Podocyte-specific overexpression of human angiotensin-converting enzyme 2 attenuates diabetic nephropathy in mice

Abstract

Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin II to angiotensin-(1-7) and is expressed in podocytes. Here we overexpressed ACE2 in podocytes in experimental diabetic nephropathy using transgenic methods where a nephrin promoter drove the expression of human ACE2. Glomeruli from these mice had significantly increased mRNA, protein, and activity of ACE2 compared to wild-type mice. Male mice were treated with streptozotocin to induce diabetes. After 16 weeks, there was no significant difference in plasma glucose levels between wild-type and transgenic diabetic mice. Urinary albumin was significantly increased in wild-type diabetic mice at 4 weeks, whereas albuminuria in transgenic diabetic mice did not differ from wild-type nondiabetic mice. However, this effect was transient and by 16 weeks both transgenic and nontransgenic diabetic mice had similar rates of proteinuria. Compared to wild-type diabetic mice, transgenic diabetic mice had an attenuated increase in mesangial area, decreased glomerular area, and a blunted decrease in nephrin expression. Podocyte numbers decreased in wild-type diabetic mice at 16 weeks, but were unaffected in transgenic diabetic mice. At 8 weeks, kidney cortical expression of transforming growth factor-β1 was significantly inhibited in transgenic diabetic mice as compared to wild-type diabetic mice. Thus, the podocyte-specific overexpression of human ACE2 transiently attenuates the development of diabetic nephropathy.

Figure 1

Characterization of transgenic (TG) mice. (a) Expression of mRNA for the human angiotensin-converting enzyme 2 (ACE2) transgene in tissues from TG mice. Graph shows relative levels of human ACE2 mRNA in various mouse tissues, determined by real-time reverse transcriptase–PCR (RT–PCR). ^*P<0.001, glomeruli vs. all other tissues; n=3 (glomeruli) and n=6–7 for other tissues. (b) Expression of endogenous mouse ACE2 mRNA in glomeruli and kidney cortex from TG mice. Graph depicts relative expression of mouse ACE2 mRNA in glomeruli and kidney cortex from TG mice, determined by real-time RT–PCR. ^*P<0.02; n=3. (c) Immunoblot analysis of ACE2 in glomeruli and cortex tissue. Shown is representative immunoblot of ACE2 protein in glomeruli and kidney cortex from wild-type (WT) and TG mice, with or without treatment with STZ, at 16 weeks (n=2). Human ACE2 transgenic protein is present as a single band at ∼120 kDa in glomeruli from TG or TG-STZ mice. Endogenous mouse ACE2 protein is observed in all lanes as a single band at ∼100 kDa. (d) Colocalization of human ACE2 transgene with podocyte protein synaptopodin. Dual-color immunofluorescence was performed on kidney sections from TG mice. Sections were stained with rabbit polyclonal antibody to hemagglutinin (HA; present in the transgene as a double epitope tag; left panel), and with rabbit polyclonal anti-synaptopodin antibody (middle panel). Merged view is shown in the panel on the right. Original magnification × 400. (e) Glomerular ACE2 activity. ACE2 activity was measured in isolated glomeruli from WT or TG mice, as described in Materials and Methods. ^*P<0.02, n=9 (WT), n=10 (TG).

Figure 2

Glomerular expression of angiotensin-converting enzyme 2 (ACE2) by immunofluorescence at 16 weeks. Graph depicting semiquantitative analysis of ACE2 immunofluorescent staining in mouse glomeruli at 16 weeks. TG, podocyte-specific ACE2 transgenic; TG-STZ, transgenic streptozotocin-treated; WT, wild type; WT-STZ, wild-type streptozotocin-treated. Values are means±s.e. ^*P<0.001 vs. WT and vs. WT-STZ; n=100 glomeruli per group, from 6 to 11 mice per group. Representative images depicting ACE2 staining in glomeruli (red) at 16 weeks are shown above graph. Blue staining represents Hoechst nuclear stain. Original magnification × 640.

Figure 3

Plasma angiotensin-converting enzyme 2 (ACE2) activity. Graph depicts plasma ACE2 enzymatic activity from wild-type (WT), transgenic (TG), wild-type streptozotocin-treated (WT-STZ), and transgenic streptozotocin-treated (TG-STZ) mice at 16 weeks. Values are means±s.e. ^*P<0.001 vs. WT and TG, ^#P<0.001 vs. WT and TG, ^$P<0.05 vs. TG-STZ; n=6.

