is a major clinical and health care problem.1, 2, 3, 4, 5, 6 It was an orphan disease until 1982 when, following the US Food and Drug Administration approval of captopril for medical use, the introduction into clinical practice of renin-angiotensin-system (RAS) inhibitors paved the way for a completely new era in which CKD became the target of disease-modifying interventions.⁷ For almost 3 decades, however, RAS inhibitors remained the only effective treatment approaches, until recent landmark trials showed that novel blood glucose lowering medications such as the SGLT2 inhibitors dapagliflozin, empagliflozin, and canagliflozin added on to RAS inhibitor therapy dramatically improved renal and cardiovascular outcomes in patients with type 2 diabetes who were at increased cardiovascular risk.8, 9, 10, 11 Canagliflozin¹² and empagliflozin^13,14 reduced renal and cardiovascular events, compared with placebo, even in type 2 diabetics with CKD. The DAPA-CKD trial showed that dapagliflozin slowed renal disease progression even in patients with nondiabetic CKD.¹⁵

SGLT2 inhibitors were developed to lower blood glucose in patients with diabetes because of their glycosuric effects related to the inhibition of glucose reuptake in the proximal tubule.¹⁶ The concomitant natriuretic effect¹⁷ was assumed to contribute to their benefit in preventing heart failure.¹⁸ The finding that slower long-term eGFR decline was preceded by an acute and reversible¹⁹ eGFR reduction suggested that, as previously reported for RAS inhibitors,20, 21, 22, 23, 24, 25 the nephroprotective effects of SGLT2 inhibitors could be sustained by direct renal hemodynamic actions, rather than their weak blood glucose lowering effect.12, 13, 14, 15 It was postulated that in diabetic rodents²⁶ and hyperfiltering type 1 diabetics,^27,28 SGLT2 inhibition-induced enhanced sodium exposure to the macula densa activated tubule-glomerular feedback, leading to preglomerular vasoconstriction, via macula densa–stimulated adenosine production,²⁹ ameliorating glomerular hyperfiltration, reducing proteinuria, and conferring consequent long-term nephroprotection. SGLT2 inhibitors were then reported to also ameliorate glomerular hyperfiltration in patients with type 2 diabetes without nephropathy, although the mechanisms were probably different.³⁰ The effects on glomerular hemodynamics and sieving function that can mediate the short-term reduction in GFR and the proteinuria achieved by SGLT2 inhibitors in nondiabetic CKD, have not yet been investigated.

Methods

ADAPT (ClinicalTrials.gov NCT04794517; EudraCT Number: 2021-000726-10) was a phase 2b, single-center, prospective, randomized, cross-over, placebo-controlled, double-blind study that evaluated the short-term renal and systemic effects of dapagliflozin on nondiabetic patients with stage 4 CKD and residual proteinuria despite optimized supportive therapy. The study was approved by the ethical committee of the Azienda Socio-Sanitaria Territoriale Papa Giovanni XXIII (Bergamo, Italy). Participants provided written informed consent. The study was conducted according to the Declaration of Helsinki guidelines and Good Clinical Practice principles. Potentially eligible patients were identified in the outpatient clinics of the Unit of Nephrology of the Azienda Socio-Sanitaria Territoriale and were referred to the “Aldo e Cele Daccò” Clinical Research Center for Rare Diseases of the Istituto di Ricerche Farmacologiche Mario Negri IRCCS (Ranica, Italy). Nondiabetic adult patients were eligible if they met the following criteria: stable CKD-Epi eGFR of 15 to 30 ml/min per 1.73 m² (< 30% changes over the last 3 months) and proteinuria > 0.5 g/24 h in at least 2 consecutive evaluations > 1 week apart and blood pressure that was < 150/90 mm Hg without changes in blood pressure–lowering medications over the previous 4 weeks. Potentially fertile subjects without acknowledged effective contraception, and pregnant or lactating women were excluded (further eligibility criteria can be found in the study protocol in Supplementary Material).

Study Design

Screening Evaluation

Data on vital signs, demographic information, medications, and medical or surgical history were recorded; and biological samples were collected for routine hematochemistry and for 2 consecutive 24-hour urinary evaluations > 1 week apart (Figure 1).

Figure 1.

Study design. HbA1c, glycated hemoglobin; OGTT, oral glucose tolerance test.

Baseline Evaluation

The vital signs recorded at the screening evaluation were reassessed. A 2-hour oral glucose tolerance test (OGTT) with a 75 g oral glucose load was performed and GFR and RPF were measured using the iohexol³¹ and para-amino-hippuric³² plasma clearance techniques. Absolute GFR and RPF values were recorded and normalized for body surface area, along with eGFR values estimated using serum creatinine-based MDRD and CKD-Epi equations, calculated filtration fraction, renal vascular resistance, preglomerular and postglomerular resistance^33,34; 24-hour urinary total protein, albumin, sodium, and glucose excretion (median of 3 consecutive 24-hour measurements); and total protein albumin, sodium, glucose fractional clearances (Supplementary Figure S1).

