Acute and chronic effects of SGLT2 blockade on glomerular and tubular function in the early diabetic rat

Abstract

Tubuloglomerular feedback (TGF) stabilizes nephron function from minute to minute and adapts to different steady-state inputs to maintain this capability. Such adaptation inherently renders TGF less efficient at buffering long-term disturbances, but the magnitude of loss is unknown. We undertook the present study to measure the compromise between TGF and TGF adaptation in transition from acute to chronic decline in proximal reabsorption (Jprox). As a tool, we blocked proximal tubule sodium-glucose cotransport with the SGLT2 blocker dapagliflozin in hyperglycemic rats with early streptozotocin diabetes, a condition in which a large fraction of proximal fluid reabsorption owes to SGLT2. Dapagliflozin acutely reduced proximal reabsorption leading to a 70% increase in early distal chloride, a saturated TGF response, and a major reduction in single nephron glomerular filtration rate (SNGFR). Acute and chronic effects on Jprox were indistinguishable. Adaptations to 10-12 days of dapagiflozin included increased reabsorption by Henle's loop, which caused a partial relaxation in the increased tone exerted by TGF that could be explained without desensitization of TGF. In summary, TGF contributes to long-term fluid and salt balance by mediating a persistent decline in SNGFR as the kidney adapts to a sustained decrease in Jprox.

Fig. 1.

Left: urine flow rate as a function of blood glucose for animals subject to acute, chronic, or no SGLT2 blockade. Blood glucose was a strong predictor of urine flow rate for each level of SGLT2 blockade (P < 0.0005). Lines are linear regressions for the individual levels of SGLT2 blockade (acute, chronic, none). Right: urine flow rate adjusted for blood glucose by ANCOVA. Intergroup comparisons are by Tukey test. Urine flow rate for each level of SGLT2 blockade was different from the other 2 levels (P < 0.001). *P < 0.001 vs. none. †P < 0.001 vs. chronic.

Fig. 2.

Single nephron glomerular filtration rate (SNGFR) at the natural tubuloglomerular feedback (TGF) operating point (SNGFRd) obtained by micropuncture downstream of the macula densa in diabetic Wistar or Wistar Froemter (MWF) rats during acute or chronic SGLT2 blockade. Means ± SE were generated by least-squares ANOVA on 137 tubular fluid samples. *P < 0.0005 vs. none. **P < 0.03 vs. none. †P < 0.05 vs. acute.

Fig. 3.

Acute and chronic SGLT2 blockade each suppress proximal reabsorption (Jprox). Data include 221 micropuncture collections from late proximal tubules of diabetic Wistar rats. Henle's loop was perfused at different rates to manipulate SNGFR by way of TGF. Left; Jprox vs. SNGFR in chronic SGLT2 blockade and respective controls. Center: Jprox vs. SNGFR during acute SGLT2 blockade and respective controls. Right: proximal reabsorption adjusted for SNGFR by least-squares ANCOVA (Jprox*). †P < 0.0005 vs. respective control. The effects of acute and chronic SGLT2 blockade on Jprox* were not different (P = 0.67).

Fig. 4.

Proximal reabsorption (Jprox) in MWF rats subjected to acute or chronic SGLT2 blockade. Left: individual late proximal tubular fluid collections showing Jprox as a function of SNGFR (n = 63). Right: proximal reabsorption adjusted for SNGFR by ANCOVA (Jprox*). Collections were made without perfusing Henle's loop, so each point represents the shoulder of a TGF curve.

Fig. 5.

Chloride concentration in early distal tubular fluid obtained by micropuncture from surface nephrons of MWF rats during acute or chronic SGLT2 blockade. *P < 0.05 vs. control.

Fig. 6.

Linear regression of SNGFRd vs. early distal chloride concentration (CED) in control (left), acute SGLT2 blockade (center), and chronic SGLT2 blockade (right) shown along with Pearson correlation coefficients (r). SGLT2 blockade introduces a negative correlation between SNGFRd and CED. This is the expected consequence if variability in SNGFRd is mainly the result of TGF responses to independent variations in proximal reabsorption. A positive correlation would result if variability in distal chloride were mainly driven by random variations in SNGFRd filtered through glomerulotubular balance.

Fig. 7.

Effects of chronic and acute SGLT2 blockade on SNGFR at both extremes of TGF activation in diabetic Wistar rats. Each experimental series has its own control group. The pooled data include 192 paired collections from 96 nephrons analyzed by repeated-measures ANOVA with experimental series and SGLT2 blockade as categorical variables and TGF activation as the repeated measure. The only significant between-subjects effect was for acute SGLT2 blockade to affect SNGFR differently than chronic blockade (P = 0.04). Within subjects, there was no effect of SGLT2 blockade on the reactivity of TGF, but TGF reactivity was less in the acute series (P = 0.003).

Fig. 8.

Left: SNGFR at shoulder, operating point and elbow of the TGF curve in Wistar rats during chronic or acute SGLT2 blockade. Each series had its own control group. Data include 3 collections in each nephron, n = 35 nephrons for chronic series, n = 21 for acute series. SNGFRd superimposed on TGF curves for illustrative purposes, with no scaling of the abscissa. Right: difference between proximal and distal SNGFR (P-D difference) normalized to the range of the TGF response. Both chronic and acute SGLT2 blockade increased the relative degree of TGF activation. Acute blockade saturated the TGF response, whereas chronic blockade did not. *P < 0.05 vs. respective control.

Fig. 9.

State of ambient TGF activation as indicated by the PD difference in MWF rats during acute or chronic SGLT2 blockade. *P < 0.05 vs. control.

Fig. 10.

Degree of TGF activation (P-D difference) as a function of the TGF stimulus (CED). Effects of acute and chronic SGLT2 blockade on P-D difference correspond to differences in SNGFR from Fig. 2. The solid curve is a hyperbolic tangent parameterized to represent the acute TGF response. The dashed lines represent the forward effects of SNGFR on CED due to glomerulotubular balance. Superimposed on this graph are the operating points (means ± SE) for the 3 phases of SGLT2 blockade in MWF rats. This figure illustrates two salient features of the transition from acute to chronic SGLT2 blockade. First, it is not possible to draw a single glomerulotubular balance curve that passes through both the acute and chronic operating points. Therefore, the transition from acute to chronic blockade requires increased reabsorption somewhere upstream of the macula densa. Since the effects of acute and chronic blockade on Jprox* were not different, this implies increased NaCl reabsorption by Henle's loop. Second, the proximity of the chronic operating point to the acute TGF curve reveals that no resetting of the TGF curve is required to explain the transition from acute to chronic SGLT2 blockade. The method for determining the slope for the TGF curve is described in the appendix.