Figure 4

Systolic blood pressure (BP). Systolic BP was measured weekly by tail-cuff plethysmography. TG, transgenic; TG-STZ, transgenic streptozotocin-treated; WT, wild type; WT-STZ, wild-type streptozotocin-treated. Values are means±s.e. ^*P<0.01 vs. WT and TG, ^**P<0.05 vs. WT and TG. There was no significant difference between WT-STZ and TG-STZ mice; n=14 mice per group.

Figure 5

Glomerular filtration rate (GFR). Graph shows GFR measurements in wild-type (WT), transgenic (TG), wild-type streptozotocin-treated (WT-STZ), and transgenic streptozotocin-treated (TG-STZ) mice after 16 weeks. Values are means±s.e. ^*P<0.05 vs. WT and TG, ^#P<0.01 vs. WT and TG; n=4–6.

Figure 6

Urinary protein excretion. (a) The 24 h urinary albumin excretion in wild-type (WT), transgenic (TG), wild-type streptozotocin-treated (WT-STZ), and transgenic streptozotocin-treated (TG-STZ) mice at 4 weeks. ^*P<0.05 vs. all other groups (WT, TG, and TG-STZ mice); n=5. (b) The 24 h urinary albumin excretion at 16 weeks. ^*P<0.01 vs. WT and TG, ^#P<0.01 vs. WT and TG. There was no significant difference between WT-STZ and TG-STZ mice; n=14. (c) Representative Coomassie blue-stained sodium dodecyl sulfate (SDS) gel of urine samples from WT, TG, WT-STZ, and TG-STZ mice at 8 weeks. Multiple protein bands are evident in urine samples from WT-STZ and TG-STZ mice. Molecular weight marker is depicted in the first and last lanes.

Figure 7

Effect of podocyte angiotensin-converting enzyme 2 (ACE2) overexpression on mesangial and glomerular area at 8 weeks. Graphs depict (a) mesangial and (b) glomerular areas at 8 weeks in wild-type (WT), transgenic (TG), wild-type streptozotocin-treated (WT-STZ), and transgenic streptozotocin-treated (TG-STZ) mice. (a) Mesangial area at 8 weeks. Values are means±s.e. (n=40 glomeruli per group of 4 mice). ^*P<0.001 vs. WT and TG, ^#P<0.02 vs. WT and TG, ^$P<0.03 vs. TG-STZ. (b) Glomerular area at 8 weeks. Values are means±s.e. (n=40 glomeruli per group of 4 mice). ^*P<0.001 vs. WT and TG, ^#P<0.001 vs. WT and TG. There was no significant difference between WT-STZ and TG-STZ mice. (c) Representative photomicrographs depicting periodic acid–Schiff (PAS)-stained glomeruli in WT, TG, WT-STZ, and TG-STZ mice at 8 weeks. Glomerular hypertrophy and increased mesangial staining are evident in STZ groups as compared with non-STZ mice. Original magnification × 640.

Figure 8

Effect of podocyte angiotensin-converting enzyme 2 (ACE2) overexpression on mesangial and glomerular area at 16 weeks. Graphs depict (a) mesangial and (b) glomerular areas at 16 weeks in wild-type (WT), transgenic (TG), wild-type streptozotocin-treated (WT-STZ), and transgenic streptozotocin-treated (TG-STZ) mice. (a) Mesangial area at 16 weeks. Values are means±s.e. (n=60 glomeruli per group of 6 mice). ^*P<0.001 vs. WT and TG, ^#P<0.001 vs. WT and TG. There was no significant difference between WT-STZ and TG-STZ mice. (b) Glomerular area at 16 weeks. Values are means±s.e. (n=60 glomeruli per group of 6 mice). ^*P<0.001 vs. WT and TG, ^#P<0.001 vs. WT and TG, ^$P<0.001 vs. TG-STZ. (c) Representative photomicrographs depicting periodic acid–Schiff (PAS)-stained glomeruli in WT, TG, WT-STZ, and TG-STZ mice at 16 weeks. Glomerular hypertrophy and increased mesangial staining is evident in STZ groups as compared with non-STZ mice. Original magnification × 640.

Figure 9

Nephrin expression at 8 and 16 weeks by immunoblot. (a) Graph depicting nephrin expression by immunoblot analysis of cortical lysates in wild-type (WT), transgenic (TG), wild-type streptozotocin-treated (WT-STZ), and transgenic streptozotocin-treated (TG-STZ) mice at 8 weeks. Representative immunoblot is shown above graph, with nephrin at ∼185 kDa, and β-actin signal below, as control for protein loading. Values are mean±s.e., n=6 per group. ^*P<0.02 vs. WT. There was no significant difference between TG and TG-STZ mice or between WT-STZ and TG-STZ mice. (b) Graph depicting nephrin expression by immunoblot analysis of cortical lysates in WT, TG, WT-STZ, and TG-STZ mice at 16 weeks. Representative immunoblot is shown above graph, with nephrin at ∼185 kDa, and β-actin signal below, as control for protein loading. Values are mean±s.e., n=6 per group. ^*P<0.05 vs. WT. There was no significant difference between TG and TG-STZ mice.