Stratification or Randomization

Following the baseline evaluation, patients were stratified according to concomitant RAS inhibitor treatment (yes/no) and randomly allocated to 2 treatment sequences within each stratum by an interactive web response system on a 1:1 basis as follows:

• •Dapagliflozin, wash-out/cross-over, placebo, wash-out/recovery
• •Placebo, wash-out/cross-over, dapagliflozin, wash-out/recovery

Treatment Periods

Patients were assigned to 6-week treatment with 10 mg dapagliflozin or placebo once a day in the morning. The parameters evaluated at baseline were reassessed at the end of the first treatment period. The GFR and 24-hour proteinuria were reassessed at the end of week 1 of the first treatment period. Then, patients entered a 6-week wash-out, cross-over period. During this period, all patients were on placebo. After reassessment of baseline parameters, patients initially randomized to dapagliflozin crossed over to the 6-week treatment with placebo. Those initially assigned to placebo crossed over to the 6-week treatment with dapagliflozin. The parameters evaluated at baseline were reassessed at the end of the second treatment period. Again, the GFR and 24-hour proteinuria were reassessed at the end of week 1 of the second treatment period. All parameters evaluated at baseline were reassessed after the 2-week wash-out or recovery period (Figure 1).

Outcomes

Changes in GFR and 24-hour proteinuria at the end of the 6-week dapagliflozin or placebo treatment periods were co–primary outcomes. Changes in other measured, calculated, and estimated renal functional parameters; as well as systemic hemodynamic, metabolic, and laboratory and safety parameters were secondary outcomes (see Study Protocol in Supplementary Material).

Sample Size Estimation

Sample size estimation was based on the expected effect of 6 weeks of treatment with dapagliflozin versus placebo on GFR and 24-hour proteinuria.

GFR

Based on data obtained from the REIN trial,^23,24,35 which involved 91 patients with nondiabetic proteinuric CKD who had a measured GFR between 15 and 30 ml/min per 1.73 m², the patients included here were expected to have a baseline mean (SD) GFR of 23.0 (3.7) ml/min per 1.73 m². Assuming there would be a GFR reduction during the active treatment period similar to that observed with dapagliflozin therapy in the DIAMOND trial,³⁶ the GFR was expected to decrease by 6.6 ml/min per 1.73 m² (from 23.0 to 16.4 ml/min per 1.73 m²) during the dapagliflozin treatment period and not to change during placebo treatment. Assuming an SD of 11.7 ml/min per 1.73 m², 14 dapagliflozin-placebo and 14 placebo-dapagliflozin treatment sequences resulting in a total of 28 patients were required (alpha = 0.05, power = 80%, 2-sided test) to detect a statistically significant difference in GFR changes between dapagliflozin and placebo treatment periods. Assuming a 15% drop-out rate, 32 patients had to be included.

24-Hour Proteinuria

Based on the REIN data,^23,24,35 from patients with nondiabetic proteinuric CKD and residual proteinuria > 0.5 g/24 h after the 3-month ramipril therapy, baseline proteinuria was expected to average 2.9 ± 1.9 g/24 h. Assuming an antiproteinuric effect similar to that of empagliflozin in patients with type 2 diabetes with nephrotic-range proteinuria included in the EMPA-REG OUTCOME trial,¹⁴ a 40% reduction in 24-hour proteinuria (from 2.9 to 1.74 g/24-hour) and an SD on a natural logarithmic scale of 0.7 (cross-over SD = 1.69 g/24 h), 10 × 2 treatment sequences (20 patients overall) were required (alpha = 0.05, power = 83%, 2-sided test) to detect a significant difference in the change in 24-hour proteinuria between dapagliflozin and placebo treatment periods. Assuming a 15% drop-out rate, 24 treatment sequences in 24 patients were needed. Thus, with a total sample size of 32 patients, the study had an 80% and a 92% power to detect a statistically significant difference in changes in GFR and 24-hour proteinuria, respectively, between the 2 treatment periods.

Statistical Analyses

Normally distributed data were presented as mean and SD, and nonnormally distributed data as median and interquartile range.

Baseline characteristics were compared according to the Wilcoxon rank sum test, t test, or Fisher exact test as appropriate to the random allocation cross-over treatment sequence. The per-protocol comparative analyses between subgroups of patients with or without background RAS inhibitor therapy were not performed because at baseline, only 3 of the 31 included patients were not on RAS inhibitor therapy. The primary analysis tested the hypothesis of no difference in pre- to post-6-week normalized GFR changes between dapagliflozin and placebo treatment periods. The analysis was performed using SAS PROC MIXED,³⁷ according to the modified intention-to-treat approach, which included all randomized participants who had postbaseline measurements available for the primary outcome.³⁸

Paired t test and Wilcoxon signed-rank tests were also carried out. The analogous statistical tests were applied to the co–primary outcome, proteinuria. The per-protocol population had to be analyzed using an approach similar to that used on the modified intention-to-treat population. Exploratory correlation analyses were carried out using Pearson’s r correlation coefficient.

Normality assumptions were assessed using the Shapiro-Wilk test. All applicable statistical tests were 2-sided and were performed using a 5% significance level and 95% confidence intervals. SAS, version 9.4, and Stata (StataCorp), version 15, were used for all analyses.

Results

Sixty-two potentially eligible candidates were identified and referred to the Clinical Research Center (Figure 2). Forty-five patients provided written informed consent and began the screening phase of the study between November 8, 2021, and July 12, 2023. Thirteen patients were excluded because of nonadherence to selection criteria, intercurrent adverse events, or previous diagnosis of type 2 diabetes. Thus, after stratification, based on RAS inhibitor therapy (yes/no), 16 patients were randomized to the dapagliflozin-placebo treatment sequence and 16 to the placebo-dapagliflozin sequence. One patient randomized to the first sequence never took the study drug and was not considered in the analyses. All the remaining 31 patients completed the study and were available for final analysis. The first patient started treatment on December 13, 2021, and the last patient to take the study drug took it on December 5, 2023.

