Angiotensin II-dependent persistent podocyte loss from destabilized glomeruli causes progression of end stage kidney disease

Abstract

Podocyte depletion is a major mechanism driving glomerulosclerosis. Progression is the process by which progressive glomerulosclerosis leads to end stage kidney disease (ESKD). In order to determine mechanisms contributing to persistent podocyte loss, we used a human diphtheria toxin transgenic rat model. After initial diphtheria toxin-induced podocyte injury (over 30% loss in 4 weeks), glomeruli became destabilized, resulting in continued autonomous podocyte loss causing global podocyte depletion (ESKD) by 13 weeks. This was monitored by urine mRNA analysis and by quantitating podocytes in glomeruli. Similar patterns of podocyte depletion were found in the puromycin aminonucleoside and 5/6 nephrectomy rat models of progressive end-stage disease. Angiotensin II blockade (combined enalapril and losartan) restabilized the glomeruli, and prevented continuous podocyte loss and progression to ESKD. Discontinuing angiotensin II blockade resulted in recurrent glomerular destabilization, podocyte loss, and progression to ESKD. Reduction in blood pressure alone did not reduce proteinuria or prevent podocyte loss from destabilized glomeruli. The protective effect of angiotensin II blockade was entirely accounted for by reduced podocyte loss. Thus, an initiating event resulting in a critical degree of podocyte depletion can destabilize glomeruli and initiate a superimposed angiotensin II-dependent podocyte loss process that accelerates progression resulting in eventual global podocyte depletion and ESKD. These events can be monitored noninvasively in real-time through urine mRNA assays.

Figure 1. Autonomous progression following initial podoctyte depletion in the hDTR Fischer 344 rat model

(A) Representative histology for sequential kidney biopsies. From left to right, micrographs are from control and at 4, 8 and 13 weeks after DT injection. Upper panels show podocytes identified using GLEPP1 / peroxidase. Lower panels show Masson’s Trichrome staining. Arrows show sclerosed and partially sclerosed glomeruli. (B) Quantitation of histology using WT1 positive podocyte nuclear number (C) Percentage of glomerular tuft area that was GLEPP1 positive. (D) Time course of urine protein:creatinine ratio, urine podocyte (podocin and nephrin) mRNA excretion and urine podocin:nephrin mRNA ratio. Note that the urine protein:creatinine ratio was increased after injury and continued at a high level until ESKD at 13 weeks (top). In the acute phase (first 21 days after injury) both podocin and nephrin mRNA levels were increased. During the chronic phase of progression (from 4 to 13 weeks) urine podocin mRNA remained elevated throughout the time course of progression showing autonomous podocyte loss over the whole time course. In contrast nephrin mRNA levels were not correspondingly increased during the chronic phase, so that the urine podocin:nephrin mRNA ratio is a marker of the chronic progression phase. n = 8, *P < 0.05 and **P < 0.01, as assessed by Kruskal-Wallis test and then Scheffe test. The serum creatinine values in these animals were 2.52 ± 0.36 mg/dl versus the normal controls 0.43 ± 0.06 mg/dl (P < 0.01, as assessed by Student’s t-test).

Figure 2. The effect of variable amounts of initial podocyte depletion in hDTR rats model

(A) Glomerular podocyte number measured by WT1 positive nuclei immunofluorescence staining in severe (n = 5), moderate (n = 13), mild (n = 5) and control (n = 4) groups at 4 and 8 weeks. (B) Percentage of glomerular tuft area that was GLEPP1 positive in severe, moderate, mild and control groups at 4 and 8 weeks. (C) Mean daily urine protein:creatinine ratio in severe, moderate, mild and control groups. (D) Mean daily urine podocin mRNA excretion in severe, moderate, mild and control groups. (E) Mean daily urine nephrin mRNA excretion in severe, moderate, mild and control groups. (F) Mean daily urine podocin:nephrin mRNA ratio in severe, moderate, mild and control groups. *P < 0.05 and **P < 0.01 vs each groups, ^##P < 0.01 vs each week, as assessed by Kruskal-Wallis test and then Scheffe test.

Figure 3. Comparison of urine biomarkers for three models of progression including the hDTR Fischer 344 rat (A), the PAN Sprague-Dawley rat (B) and the 5/6 Nephrectomy Sprague-Dawley rat (C)

Biomarkers include urine protein:creatinine ratio, urine podocin and podocin:nephrin ratio as measures of podocyte loss and stress, TGFβ1 mRNA as a marker of the profibrotic process, and aquaporin 2 (AQP2) as a kidney tubular cell marker. During progression similar events were observed in all three model systems compatible with the conclusion that similar processes were taking place during progression. Experimental data are shown in closed circles and control data are shown in open circles for different rat strains used.