Figure 10

Nephrin expression at 8 and 16 weeks by immunofluorescence. (a) Graph depicting semiquantitative analysis of nephrin expression by immunofluorescence in wild-type (WT), transgenic (TG), wild-type streptozotocin-treated (WT-STZ), and transgenic streptozotocin-treated (TG-STZ) mice at 8 weeks. Values are means±s.e., n=100 glomeruli per group, from 7 to 8 mice per group. ^*P<0.001 vs. WT. There was no significant difference between TG and TG-STZ mice. Representative images depicting glomerular nephrin expression (red) at 8 weeks are shown above graph. Nuclear Hoechst staining (blue) is also shown. Original magnification × 640. (b) Graph depicting nephrin expression by immunofluorescence at 16 weeks. Values are means±s.e., n=100 glomeruli per group, from 6 to 11 mice per group. ^*P<0.001 vs. WT. Representative images depicting glomerular nephrin expression (red) at 16 weeks are shown above graph. Nuclear Hoechst staining (blue) is also shown. Original magnification × 640.

Figure 11

Synaptopodin expression at 8 and 16 weeks by immunofluorescence. (a) Graph depicting semiquantitative analysis of synaptopodin expression by immunofluorescence in wild-type (WT), transgenic (TG), wild-type streptozotocin-treated (WT-STZ), and transgenic streptozotocin-treated (TG-STZ) mice at 8 weeks. Values are means±s.e., n=100 glomeruli per group, from 7 to 8 mice per group. There was no significant difference among the groups. Representative images depicting glomerular synaptopodin expression (green) at 8 weeks are shown above graph. Nuclear Hoechst staining (blue) is also shown. Original magnification × 640. (b) Graph depicting synaptopodin expression at 16 weeks. Values are means±s.e., n=100 glomeruli per group, from 6 to 11 mice per group. ^*P<0.001 vs. WT and TG, ^**P<0.001 vs. WT-STZ. There was no significant difference between TG and TG-STZ mice. Representative images depicting glomerular synaptopodin expression (green) at 16 weeks are shown above graph. Nuclear Hoechst staining (blue) is also shown. Original magnification × 640.

Figure 12

Podocyte number at 8 and 16 weeks. (a) Graph shows average number of podocytes per glomerulus in wild-type (WT), transgenic (TG), wild-type streptozotocin-treated (WT-STZ), and transgenic streptozotocin-treated (TG-STZ) mice at 8 weeks, as determined by counting of WT-1-positive nuclei in kidney sections. Values are means±s.e., n=78–106 glomeruli per 7 to 8 mice in each group. ^*P<0.001 vs. WT and TG, ^#P<0.01 vs. TG, ^$P<0.01 vs. WT. (b) Podocyte numbers at 16 weeks. Values are means±s.e., n=120 glomeruli per 6 mice in each group. ^*P<0.01 vs. WT and TG. There was no significant difference between TG-STZ and WT, TG, or WT-STZ mice. (c) Representative photomicrograph depicting glomerular WT-1 staining in glomeruli from WT-STZ vs. TG-STZ mice at 16 weeks. Original magnification × 400.

Figure 13

Expression of transforming growth factor-β1 (TGF-β1) at 8 and 16 weeks. (a) Graph shows expression of TGF-β1 in kidney cortical lysates from wild-type (WT), transgenic (TG), wild-type streptozotocin-treated (WT-STZ), and transgenic streptozotocin-treated (TG-STZ) mice at 8 weeks, by immunoblot. Values are means±s.e., n=6 per group. ^*P<0.02 vs. all other groups (WT, TG, and TG-STZ). Above graph is a representative immunoblot for TGF-β1, depicting bands at 12.5 and 25 kDa, the latter representing the TGF-β1 dimer. Blot for β-actin, as loading control, is depicted below. (b) Graph shows expression of TGF-β1 from cortical lysates at 16 weeks, with representative immunoblot above graph. Values are means±s.e., n=6 per group. ^*P<0.05 vs. WT and TG. ^**P<0.01 vs. WT and TG. There was no significant difference between WT-STZ and TG-STZ mice.