Figure 2.

Study flow chart. ACEi, angiotensin-converting enzyme inhibitor; ARB, angiotensin 2 receptor blocker; CRC, clinical research center; eGFR, estimated glomerular filtration rate; HBV, hepatitis B virus; OGTT, oral glucose tolerance test.

The characteristics (Table 1) and treatments (Supplementary Table S1) of patients allocated to the 2 treatment sequences were similar. Twenty-one patients were treated with angiotensin-converting enzyme inhibitors and 18 with angiotensin 2 receptor blockers; 11 patients were receiving both medications. The 11 patients with OGTT >140 mg/dl were older and had higher systolic blood pressure values and lower renin activity than those with normal OGTT (Supplementary Table S2).

Table 1.

Baseline parameters of all randomized patients considered as a whole (overall) and according to the randomization to the 2 sequences of treatment with dapagliflozin followed by placebo or with placebo followed by dapagliflozin

Parameters	Overall N = 31	Dapagliflozin-placebo n = 15	Placebo- dapagliflozin n = 16	P-valuea
Demographic parameters
Age (yrs)	59.81 ± 14.87	57.87 ± 15.44	61.63 ± 14.59	0.491
Male sex, n (%)	26 (83.87)	12 (80.0)	14 (87.50)	0.654
Caucasian, n (%)	31 (100)	15 (100.0)	16 (100.0)	-
Current smoker, n (%)	6 (19.35)	2 (13.33)	4 (25.0)	0.654
Clinical parameters
Body-mass index (kg/m2)	26.06 ± 3.60	25.25 ± 3.61	26.82 ± 3.53	0.231
Systolic BP (mm Hg)	132.74 ± 9.59	129.27 ± 9.03	135.98 ± 9.19	0.050
Diastolic BP (mm Hg)	80.24 ± 7.13	78.74 ± 7.45	81.64 ± 6.75	0.264
Mean BP (mm Hg)	97.75 ± 7.21	95.58 ± 7.21	99.78 ± 6.81	0.106
Laboratory parameters
Sodium (mEq/dl)	139.44 ± 2.33	139.77 ± 2.39	139.13 ± 2.31	0.453
Potassium (mEq/dl)	4.54 ± 0.49	4.58 ± 0.64	4.51 ± 0.31	0.713
Glucose (mg/dl)	97.90 ± 10.12	98.33 ± 12.34	97.50 ± 7.89	0.823
HbA1C (mmol/mol)	38.20 ± 4.39	38.68 ± 4.46	37.75 ± 4.42	0.564
Total serum protein (g/dl)	6.45 ± 0.32	6.48 ± 0.31	6.42 ± 0.33	0.599
Serum albumin (g/dl)	3.77 ± 0.33	3.82 ± 0.31	3.73 ± 0.35	0.459
Total cholesterol (mg/dl)	186.52 ± 38.44	184.87 ± 32.88	188.06 ± 44.06	0.821
Serum active renin (μU/ml)	50.5 (10.9–76.8)	19.3 (7.6–63.8)	56.4 (14.1–91.0)	0.304
Urine parameters
Proteins (g/24 h)	1.80 (1.30–2.7])	1.60 (1.00–2.70)	1.85 (1.40–3.25)	0.423
Albumin (μg/min)	820 (620–1260)	727 (540–1260)	844 (691–1510)	0.447
Glucose (mg/24 h)	301.5 ± 472.7	153.1 ± 71.7	461.2 ± 652.0	0.115
Sodium (mEq/24 h)	157.4 ± 55.3	149.4 ± 54.0	164.8 ± 57.1	0.446
Protein fractional clearance (× 10−5)	78.1 (47.4–122.7)	82.9 (40.6–122.7)	74.9 (55.7–143.9)	0.571
Albumin fractional clearance (×10−5)	86.4 (56.2–152.1)	82.6 (42.3–152.1)	88.3 (59.6–150.9)	0.597
Glucose fractional clearance (× 10−5)	461 (304–774)	394 (304–511)	659 (371–1265)	0.164
Kidney function parameters
Serum creatinine (mg/dl)	3.02 ± 0.60	3.03 ± 0.62	3.01 ± 0.59	0.942
GFR (ml/min per 1.73 m2)	24.83 ± 5.04	24.78 ± 5.35	24.88 ± 4.90	0.955
CKD-EPI (ml/min per 1.73 m2)	21.42 ± 4.71	21.33 ± 4.31	21.51 ± 5.19	0.921
MDRD (ml/min per 1.73 m2)	22.62 ± 4.52	22.36 ± 4.07	22.86 ± 5.02	0.766
RPF (ml/min per 1.73 m2)	324.68 ± 77.10	328.08 ± 100.20	321.70 ± 52.64	0.833
Glomerular resistances Ra (dyn·s/cm5)	9660 ± 2375	9333 ± 2549	9946 ± 2256	0.491
Glomerular resistances Re (dyn·s/cm5)	889.5 ± 176.8	891.4 ± 216.6	887.8 ± 140.6	0.957

BP, blood pressure; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; GFR, glomerular filtration rate; HbA1c, glycated hemoglobin; MDRD, Modification of Diet in Renal Disease; RPF, renal plasma flow.

