Kidney aging is associated with an increasing proportion of globally scarred glomeruli, decreasing renal function, and exponentially increasing ESRD prevalence. In model systems, podocyte depletion causes glomerulosclerosis, suggesting age-associated glomerulosclerosis could be caused by a similar mechanism. We measured podocyte number, size, density, and glomerular volume in 89 normal kidney samples from living and deceased kidney donors and normal poles of nephrectomies. Podocyte nuclear density decreased with age due to a combination of decreased podocyte number per glomerulus and increased glomerular volume. Compensatory podocyte cell hypertrophy prevented a change in the proportion of tuft volume occupied by podocytes. Young kidneys had high podocyte reserve (podocyte density .300 per 10 6 mm 3 ), but by 70-80 years of age, average podocyte nuclear density decreased to, ,100 per 10 6 mm 3 , with corresponding podocyte hypertrophy. In older age podocyte detachment rate (urine podocin mRNA-to-creatinine ratio) was higher than at younger ages and podocytes were stressed (increased urine podocin-to-nephrin mRNA ratio). Moreover, in older kidneys, proteinaceous material accumulated in the Bowman space of glomeruli with low podocyte density. In a subset of these glomeruli, mass podocyte detachment events occurred in association with podocytes becoming binucleate (mitotic podocyte catastrophe) and subsequent wrinkling of glomerular capillaries, tuft collapse, and periglomerular fibrosis. In kidneys of young patients with underlying glomerular diseases similar pathologic events were identified in association with focal global glomerulosclerosis. Podocyte density reduction with age may therefore directly lead to focal global glomerulosclerosis, and all progressive glomerular diseases can be considered superimposed accelerators of this underlying process.

Podocyte depletion is a major mechanism driving glomerulosclerosis. Progression is the process by which progressive glomerulosclerosis leads to End Stage Kidney Disease (ESKD). We therefore tested the hypothesis that progression to ESKD can be caused by persistent podocyte loss using the human diphtheria toxin transgenic rat model. After initial podocyte injury causing >30% loss of podocytes glomeruli became destabilized, resulting in continuous podocyte loss over time until global podocyte depletion (ESKD) occured. Similar patterns of podocyte depletion were observed in the puromycin aminonucleoside and 5/6 nephrectomy rat models of progression. Angiotensin II blockade prevented continuous podocyte loss and progression (restabilized glomeruli). Discontinuing angiotensin II blockade resulted in recurrent glomerular destabilization, podocyte loss and progression. Reduction in blood pressure alone did not reduce proteinuria or prevent podocyte loss from destabilized glomeruli. The protective effect of angiotensin II blockade could be entirely accounted for by reduction in podocyte loss. These data demonstrate that an initiating event that results in a critical degree of podocyte depletion can destabilize glomeruli by setting in train a superimposed angiotensin II-dependent podocyte loss cycle that accelerates the progression process and results in global podocyte depletion and progression to ESKD. These events can be monitored non-invasively through urine mRNA assays.

Podocyte depletion leads to glomerulosclerosis, but whether an impaired capacity of podocytes to respond to hypertrophic stress also causes glomerulosclerosis is unknown. We generated transgenic Fischer 344 rats that express a dominant negative AA-4E-BP1 transgene driven by the podocin promoter; a member of the mammalian target of rapamycin complex 1 (mTORC1) pathway, 4E-BP1 modulates cap-dependent translation, which is a key determinant of a cell's hypertrophic response to nutrients and growth factors. AA-4E-BP1 rat podocytes expressed the transgene and had normal kidney histology and protein excretion at 100 g of body weight but developed ESRD by 12 months. Proteinuria and glomerulosclerosis were linearly related to both increasing body weight and transgene dose. Uni-nephrectomy reduced the body weight at which proteinuria first developed by 40%-50%. The initial histologic manifestation of disease was the appearance of bare areas of glomerular basement membrane from the pulling apart of podocyte foot processes, followed by adhesions to the Bowman capsule. Morphometric analysis confirmed the mismatch between glomerular tuft volume and total podocyte volume (number 3 size) per tuft in relation to weight gain and nephrectomy. Proteinuria and glomerulosclerosis did not develop if dietary calorie restriction prevented weight gain and glomerular enlargement. In summary, failure of podocytes to match glomerular tuft growth in response to growth signaling through the mTORC1 pathway can trigger proteinuria, glomerulosclerosis, and progression to ESRD. Reducing body weight and glomerular growth may be useful adjunctive therapies to slow or prevent progression to ESRD.

