Allelic depletion of grem1 attenuates diabetic kidney disease

Abstract

Objective: Gremlin (grem1) is an antagonist of the bone morphogenetic protein family that plays a key role in limb bud development and kidney formation. There is a growing appreciation that altered grem1 expression may regulate the homeostatic constraints on damage responses in diseases such as diabetic nephropathy.

Research design and methods: Here we explored whether knockout mice heterozygous for grem1 gene deletion (grem1(+/-)) exhibit protection from the progression of diabetic kidney disease in a streptozotocin-induced model of type 1 diabetes.

Results: A marked elevation in grem1 expression was detected in the kidneys and particularly in kidney tubules of diabetic wild-type mice compared with those of littermate controls. In contrast, diabetic grem1(+/-) mice displayed a significant attenuation in grem1 expression at 6 months of diabetes compared with that in age- and sex-matched wild-type controls. Whereas the onset and induction of diabetes were similar between grem1(+/-) and wild-type mice, several indicators of diabetes-associated kidney damage such as increased glomerular basement membrane thickening and microalbuminuria were attenuated in grem1(+/-) mice compared with those in wild-type controls. Markers of renal damage such as fibronectin and connective tissue growth factor were elevated in diabetic wild-type but not in grem1(+/-) kidneys. Levels of pSmad1/5/8 decreased in wild-type but not in grem1(+/-) diabetic kidneys, suggesting that bone morphogenetic protein signaling may be maintained in the absence of grem1.

Conclusions: These data identify grem1 as a potential modifier of renal injury in the context of diabetic kidney disease.

FIG. 1.

Induction of type 1 diabetes in wild-type (WT) and grem1^+/− mice. A: grem1 promoter activity was examined in embryonic fibroblasts from embryonic day 13.5 mouse embryos. Lysates from wild-type (^+/+), grem1^+/−, or grem1^−/− cells were assayed for β-galactosidase activity as described. Results are representative of four experiments carried out in duplicate. B: Wild type and grem1^+/− mice were injected intraperitoneally with either citrate buffer (control [ctrl]) or 50 mg/kg STZ for 5 consecutive days (week 0) according to established procedures (research design and methods). Fasting blood glucose levels were monitored biweekly for 27 weeks using a glucometer and a drop of blood from the tail vein. Significant increases in blood glucose levels developed in both groups after 2 weeks (P < 0.001, n = 10–12 mice per group) and were maintained over the 27 weeks study time course. □, wild-type control; ●, grem1^+/− control; ■, wild-type diabetic; ○, grem1^+/− diabetic. C: Whole blood was collected via cardiac puncture at time of sacrifice in both cohorts of mice. Percent A1C was assessed via ELISA as described in research design and methods. A significant increases in percent A1C were detected in both cohorts at 18 and 27 weeks of diabetes (mean ± SE). ***P < 0.001, using one-way ANOVA and Tukey-Kramer multiple comparison test, n = 6–11 in each group.

FIG. 2.

Diabetes-mediated induction of grem1 expression is attenuated in grem1^+/− mice. A: Total RNA was extracted from renal poles of control (C) and diabetic (D) wild-type (□) and grem1^+/− (●) mice at each time point indicated. A quantitative TaqMan PCR was performed using mouse grem1 specific oligonucleotides as described. ΔΔCt values were calculated by subtracting the Ct values for the 18S control from the corresponding grem1 value obtained in the same tube, and altered mRNA levels were then calculated by setting the control in each age-group to 1. Data are plotted as mean ± SE. *P < 0.05, Student's unpaired t test, n = 4–6 for each group. B. Fold change in grem1 mRNA was calculated by dividing the ΔΔCt value for diabetic mice by the mean of the corresponding age-matched control group. Data are plotted as mean fold change ± SE. *P < 0.05, Student's unpaired t test, n = 4–6 per group. C: Protein extracts (20 μg) from control and diabetic wild-type and grem1^+/− renal poles were probed by Western blot with grem1 antibody (R&D Systems) and β-actin (Sigma). An approximately 25-kDa band corresponding to grem1 was detected. D: Densitometry was performed using Scion Image software, and the intensity of grem1 expression was normalized to the β-actin loading control. Data were then plotted as diabetic/control fold change for both wild-type and grem1^+/− mice. **P < 0.01, Student's t test, n = 3.

FIG. 3.

Glomerular basement membrane thickening is attenuated in diabetic grem1^+/− mice compared with wild-type. Kidney pieces were processed as described in research design and methods, and 100-nm sections were cut from the renal pole harvested from control and diabetic wild-type and grem1 ± mice at 27 weeks. Sections were viewed with an FEI CM-12 transmission electron microscope operated at 80 keV. Glomeruli were randomly selected, viewed at ×15,000 magnification and serial measurements along the GBM were assessed. Arrows indicate the position of the glomerular basement membrane (A). Top left, nondiabetic wild-type (^+/+) control; top right, diabetic wild-type (^+/+); bottom left, nondiabetic grem1^+/− control; bottom right, diabetic grem1^+/−. Arrows indicate the thickness of the GBM. B and C: Quantitation of GBM thickness from all groups of mice. Up to 60 serial measurements were made from each individual glomerulus, and a mean value per mouse was calculated. Data are plotted as group means ± SE. GBM thickness was significantly higher in wild-type diabetic mice compared with nondiabetic controls (P < 0.001 using one-way ANOVA and Tukey-Kramer multiple comparison test, n = 7–11 per group). The increase observed in diabetic grem1^+/− mice compared with controls did not reach significance (P = 0.224). Fold change in GBM thickening. Mean GBM thickness values for each diabetic animal were divided by the mean thickness for control mice for both wild-type and grem1^+/− groups. Mean fold change values were calculated for both wild-type and grem1^+/− mice at 27 weeks. *P < 0.05, Student's two-tailed t test. □, wild type; ■, grem1^+/−.