Data are mean ± SD or median with interquartile range (square brackets) or numbers and percentages (round brackets). Chemistries are serum values unless stated otherwise. Conversion factors for units: creatinine in mg/dl to μmol/l, × 88.4; cholesterol (total) in mg/dl to mmol/l, × 0.02586.

^aPaired t test and Wilcoxon signed-rank test used for normally and non-normally distributed variables as necessary to calculate Δ dapagliflozin-placebo versus placebo-dapagliflozin.

Primary Outcome and Confirmatory Outcomes

Normalized GFR decreased significantly, by 2.81 ± 2.35 ml/min per 1.73 m², during the 6-week dapagliflozin treatment (P < 0.001), whereas it did not significantly change while patients were taking the placebo (−0.96 ± 3.41 ml/min per 1.73 m², P = 0.135) (Table 2, Figure 3a). GFR changes during the 2 treatment periods differed by 1.88 ± 5.00 ml/min per 1.73 m² (P = 0.022). Consistent with this, GFR significantly increased (1.58 ± 2.24 ml/min per 1.73 m², P < 0.001) during the post-dapagliflozin wash-out period, but did not change appreciably (−0.52 ± 2.28 ml/min per 1.73 m², P = 0.219) after placebo-wash-out (Figure 3). Similar changes were observed following just 1 week of treatment with a significant reduction in dapagliflozin (−1.66 ± 2.14 ml/min per 1.73 m², P < 0.001), whereas there were no significant changes with placebo (−0.32 ± 3.18 ml/min per 1.73 m², P = 0.590).

Table 2.

Clinical, laboratory, and instrumental parameters before and after 6 weeks of treatment with dapagliflozin and placebo

Parameters	Dapagliflozin (n = 31)		Δ Pre-post dapagliflozin	Placebo (n = 31)		Δ Pre-post placebo	Δ Dapagliflozin vs. Placebo
Clinical parameters	Pre	Post	P-valuea	Pre	Post	P-valuea	P-valueb
BMI (kg/m2)	26.01 ± 3.57	25.92 ± 3.57	0.292	26.10 ± 3.67	26.03 ± 3.55	0.493	0.862
Systolic (mm Hg)	129.62 ± 8.38	127.61 ± 11.05	0.145	133.10 ± 10.39	131.76 ± 8.91	0.391	0.648
Diastolic (mm Hg)	78.75 ± 6.88	77.23 ± 6.53	0.178	80.62 ± 7.16	79.95 ± 6.38	0.685	0.504
MAP (mm Hg)	95.70 ± 6.66	94.01 ± 7.15	0.112	98.12 ± 7.34	97.22 ± 6.06	0.537	0.517
Hematochemistry
Sodium (mEq/dl)	139.73 ± 2.03	139.55 ± 1.59	0.610	139.21 ± 2.20	139.41 ± 2.01	0.537	0.346
Potassium (mEq/dl)	4.61 ± 0.55	4.54 ± 0.45	0.483	4.50 ± 0.37	4.62 ± 0.38	0.090	0.120
Glucose (mg/dl)	97.58 ± 11.01	93.97 ± 11.61	0.027	99.71 ± 22.07	95.23 ± 9.50	0.288	0.800
OGTT (glucose after 120 min)	133.65 ± 46.39	128.71 ± 41.45	0.418	134.19 ± 39.64	129.90 ± 38.72	0.407	0.989
Total serum protein (g/dl)	6.39 ± 0.36	6.49 ± 0.38	0.186	6.36 ± 0.40	6.35 ± 0.27	0.728	0.222
Serum albumin (g/dl)	3.76 ± 0.30	3.92 ± 0.36	0.003	3.72 ± 0.43	3.75 ± 0.35	0.533	0.034
Total cholesterol (mg/d)	181.74 ± 34.57	180.29 ± 41.61	0.804	178.35 ± 38.50	185.10 ± 43.62	0.169	0.278
Serum active renin (μU/ml)	35.10 (10.10–89.70)	55.80 (17.60–213.20)	< 0.001	51.00 (13.40–76.80)	37.35 (11.30–97.50)	0.760	0.020
Urinary parameters
Protein (g/24 h)	1.80 (1.20–2.70)	1.50 (1.00–2.50)	0.007	1.80 (1.20–2.90)	2.00 (1.10–3.30)	0.391	0.026
Albumin (μg/min)	820.3 (540.7–1377.7)	721.0 (397.0–1078.0)	< 0.001	829.3 (601.3–1233.3)	981.0 (544.0–1308.3)	0.730	0.001
Glucose (mg/24 h)	144.4 (110.8–226.1)	7063.2 (4480.8–10733.6)	< 0.001	172.9 (107.8–301.4)	116.9 (65.2–247.9)	0.702	< 0.001
Sodium (mEq/24 h)	161.59 ± 58.79	159.57 ± 43.46	0.822	161.33 ± 51.05	169.74 ± 55.65	0.228	0.261
Protein fractional clearance (× 10-5)	83.0 (40.6–122.7)	68.4 (50.7–129.2)	0.181	72.9 (52.8–129.6)	90.71 (55.3–144.9)	0.327	0.057
Albumin fractional clearance (× 10-5)	92.9 (42.3–152.1)	72.1 [45.0–140.9)	0.012	84.4 (57.1–134.4)	102.7 (59.9–165.2)	0.288	0.009
Glucose fractional clearance (× 10-5)	410 (290–999)	26,824 (19,503–34,760)	< 0.001	521 (268–775)	458 (210–968)	0.705	< 0.001
Kidney function parameters
Serum creatinine (mg/dl)	3.14 ± 0.65	3.53 ± 0.65	< 0.001	3.11 ± 0.66	3.19 ± 0.70	0.147	0.001
GFR (ml/min per 1.73 m2)	23.80 ± 5.08	21.03 ± 4.93	< 0.001	24.25 ± 5.62	23.29 ± 5.12	0.136	0.022
GFR (ml/min)	25.94 ± 6.78	22.89 ± 6.73	< 0.001	26.43 ± 7.64	25.38 ± 6.98	0.135	0.026
CKD-EPI (ml/min per 1.73 m2)	20.48 ± 4.53	17.72 ± 3.76	< 0.001	20.81 ± 4.73	20.19 ± 4.39	0.151	0.003
MDRD EP (ml/min per 1.73 m2)	21.67 ± 4.46	18.84 ± 3.50	< 0.001	21.99 ± 4.53	21.39 ± 4.33	0.162	0.002
RPF (ml/min per 1.73 m2)	172.37 ± 36.36	162.98 ± 29.83	0.052	172.59 ± 31.31	174.47 ± 31.41	0.937	0.138
RPF (ml/min)	324.67 ± 78.56	305.58 ± 63.98	0.041	323.74 ± 66.61	328.05 ± 71.86	0.870	0.105
RVR (mmHg/ml/min per 1.73 m2)	0.21 ± 0.04	0.22 ± 0.04	0.403	0.22 ± 0.04	0.21 ± 0.04	0.557	0.356
FF	0.08 ± 0.02	0.07 ± 0.02	0.017	0.08 ± 0.02	0.08 ± 0.02	0.202	0.318
Pre-glomerular resistance (dyn·s/cm5)	9491 ± 2478	9740 ± 2442	0.593	9886 ± 2260	9682 ± 2621	0.565	0.463
Post-glomerular resistance (dyn·s/cm5)	855.5 ± 192.4	778.2 ± 152.8	0.027	858.3 ± 141.1	823.1 ± 153.2	0.274	0.264