Model systems demonstrate that progression to ESRD is driven by progressive podocyte depletion (the podocyte depletion hypothesis) and can be noninvasively monitored through measurement of urine pellet podocyte mRNAs. To test these concepts in humans, we analyzed urine pellet mRNAs from 358 adult and pediatric kidney clinic patients and 291 controls (n=1143 samples). Compared with controls, urine podocyte mRNAs increased 79-fold (P,0.001) in patients with biopsy-proven glomerular disease and a 50% decrease in kidney function or progression to ESRD. An independent cohort of patients with Alport syndrome had a 23-fold increase in urinary podocyte mRNAs (P,0.001 compared with controls). Urinary podocyte mRNAs increased during active disease but returned to baseline on disease remission. Furthermore, urine podocyte mRNAs increased in all categories of glomerular disease evaluated, but levels ranged from high to normal, consistent with individual patient variability in the risk for progression. In contrast, urine podocyte mRNAs did not increase in polycystic kidney disease. The association between proteinuria and podocyturia varied markedly by glomerular disease type: a high correlation in minimalchange disease and a low correlation in membranous nephropathy. These data support the podocyte depletion hypothesis as the mechanism driving progression in all human glomerular diseases, suggest that urine pellet podocyte mRNAs could be useful for monitoring risk for progression and response to treatment, and provide novel insights into glomerular disease pathophysiology.

The attrition rate of functioning allografts beyond the first year has not improved despite improved immunosuppression, suggesting that nonimmune mechanisms could be involved. Notably, glomerulopathies may account for about 40% of failed kidney allografts beyond the first year of engraftment, and glomerulosclerosis and progression to ESRD are caused by podocyte depletion. Model systems demonstrate that nephrectomy can precipitate hypertrophic podocyte stress that triggers progressive podocyte depletion leading to ESRD, and that this process is accompanied by accelerated podocyte detachment that can be measured in urine. Here, we show that kidney transplantation "reverse nephrectomy" is also associated with podocyte hypertrophy and increased podocyte detachment. Patients with stable normal allograft function and no proteinuria had levels of podocyte detachment similar to levels in two-kidney controls as measured by urine podocyte assay. By contrast, patients who developed transplant glomerulopathy had 10-to 20-fold increased levels of podocyte detachment. Morphometric studies showed that a subset of these patients developed reduced glomerular podocyte density within 2 years of transplantation due to reduced podocyte number per glomerulus. A second subset developed glomerulopathy by an average of 10 years after transplantation due to reduced glomerular podocyte number and glomerular tuft enlargement. Reduced podocyte density was associated with reduced eGFR, glomerulosclerosis, and proteinuria. These data are compatible with the hypothesis that podocyte depletion contributes to allograft failure and reduced allograft half-life. Mechanisms may include immune-driven processes affecting the podocyte or other cells and/or hypertrophy-induced podocyte stress causing accelerated podocyte detachment, which would be amenable to nonimmune therapeutic targeting. Podocytes are complex neuron-like postmitotic cells adherent to the underlying glomerular basement membrane via foot processes that must contiguously cover the filtration surface area to maintain the normal filtration barrier. Podocytes cannot divide in situ and have limited capacity for replacement. 1 This means that when podocytes are lost, or the glomerular surface area increases due to glomerular growth, the major adaptive response is by hypertrophy. At the same time, the podocyte's structural complexity means that its capacity to hypertrophy is limited. Inability to maintain contiguous coverage of the filtration surface by foot processes results in protein leak into the filtrate. If podocyte detachment exceeds hypertrophic capacity, other glomerular cells adapt by proliferating and laying down matrix resulting in glomerulosclerosis. [2][3][4][5][6]