Accelerated podocyte detachment and progressive podocyte loss from glomeruli with age in Alport Syndrome

Abstract

Podocyte depletion is a common mechanism driving progression in glomerular diseases. Alport Syndrome glomerulopathy, caused by defective α3α4α5 (IV) collagen heterotrimer production by podocytes, is associated with an increased rate of podocyte detachment detectable in urine and reduced glomerular podocyte number suggesting that defective podocyte adherence to the glomerular basement membrane might play a role in driving progression. Here a genetically phenotyped Alport Syndrome cohort of 95 individuals [urine study] and 41 archived biopsies [biopsy study] were used to test this hypothesis. Podocyte detachment rate (measured by podocin mRNA in urine pellets expressed either per creatinine or 24-hour excretion) was significantly increased 11-fold above control, and prior to a detectably increased proteinuria or microalbuminuria. In parallel, Alport Syndrome glomeruli lose an average 26 podocytes per year versus control glomeruli that lose 2.3 podocytes per year, an 11-fold difference corresponding to the increased urine podocyte detachment rate. Podocyte number per glomerulus in Alport Syndrome biopsies is projected to be normal at birth (558/glomerulus) but accelerated podocyte loss was projected to cause end-stage kidney disease by about 22 years. Biopsy data from two independent cohorts showed a similar estimated glomerular podocyte loss rate comparable to the measured 11-fold increase in podocyte detachment rate. Reduction in podocyte number and density in biopsies correlated with proteinuria, glomerulosclerosis, and reduced renal function. Thus, the podocyte detachment rate appears to be increased from birth in Alport Syndrome, drives the progression process, and could potentially help predict time to end-stage kidney disease and response to treatment.

Keywords: Alport syndrome; podocyte; podometrics; progression.

Figure 1. Increased urine podocyte detachment rate in AS

Measurements of the rate of podocyte detachment measured in the urine pellet were expressed either as the urine podocin mRNA: creatinine ratio (analogous to the urine protein:creatinine ratio) (A) or as the Estimated 24hr excretion of podocin mRNA (B). Urine from normal (n=37) and AS patients (n=94) were compared. AS patients had 10.8-fold (95% confidence limits 7.5-15.3) and 11.4-fold (95% confidence limits 8.0-16.4) increased podocin mRNA in the urine pellet as measured by the two methods (P<0.001).

Figure 2. AS patients without proteinuria have increased podocin mRNA in the urine pellet

The 94 AS patients shown in Figure 1 were divided into 4 groups based on the 24hr urine protein excretion and microalbuminuria assay as shown in Table 1. AS group 1 had 24hr urine protein <0.2g and microalbuminuria at <30ug/ml (n=12), AS group 2 had 24hr urine protein <0.2g with microalbuminuria >100mg (n=20), AS group 3 had 24hr urine protein 0.2-3g (n=45), and AS group 4 had 24hr urine protein >3g (n=17). All four groups had statistically increased urine podocin mRNA excretion in the urine pellet with no statistical difference between any of the AS groups.

Figure 3. Biopsy podometrics

Panels show examples of a control glomerulus (A), an AS glomerulus with relatively normal number of podocytes (B), an AS glomerulus with an intermediate number of podocytes (C) and an AS glomerulus with reduced podocyte number (D). Left panels show TLE4 immunofluorescence delineating podocyte nuclei (red) with background green fluorescence showing erythrocytes as light green. Right panels show the same glomeruli from the same section in which the coverslip has been removed and the section stained by Glepp1 immunoperoxidase (brown) and counterstained with hematoxylin (33,34). The bar represents 100um.

Figure 4. Podocyte number per glomerulus in relation to age

(A) Observed relationship of podocyte number per glomerulus to age. The normal podocyte number per glomerulus with age is shown with coordinates y = -2.3x + 580 derived from prior reports (19,31). This data shows that glomeruli normally start life with about 580 podocytes and lose 2.3 podocytes per glomerulus per year throughout life. From Table 4 AS groups A, B and C comprise biopsy podocyte number per glomerulus data from AS patients with 24hr urine protein <0.2g, 0.2-3g and >3g respectively are plotted against age. This resultant line has coordinates y = -26x + 557.5 indicating that at time 0 (birth) AS patients are projected to have 558 podocytes per glomerulus (within the normal range). The slope of this line at -26x is 26/2.3 = 11.3-fold steeper than the normal value, thereby indicating that AS podocytes are lost from glomeruli about 11-fold faster than normal. Projection of this line to intersect with the x axis shows that AS patients in this cohort would be expected to reach low levels of podocyte per glomerulus (i.e. ESKD) by about 20 years of age. (B) Projected decrease in podocyte number per glomerulus with age based on the urine pellet podocin assay. The normal decrease in podocyte number per glomerulus with age is shown derived from prior studies (19,31). From Figure 1A and B the average rate of podocyte detachment in AS patients was measured at 10.8-11.4-fold (average about 11-fold) above the normal rate. The dashed line was generated by assuming that AS patients start with a normal podocyte number per glomerulus (580) and lose podocytes 11-fold faster than normal. This line intersects with the x axis at about 20 years as the projected time at which ESKD will occur. AS biopsy data (open circles) is shown for podocyte number per glomerulus in relation to the age at time of biopsy demonstrating reasonable correspondence between the average projected podocyte number based on the urine assay and the observed values.

Figure 5. Relationship of podocyte depletion to clinical parameters in AS

Data were used from all PUFH AS patients (n=41) shown in Table 2 (closed triangles) including those with <8 glomerular profiles per biopsy as well as data previously reported for an Ann Arbor AS cohort (n=21) (closed circles) (18). Panels A and B. Urine protein:creatinine ratio (UPCR). UPCR is related to podocyte density measured by two independent methods either as Podocyte nuclear density (A) or Glepp1 area density (B). The ranges for normal for urine PCR and the density range (mean+1SD) are shown in the dashed line box. Decreasing poodcyte density is associated with an increasing level of proteinuria. Panel C and D. Glomerulosclerosis. Glomerulosclerosis was measured as the sum of all abnormal glomeruli containing adhesions, FSGS lesions or globally sclerotic glomeruli as a percentage of all tufts in biopsies containing at least 10 tufts. Podocyte depletion as measured by two independent methods was linearly related to proportion of scarred glomeruli. Panel C shows that decreased podocyte number (shown as % nuclear depletion below the normal mean value for the control group) at <30% of normal was not associated with increased glomerulosclerosis. In contrast after >30% of podocytes are lost there is a linear increase in proportion of glomeruli with glomerulosclerosis. Panels E and F. Estimated GFR (eGFR). eGFR was measured as creatinine clearance per 1.73m². The dashed line box shows the normal ranges with the lower limit of normal eGFR for the pediatric population shown as 75 ml/min/1.73m². Reduced eGFR was associated with podocyte depletion measured by two independent methods either as Podocyte number per glomerulus (Panel E) or Glepp1 Area density (Panel F). Reduced eGFR was associated with podocyte depletion.