BMI, body mass index; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; FF, filtration fraction; GFR, glomerular filtration rate; MAP, mean arterial pressure; MDRD, Modification of Diet in Renal Disease; OGTT, oral glucose tolerance test; RPF, renal plasma flow; RVR, renal vascular resistance.

All data expressed as mean ± SD or as median [interquartile range]. Conversion factors for units: creatinine in mg/dl to μmol/l, × 88.4; Total cholesterol in mg/dl to mmol/l, × 0.02586.

^aPaired t test and Wilcoxon signed-rank test used for normally and non-normally distributed variables as necessary to calculate Δ pre-post dapagliflozin and Δ pre-post placebo.

^bLinear mixed effect model used to calculate Δ dapagliflozin vs. placebo.

Figure 3.

Changes in the primary outcome and confirmatory outcomes. Changes in (a) normalized GFR (primary outcome), (b) absolute GFR, (c) CKD-Epi, and (d) MDRD estimated GFR (confirmatory outcomes), during the different study periods. Boxes indicate mean values. Points represent individual patients’ data. CKD-Epi, CKD-Epidemiology Collaboration equation; Dapa, changes between pre-post-dapagliflozin treatment periods; GFR, glomerular filtration rate; MDRD, Modification of Diet in Renal Disease equation; Plac, changes between pre-post placebo treatment periods; WoD, changes between pre-and post-wash-out periods after dapagliflozin treatment; WoP, changes between pre- and post-wash-out periods after placebo treatment (WoP). All P values are calculated by paired t test with the exception of P-values between changes during dapagliflozin and placebo treatment periods that are calculated by Linear Mixed Effect model.

Changes in confirmatory outcomes, including absolute GFR, CKD-Epi eGFR, and MDRD eGFR closely paralleled the changes in normalized GFR (Table 2, Figure 3b–d), whereas changes in serum creatinine were specular to those in GFR (Table 2).

Co–Primary Outcome and Confirmatory Outcomes

Median (interquartile range) proteinuria decreased significantly, by 0.30 (−0.60 to 0.10) g/24 h with dapagliflozin (P = 0.007), but did not change appreciably with placebo (0.20 [−0.40 to 0.40] g/24 h, P = 0.39) (Table 2, Figure 4a). Proteinuria changes during the 2 treatment periods differed by −0.50 (−0.80 to 0.10) g/24 h (P = 0.026). Proteinuria decreased significantly following just 1 week of dapagliflozin treatment (−0.20 [−0.40 to 0.00] g/24 h, P = 0.003), whereas it did not change appreciably after 1 week of placebo treatment (0.00 [−0.30 to 0.30] g/24 h, P = 0.923). Changes in 24-hour albuminuria, and total protein and albumin fractional clearances closely paralleled changes in 24-hour proteinuria (Table 2, Figure 4b–d).

Figure 4.