Figure 4. Prevention of autonomous podocyte depletion by angiotensin II blockade

(A) Representative histology with or without angiotensin II blockade in hDTR rat model. Upper panels show podocytes identified using GLEPP1 / peroxidase. Lower panels show Masson’s Trichrome staining. Arrows show sclerosed and partially sclerosed glomeruli. (B) Quantitation of histology with or without angiotensin II blockade by WT1 positive nuclei immunofluorescence staining (C) Percentage of glomerular tuft area that was GLEPP1 positive. (D) Time course of urine protein:creatinine ratio, urine podocyte (podocin and nephrin) mRNA excretion and urine podocin:nephrin mRNA ratio with or without angiotensin II blockade. Note that the drug-treated group showed reduced proteinuria by about 5 days after the start of drug treatment. In the acute phase (the first 21 days after injury), urine podocyte mRNAs (podocin and nephrin) excretion were not significantly different, but in the chronic phase (after 21 days), urine podocyte mRNAs and urine podocin:nephrin mRNA ratio were significantly reduced in the drug-treated group. (E) Acute and chronic phase analysis of the urine protein:creatinine ratio, urine podocyte mRNAs and urine podocin:nephrin mRNA ratio. (F) Time course of systolic blood pressure with or without angiotensin II blockade. n = 7 per group, *P < 0.05 and **P < 0.01, as assessed by Student’s t-test. ^##Z > 3 as assessed by Z-statistical analysis. The serum creatinine values in non-drug treated animals were 2.60 ± 0.14 mg/dl versus the drug treated animals 0.73 ± 0.04 mg/dl, (P < 0.01, as assessed by Student’s t-test).

Figure 5. Discontinuation of angiotensin II blockade causes glomerular destabilization in the hDTR rat model

(A) Representative histology for angiotensin II blockade continued for 19 weeks or discontinued after 8 weeks. Upper panels show podocytes were identified using GLEPP1 / peroxidase. Lower panels show Masson’s Trichrome staining. Arrows show sclerosed and partially sclerosed glomeruli. (B) Quantitation of histology measured by WT1 positive podocyte number. (C) Percentage of glomerular tuft area that was GLEPP1 positive. (D) Time course of urine protein:creatinine ratio, urine podocyte (podocin and nephrin) mRNA excretion and urine podocin:nephrin mRNA ratio. Note that proteinuria was reduced throughout the time course in the continuous drug group, but increased after drug discontinuation. Urine podocyte mRNAs (podocin and nephrin) and urine podocin:nephrin mRNA ratio also increased after drug discontinuation. (E) Time course of systolic blood pressure. For D and E a two-sample Z-statistic was used to compare the mean levels in the two arms of the study. The number and percentage of p-values below 0.05 prior to and after drug discontinuation respectively were: Systolic blood pressure 0/6 (0%) and 9/9 (100%), urine protein:creatinine ratio 4/35 (11.4%) and 40/40 (100%), urine podocin mRNA 1/35 (2.9%) and 38/40 (95%), urine nephrin mRNA 2/35 (5.7%) and 23/40 (58%), and urine podocin:nephrin mRNA ratio 2/35 (5.7%) and 33/40 (82.5%). If there was no difference between the two groups, on average 5% of p-values will be below 0.05. For 35 independent measurements, the 95% range of the percentage of p-values below 0.05 is 0% to 14%. Thus all results above are consistent with there being no difference between the two groups of rats prior to discontinuation of drug, and a strong difference after discontinuation of drug.

Figure 6. Blood pressure reduction in the acute phase using triple therapy did not prevent autonomous podocyte depletion in the hDTR rat model

(A) Representative histology with or without triple therapy (hydralazine, reserpine and hydrochlorothiazide). Upper panels show podocytes identified using GLEPP1 / peroxidase staining. Lower panels show Masson’s Trichrome staining. Arrows show sclerosed and partially sclerosed glomeruli. (B) Quantitation of histology with or without triple therapy showing no difference in glomerular podocyte number by WT1 positive nuclear immunofluorescence. (C) Percentage of glomerular tuft area that was GLEPP1 positive. (D) Time course of urine protein:creatinine ratio, urine podocyte (podocin and nephrin) mRNA excretion and urine podocin:nephrin mRNA ratio. Proteinuria, urine podocyte mRNAs (podocin and nephrin) and urine podocin:nephrin mRNA ratio showed no difference between drug-treated and control groups. (E) Acute and chronic phase analysis of the urine protein:creatinine ratio, urine podocyte (podocin and nephrin) mRNA excretion and urine podocin:nephrin mRNA ratio. (F) Time course of systolic blood pressure with or without triple therapy showing reduction of blood pressure only in the acute phase. n = 7 for the drug-treated group, n = 8 for the non drug-treated group *P < 0.05 and **P < 0.01, as assessed by Student’s t-test. ^##Z > 3 as assessed by Z-statistical analysis. The serum creatinine values in non-drug treated animals were 1.19 ± 0.17 mg/dl versus the drug treated animals 1.47 ± 0.28 mg/dl, (P = 0.40, as assessed by Student’s t-test.