FIG. 4.

Mild structural changes are evident in diabetic wild-type and grem1^+/− mice by light microscopy. Post mortem, mouse kidneys were fixed by perfusion fixation in situ using 4% (wt/vol) paraformaldehyde, and 3-μm paraffin-embedded sections were stained with (A) Picrosirius red to detect interstitial collagen or (C) periodic acid Schiff to assess glomerular matrix secretion. Slides (n = 5 for each group) were scored blindly by an independent renal pathologist on a scale of 0–4. Data are plotted as mean scores ± SE for control (C) or diabetic (D) mice in wild-type (□) or grem1^+/− (■) mice. Observed increases in both glomerular matrix secretion (B) or collagen staining (D) did not reach significance using Student's two-tailed t test. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Renal function impairment is attenuated in diabetic grem1^+/− mice compared with that in wild-type mice. A: Twenty-four–hour urine volumes were measured and levels of microalbumin were measured using an Albuwell M ELISA as described. Values for control nondiabetic (27 weeks), 18-week, and 27-week diabetic groups of wild-type (□) and grem1^+/− mice (■) were plotted (n = 6–11 per group except grem1^+/− 18 weeks, n = 3). Data were analyzed using a one-way ANOVA and Tukey-Kramer post hoc analysis. *P < 0.05; **P < 0.01; ***P < 0.001. B: Fold change in 24-h microalbuminuria was calculated by divided microalbumin values for individual diabetic wild-type and grem1^+/− mice by the mean microalbumin value for the corresponding 18- or 27-week control group. Mean fold change values ± SE were plotted. *P < 0.05, using a two-tailed t test. C: The ACR was calculated by dividing urinary microalbumin by urinary creatinine and is plotted as micrograms per milliliter. Values from control (nondiabetic), 18-week, and 27-week diabetic wild-type and grem1^+/− mice were plotted (n = 6–11). *P < 0.05, one-way ANOVA with post hoc Tukey-Kramer multiple comparison test. D: Fold change in ACR was calculated by dividing ACR values for individual diabetic wild-type and grem1^+/− mice by the mean ACR value for the corresponding 18- or 27-week control group. Mean fold change values ± SE were plotted. *P < 0.05, using Student's two-tailed t test. E: Creatinine clearance was calculated as microliter per minute per gram body weight. Data from both control and diabetic wild-type and grem1^+/− mice at time 0, 18 weeks, and 27 weeks of diabetes were plotted (n = 6–11). *P < 0.05; ***P < 0.001, using one-way ANOVA with post hoc Tukey-Kramer multiple comparison test. F: Fold change in creatinine clearance was calculated by dividing creatinine clearance values for individual diabetic wild-type and grem1^+/− mice by the mean creatinine clearance value for the corresponding 18- or 27-week control group. Mean fold change values ± SE were plotted. *P < 0.05 using Student's two-tailed t test. □, wild type; ■, grem1^+/−.

FIG. 6.

grem1 mRNA levels correlate with indexes of renal damage. A: Twentyfour microalbumin values (micrograms) were plotted against ACR values (micrograms of microalbumin per milligrams of creatinine) for wild-type and grem1^+/− control and diabetic mice over the course of the study (n = 50). R² value of the trendline was calculated at 0.5846 (n = 49), two-tailed P < 0.0001 using Spearman's rank correlation analysis. B: grem1 mRNA levels were plotted against ACR values for wild-type and grem1^+/− mice. The cohort of control and diabetic mice of both genotypes at 18 and 27 weeks of diabetes were plotted (n = 30). Spearman rank correlation analysis revealed an R² value of 0.5704 with a two-tailed P < 0.0001. (C). grem1 mRNA values were plotted against mean GBM thickness for control and diabetic wild-type and grem1^+/− mice at 27 weeks of diabetes (n = 17). R² = 0.533, two-tailed P < 0.001, using Spearman's rank correlation analysis.

FIG. 7.

Upregulation of gene markers of diabetic nephropathy is attenuated in grem1^+/− mice. Total RNA was extracted from renal poles of control (C) and diabetic (D) wild-type (□) and grem1^+/− (■) mice at 27 weeks of diabetes. A quantitative TaqMan PCR was performed using mouse fibronectin (A), vimentin (B), or CTGF (C) specific oligonucleotides as described. ΔΔCt values were calculated by subtracting the Ct values for the 18S control from the corresponding test gene value obtained in the same tube, and altered mRNA levels were then calculated by setting the control in each age-group to 1. Data are plotted as means ± SE. *P < 0.05, Student's unpaired t test, n = 4–6 for each group. Increases in vimentin in wild-type diabetic kidney just failed to reach significance (P = 0.079).

FIG. 8.

Decreased pSmad1/5/8 phosphorylation is evident in wild-type but not grem1^+/− diabetic kidney. A: Protein extracts from renal poles of control (C) and 27-week diabetic (D) wild-type (□) and grem1^+/− (■) mice were probed via Western blot using phospho-Smad1/5/8, total Smad1/5/8, and β-actin antibodies as described. n = 3 mice per group. B: Densitometry was performed using Scion Image software. pSmad1/5/8 intensities were normalized to total Smad1/5/8 levels and plotted as relative intensity. *P < 0.05 using Student's unpaired t test.