Changes in co–primary outcome and confirmatory outcomes. Changes in (a) 24-hour proteinuria (co–primary outcome), (b) 24-hour albuminuria, (c) total protein fractional clearance, and (d) albumin fractional clearance (confirmatory outcomes), during the different study periods. Boxes indicate median values. Points represent individual patients’ data. Dapa, changes between pre- and post-dapagliflozin treatment periods; F.C., fractional clearance; Plac, changes between pre- and post-placebo treatment period; WoD, changes between pre- and post-wash-out period after dapagliflozin treatment; WoP, changes between pre- and post-wash-out periods after placebo treatment. All P values are calculated using the Wilcoxon signed-rank test with the exception of P values between changes during dapagliflozin and placebo treatment periods that are calculated using Linear Mixed Effect model.

Secondary Outcomes

Absolute RPF decreased significantly, by 15.83 ± 39.98 ml/min, (P = 0.041) during dapagliflozin treatment, whereas the 7.94 ± 20.73 ml/min per 1.73 m² reduction in normalized RPF was borderline significant (P = 0.052). Filtration fraction and postglomerular resistances decreased significantly (0.01 ± 0.02, P = 0.017 and 78.7 ± 178.2 dyn·s/cm,⁵ P = 0.027, respectively) during dapagliflozin treatment (Table 2). There were never any significant differences between the 2 treatment periods regarding any of the considered hemodynamic parameters (Table 2).

Twenty-four–hour urinary glucose excretion and glucose fractional clearance increased significantly during dapagliflozin treatment (6905 [4736–10,664] mg/24 h and 28,633 [19,981–34,291] × 10⁻⁵, P < 0.001 for both), but did not change with placebo treatment (−7 [−74 to 45] mg/24 h, P = 0.702 and 23 [−221 to 298] × 10⁻⁵, P = 0.705) (Table 2, Figure 5a and b). Differences in changes in both parameters during dapagliflozin and placebo treatment were significant (7004 [5343–10697] mg/24 h and 29,165 [20,124–34,390] × 10⁻⁵, P < 0.001 for both). Sodium fractional clearance tended to increase during dapagliflozin treatment, by 297 (−325 to 753) × 10⁻⁵ (P = 0.099), and did not change during placebo treatment (−40 [−349 to 1290] × 10⁻⁵, P = 0.450) (Table 2).

Figure 5.

Changes in (a) 24-hour glycosuria, (b) glucose fractional clearance, and (c) serum active renin, during the different study periods. Boxes indicate median values. Points represent individual patients’ data. Dapa, changes between pre- and post-dapagliflozin treatment period; F.C., fractional clearance; Plac, changes between pre- and post-placebo treatment period; WoD, changes between pre- and post-wash-out period after dapagliflozin treatment; WoP, changes between pre- and post-wash-out period after placebo treatment. All P-values are calculated using Wilcoxon signed-rank test, with the exception of P-values between changes during dapagliflozin and placebo treatment periods that are calculated using Linear Mixed Effect model.

Blood glucose decreased significantly during dapagliflozin treatment, by 3.61 ± 8.67 mg/dl, (P = 0.027), whereas it did not change appreciably with placebo treatment (−5.07 ± 33.80 mg/dl, P = 0.418). Serum albumin increased significantly, by 0.16 ± 0.27 g/dl (P = 0.003) during dapagliflozin treatment, but did not change during placebo treatment (0.02 ± 0.20 g/dl, P = 0.533). There were significant differences between the 2 treatment periods regarding serum albumin changes (0.14 ± 0.37 mg/dl, P = 0.034). Serum renin activity increased significantly, by 12.7 (3.4–80.1) μU/ml, (P < 0.001), with dapagliflozin, but did not change with placebo treatment (−0.4 [−5.3 to 12.3] μU/ml, P = 0.760). Changes during the 2 treatment periods were significantly different (21.0 [4.4–68.0] μU/ml, P = 0.020, Figure 5c). Changes in other laboratory parameters are shown in Table 2.

Correlation Analyses

During dapagliflozin treatment, changes in GFR correlated negatively with concomitant changes in RPF (ρ = −0.476, P = 0.010) and correlated positively with concomitant changes in postglomerular resistance (ρ = 0.723, P < 0.001), but did not correlate with concomitant changes in preglomerular resistance (Figure 6a and b). The correlations between dapagliflozin-induced changes in filtration fraction and concomitant changes in RPF (ρ = −0.810, P < 0.001) or postglomerular resistances (ρ = 0.958, P < 0.001) were even stronger (Figure 6c and d).

Figure 6.

Linear regression model and Pearson correlation coefficient between changes in normalized GFR (or filtration fraction [FF]) and corresponding changes in (a and c) normalized renal plasma flow (RPF), and (b and d) efferent glomerular vascular resistance (GRRE) during the dapagliflozin treatment period. GFR, glomerular filtration rate.

Safety

There were no serious adverse events (Supplementary Table S3). The number of nonserious adverse events and of patients affected by events tended to be higher during the placebo treatment period. Three hypoglycemic events were described as possibly being related to the study drug as follows: 1 with dapagliflozin and 2 with the placebo.