Figure 7. Blood pressure reduction throughout the time course using aliskiren (delivered at 25 mg/kg/day by osmotic minipump) did not reduce proteinuria or prevent autonomous podoyte depletion. In contrast similar blood pressure reduction using the angiotensin II receptor blocker losartan reduced proteinuria and podocyte depletion

(A) Representative histology in saline, aliskiren, losartan and aliskiren+losartan groups. Upper panels show podocytes identified using GLEPP1 / peroxidase staining. Lower panels show Masson’s Trichrome staining. Arrows show sclerosed and partially sclerosed glomeruli. Color coding is shown by the box at upper right for the saline, aliskiren, losartan and aliskiren+losartan groups. (B) Quantitation of glomerular podocyte number by WT1 positive nuclear immunofluorescence. (C) Percentage of glomerular tuft area that was GLEPP1 positive. (D) Quantitation of glomerular sclerosis score. (E) Quantitation of interstitial fibrosis score. (F) Time course of urine protein:creatinine ratio, urine podocyte (podocin and nephrin) mRNA excretion, and urine podocin:nephrin mRNA ratio. Aliskiren alone did not reduce proteinuria or urine podocyte markers. In contrast losartan alone and aliskiren+losartan reduced proteinuria and urine mRNA markers. (G). Acute and chronic phase analysis of the urine protein:creatinine ratio, urine podocyte mRNAs and urine podocin:nephrin mRNA ratio. (H). Time course of systolic blood pressure showing significant reduction of blood pressure into the normal range throughout the time course in aliskiren, losartan and aliskiren+losartan group rats. n = 8 (saline and aliskiren groups), n = 6 (losartan and aliskiren+losartan groups). *P < 0.05 and **P < 0.01, as assessed by Kruskal-Wallis test and then Scheffe test. ^#Z > 2 and ^##Z > 3 as assessed by Z-statistical analysis. The serum creatinine values in these four groups were 2.64 ± 0.29 mg/dl, 2.14 ± 0.13 mg/dl, 1.29 ± 0.07 mg/dl and 1.33 ± 0.10 mg/dl, (P < 0.01, saline vs losartan and saline vs aliskiren+losartan, P < 0.05, aliskiren vs losartan, as assessed by Kruskal-Wallis test and then Scheffe test.)

Figure 8. Relationship between glomerular sclerosis or interstitial fibrosis score and % depletion of the WT1 podocyte nuclear count

(A) Relationship between glomerular sclerosis score and % depletion of WT1 podocyte nuclear count for all animals (n = 87, R² = 0.79, P < 0.001). (B) Relationship between interstitial fibrosis score and % depletion of WT1 podocyte nuclear count for all animals (n = 87, R² = 0.61, P < 0.001). (C) Relationship between glomerular sclerosis score and % depletion of podocyte count with (closed squares thick line) or without (open triangles thin line) angiotensin II blockade. Angiotensin II blockade caused reduced podocyte loss but did not change either the slope or the coordinates of the correlation (Non-drug group: n = 57, R² = 0.81, P < 0.001, Drug group: n = 30, R² = 0.63, P < 0.001). (D) Relationship between interstitial fibrosis score and % depletion of WT1 podocyte nuclear count with (closed squares and thick line) or without (open triangles and thin line) angiotensin II blockade. Angiotensin II blockade reduced podocyte loss but did not change the slope of the relationship (Non-drug group: n = 57, R² = 0.57, P < 0.001, Drug group: n = 30, R² = 0.44, P < 0.001).

Figure 9. Diagrammatic summary of the glomerular destabilization and progression hypothesis

Many factors can cause podocyte injury. If as a result of this initiating injury there is a critical level of podocyte depletion then the glomerulus can become destabilized such that further podocyte loss occurs independent of the initiating event and driven by the renin-angiotensin system until glomeruli become globally depleted of podocytes (ESKD). This progression accelerating mechanism can be retarded or prevented by angiotensin II blockade (glomerular restabilization), but discontinuation of angiotensin II blockade results in relapse to the destabilized glomerular phenotype with recurrence of podocyte loss. These events can be monitored non-invasively by measuring the podocyte mRNA products in urine.