Discussion

In this prospective, randomized, double-blind, cross-over, placebo-controlled clinical trial conducted in 31 nondiabetic patients with severe renal insufficiency we found that 1- and 6-week treatment with 10 mg/d of the SGLT2 inhibitor, dapagliflozin significantly decreased directly measured, normalized GFR, whereas no appreciable effect on GFR was observed after 1- or 6-week treatment with placebo. The treatment effects of dapagliflozin and placebo were significantly different. The finding that dapagliflozin-induced GFR reduction was already evident following just 1 week of treatment, and fully recovered during the wash-out period, confirmed that GFR reduction reflected a functional and reversible effect. The finding that GFR reduction during dapagliflozin treatment was paralleled by a concomitant filtration fraction reduction, further corroborated the hypothesis that, as previously observed for RAS inhibitors,20, 21, 22 these acute and reversible effects reflected an amelioration of compensatory hyperfiltration by residual functioning glomeruli in patients with reduced nephron number,³⁶ rather than a nephrotoxic effect. This short-term functional effect is expected to translate into long-term nephroprotection, as demonstrated by long-term clinical trials in patients with diabetes and nondiabetic patients with less severe CKD12, 13, 14, 15; and recent observational evidence that, compared with no SGLT2 inhibitor use, SGLT2 inhibitor use was associated with lower risk for dialysis in a large cohort of patients with type 2 diabetes and stage 5 CKD.³⁹

Virtually identical treatment effects were observed when the analyses considered nonnormalized GFR values—to rule out a potentially confounding effect of weight loss during dapagliflozin therapy—or GFR values that were estimated indirectly with CKD-Epi and MDRD equations. These findings were mirrored by concomitant changes in serum creatinine levels. Thus, in clinical practice an early increase in serum creatinine shortly after the initiation of SGLT2 inhibitor therapy should not be taken as a safety signal, but rather as a marker of treatment effect.

The finding that, during dapagliflozin treatment, changes in GFR correlated inversely with concomitant changes in RPF, and positively and strongly correlated with concomitant changes in postglomerular vascular resistance, but did not correlate with changes in preglomerular resistance, challenges the common belief^27,28 that dapagliflozin-induced amelioration of glomerular hyperfiltration is mediated by increased preglomerular resistance, resulting in parallel reductions in GFR and RPF. Conversely, our findings extend previous evidence that in type 2 diabetes, the amelioration of glomerular hyperfiltration is mediated by a dapagliflozin-induced reduction in postglomerular resistance,³⁰ to nondiabetic patients with CKD. Notably, almost all patients in this cohort, as well as in our present study population, were on concomitant RAS inhibitor therapy. It is conceivable that dapagliflozin-induced postglomerular vasodilation could be mediated by decreased reactive oxygen species⁴⁰ and/or increased prostaglandin production.³⁰ In addition, a concomitant increase in adenosine production might have further contributed to postglomerular vasodilation,^41,42 in particular with concomitant RAS inhibitor therapy.⁴³

Notably, the aforementioned hemodynamic effects were associated with a significant reduction in 24-hour proteinuria, achieved after either 1- or 6-week dapagliflozin treatment, whereas proteinuria did not change with placebo treatment. The antiproteinuric effects of dapagliflozin and placebo were significantly different. Treatment effects on 24-hour albuminuria were similar. Notably, the additional antialbuminuric effect of dapagliflozin in patients with background dual RAS blockade with an angiotensin-converting enzyme inhibitor and an angiotensin 2 receptor blocker therapy (in 11 of the 31 study patients) recalled the additional antialbuminuric effect of dapagliflozin in patients with less severe CKD and dual RAS blockade with the mineralocorticoid receptor antagonist, eplerenone added on an angiotensin-converting enzyme inhibitor or an angiotensin 2 receptor blocker.⁴⁴ Independently of the underlying mechanisms, amelioration of glomerular hyperfiltration and sieving function are expected to translate into clinically relevant, long-term nephroprotection.12, 13, 14, 15^,21, 22, 23, 24, 25^,35 The finding that changes in proteinuria and albuminuria were associated with significantly different treatment effects from dapagliflozin compared with placebo, on total protein and albumin fractional clearances, suggests that the antiproteinuric effect of dapagliflozin was not a simple consequence of GFR reduction, but was also due to significantly reduced protein ultrafiltration secondary to improved sieving function of the glomerular barrier, as previously reported with RAS inhibitors in experimental and human CKD.^21,22,45, 46, 47, 48, 49, 50, 51 These effects translate into effective long-term nephroprotection even in patients who have severe renal insufficiency to start with⁵² and could explain the well-documented, long-term nephroprotective effects of SGLT2 inhibitors,12, 13, 14, 15 even in patients with type 2 diabetes and stage 5 CKD.³⁹ Notably, serum albumin levels increased significantly during dapagliflozin treatment, most likely because of treatment-induced proteinuria reduction.

The highly significant 1- and 6-week increase in glucosuria and glucose fractional clearance achieved by dapagliflozin while the placebo had no appreciable effects, confirms that SGLT2 inhibitors exert their biological effect even in patients with nondiabetic CKD and provides novel evidence that this effect can be extended to patients with severe renal insufficiency. In contrast, evidence that increased glycosuria had a reducing effect on fasting blood glucose levels without appreciably affecting glucose tolerance after a standardized OGTT, confirmed the weak metabolic effects of SGLT2 inhibitors.

An ancillary but clinically relevant finding was the highly significant increase in serum active renin levels achieved by dapagliflozin. This finding provides a strong rationale for combined treatment with SGLT2 and RAS inhibitors in order to maximize nephroprotection and cardioprotection in patients with diabetes and patients without diabetes with CKD.12, 13, 14, 15 SGLT2 inhibitors can therefore be an additional therapeutic option for multimodal treatment (remission clinic) for patients with CKD,⁵³ including those with more severe renal insufficiency.

As expected, at baseline evaluation, patients with impaired glucose tolerance were significantly older than those with normal glucose OGTT, which is consistent with evidence that there is progressive impairment of glucose metabolism with age. Older age was also associated with higher systolic blood pressure, possibly related to aging-related vascular stiffness⁵⁴ and lower renin activity, which could be consistent with the reduced plasma renin activity reported in patients with diabetes.⁵⁵ Renal insufficiency is per se an independent pathogenic factor for increased insulin resistance and impaired glucose metabolism,⁵⁶ which is another indication to extend SGLT2 inhibitor therapy to patients with severe renal insufficiency.

Safety

Dapagliflozin was safe and very well-tolerated, even in this particularly frail population. There were no serious adverse events and all nonserious adverse events were transient, self-limited, and even tended to be more frequent with placebo than with dapagliflozin.

Limitations and Strengths

The relatively small sample size is typical of a pilot, single-center, highly demanding pathophysiology study and the short observation period is explained by the need to investigate acute, short-term treatment effects. Treatment effect was observed on top of dual RAS blockade in 11 out of 31 study patients. Dual RAS blockade is part of our common practice but is seldom applied in most nephrology units for the treatment of patients with proteinuric CKD. This approach can be a limitation to the generalizability of our study findings; however, it provides the evidence of an additional, specific antiproteinuric effect of SGLT2 inhibitors even in patients with maximized proteinuria reduction achieved by dual RAS blockade. The use of gold-standard procedures,^31,32 to directly measure GFR and RPF and the evaluation of 24-hour proteinuria in 3 consecutive 24-hour urine collections increased the accuracy of study evaluations. The improved accuracy reduced random data fluctuations which, remarkably, increased the statistical power of the analyses. Moreover, because of the cross-over design, each patient received either dapagliflozin or placebo in a random order. Thus, each patient served as their own control: this enabled within-patient comparisons and made it possible to avoid interindividual data variability. Baseline characteristics of patients randomized to the 2 treatment sequences were comparable. The high patient retention rate, despite the demanding study design, was another strength.

Conclusion

Dapagliflozin is glycosuric and ameliorates hyperfiltration and proteinuria in patients with severe, nondiabetic CKD. However, the amelioration of glomerular hyperfiltration appears to be predominantly mediated by postglomerular vasodilation rather than preglomerular vasoconstriction. Because of its remarkably good short-term risk-benefit profile, the potential benefits of long-term dapagliflozin therapy is worth investigating, even in this particularly frail population.

Appendix

List of the members of the ADAPT Study Group

Coordinating Centre: Mario Negri Institute for Pharmacological Research IRCCS (MNI), Clinical Research Centre for Rare Diseases Aldo e Cele Daccò, Ranica (Bergamo); Research Coordinator: Giuseppe Remuzzi (Bergamo), Trial coordinator: Norberto Perico (Bergamo).

Centre including patients: Aldo e Cele Daccò Clinical Research Centre for Rare Diseases, Ranica (Piero Ruggenenti Principal Investigator, Norberto Perico, Matias Trillini, Erica Daina, Elena Bresin, Diego Curtò, Giulia Gherardi, Daniela Cugini, Silvia Prandini, Veruscka Lecchi, Diana Cadè, Sara Gamba, Angelo Diffideni, Chiara Guarinoni). Centre for screening only: Azienda Socio-Sanitaria Territoriale Papa Giovanni XXIII Nephrology Unit (Alessia Gennarini, Maria Rosa Caruso, Valentina Leone, Stefano Rota).

Centralized activities: Monitoring, Drug Distribution and Pharmacovigilance (Nadia Rubis, Wally Calini, Davide Villa, Olimpia Diadei MNI); Database and Data Validation (Davide Martinetti, Sergio Carminati at MNI); Randomization (Giovanni Antonio Giuliano MNI); Data Analysis (Annalisa Perna, Diego Fidone, Tobia Peracchi MNI); Medical Imaging (Anna Caroli, Giulia Villa, Andrea Remuzzi MNI and Paolo Brambilla, Francesca Abbadini, Sandro Sironi, Azienda Socio-Sanitaria Territoriale Papa Giovanni XXIII Radiology Unit); Laboratory Measurements (Daniela Cugini, Flavio Gaspari, Fabiola Carrara, Silvia Ferrari, Nadia Stucchi, Antonio Nicola Cannata MNI); Regulatory Affairs (Paola Boccardo, Sara Peracchi, Jennifer Piffari MNI); IMP management (Francesco Gregis, Simone Borchetto, Bianca Taddei, Erika Diani at Azienda Socio-Sanitaria Territoriale Papa Giovanni XXIII Pharmacy).

Disclosure

All the authors declared no competing interests.

Supplementary Material

Supplementary File (PPT and PDF)

Study protocol. (PDF). Figure S1. Outline of standardized medical examinations before and after dapagliflozin and placebo treatment periods. (PPT). Table S1. Concomitant treatment in the study group considered as a whole (overall) and according to treatment sequence. (PDF). Table S2. Baseline characteristics of all randomized patients according to stratification to OGTT > 140 mg/dl or < 140 mg/dl independently of the study treatment sequence. (PDF). Table S3. Adverse events during the different treatment periods. (PDF). CONSORT checklist. (PDF)