Critical timing of ACEi initiation prevents compensatory glomerular hypertrophy in the remaining single kidney.

Increasing evidence suggests that single in kidney states (e.g., kidney transplantation and living donation) progressive glomerulosclerosis limits kidney lifespan. Modeling shows that post-nephrectomy compensatory glomerular volume (GV) increase drives podocyte depletion and hypertrophic stress resulting in proteinuria and glomerulosclerosis, implying that GV increase could serve as a therapeutic target to prevent progression. In this report we examine how Angiotensin Converting Enzyme inhibition (ACEi), started before uninephrectomy can reduce compensatory GV increase in wild-type Fischer344 rats. An unbiased computer-assisted method was used for morphometric analysis. Urine Insulin-like growth factor-1 (IGF-1), the major diver of body and kidney growth, was used as a readout. In long-term (40-week) studies of uni-nephrectomized versus sham-nephrectomized rats a 2.2-fold increase in GV was associated with reduced podocyte density, increased proteinuria and glomerulosclerosis. Compensatory GV increase was largely prevented by ACEi started a week before but not after uni-nephrectomy with no measurable impact on long-term eGFR. Similarly, in short-term (14-day) studies, ACEi started a week before uni-nephrectomy reduced both GV increase and urine IGF-1 excretion. Thus, timing of ACEi in relation to uni-nephrectomy had significant impact on post-nephrectomy "compensatory" glomerular growth and outcomes that could potentially be used to improve kidney transplantation and live kidney donation outcomes.

Urine marker analysis identifies evidence for persistent glomerular podocyte injury across allograft lifespan.

Long-term kidney transplant (KT) survival has remained relatively stagnant. Protocol biopsy studies suggest that glomerulosclerosis is a significant contributor to long-term graft failure. We previously demonstrated that podocyte loss in the first year post-transplantation predicted long-term allograft survival. However, whether increased podocyte loss continues over the lifespan of a KT remains unclear. We performed a cross-sectional analysis of 1182 urine samples from 260 KT recipients up to 19-years after transplantation. Urine pellet (UP) mRNAs were assayed for podocyte (NPHS2/podocin and nephrin/NPHS1), distal tubule (aquaporin2), and profibrotic cytokine (TGFbeta1). Multivariable generalized estimating equations were used to obtain "population-averaged" effects for these markers over time post-KT. Consistent with early stresses both podocyte and tubular markers increased immediately post-KT. However, only podocyte markers continued to increase long-term. A role for hypertrophic stresses in driving podocyte loss over time is implied by their association with donor BMI, recipient BMI, and donor-recipient BMI mismatch at transplantation. Furthermore, UP podocin mRNA was associated with urine TGFbeta1, proteinuria, and reduced estimated glomerular filtration rate, thereby linking podocyte injury to allograft fibrosis and survival. In conclusion we observed that podocyte loss continues long-term post-KT suggesting an important role in driving late graft loss.

Podocyte stress and detachment measured in urine are related to mean arterial pressure in healthy humans.

Hypertension-associated progressive glomerulosclerosis is a significant driver of both de novo and all-cause chronic kidney disease leading to end-stage kidney failure. The progression of glomerular disease proceeds via continuing depletion of podocytes from the glomeruli into the ultrafiltrate. To non-invasively assess injury patterns associated with mean arterial pressure (MAP), we conducted an observational study of 87 healthy normotensive individuals who were cleared for living kidney donation. Urine pellet podocin and aquaporin2 mRNAs normalized to the urine creatinine concentration (UPod:Creat ratio and UAqp2:Creat ratio) were used as markers of podocyte detachment and tubular injury, respectively. The ratio of two podocyte mRNA markers, podocin to nephrin (UPod:Neph) as well as the ratio of podocin to the tubular marker aquaporin2 (UPod:Aqp2) estimated the relative rates of podocyte stress and glomerular vs. tubular injury. The MAP was positively correlated with the UPod:Neph and UPod:Aqp2, thereby confirming the relationship of MAP with podocyte stress and the preferential targeting of the glomerulus by higher MAP. In multivariable linear regression analysis, both UPod:Neph and UPod:Creat, but not UAqp2:Creat or proteinuria, were both significantly related to a range of normal MAP (70 to 110 mm Hg). Systolic, as opposed to diastolic or pulse pressure was associated with UPod:Creat. Thus, higher podocyte stress and detachment into the urine are associated with MAP even in a relatively "normal" range of MAP. Hence, urine pellet mRNA monitoring can potentially identify progression risk before the onset of overt hypertension, proteinuria or chronic kidney disease.

Accelerated podocyte detachment early after kidney transplantation is related to long-term allograft loss of function.

BACKGROUND: Kidney allograft half-life has not improved despite excellent short-term survival. Recent long-term surveillance biopsy studies identify accumulating glomerulosclerosis (GS) to be associated with late allograft loss. While podocyte depletion is well known to drive proteinuria and GS in animal models and human glomerular diseases, its role in renal allograft loss of function is generally not recognized. METHODS: To address these questions, we collected urine from 125 kidney allograft recipients in the first posttransplant year for urine pellet messenger RNA (mRNA) and protein analysis, with a median follow up of 4.5 years. RESULTS: Using multivariable linear models adjusted for proteinuria, transplant, recipient and donor factors, we observed that the average urine pellet podocin mRNA normalized to urine creatinine (UPodCR) in the first posttransplant year was significantly associated with an estimated glomerular filtration rate (eGFR) decline (P = 0.001). The relationship between UPodCR and eGFR decline persisted even among recipients who were nonproteinuric and who had no recurrent or de novo glomerular disease identified on 1-year protocol biopsy. Finally, we identified recipient, donor and recipient:donor body surface area mismatch ratio to be independently associated with UPodCR early after transplantation. A larger donor was protective, while a larger recipient and increased recipient:donor size mismatch ratio were associated with increased UPodCR. CONCLUSIONS: These findings support the concept that in kidney allografts, accelerated podocyte loss precedes proteinuria and is associated with inferior long-term allograft outcomes as measured by eGFR decline and may be initiated by recipient:donor size mismatch. Modulating factors driving early podocyte detachment after kidney transplantation may help improve long-term outcomes.

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

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.

FSGS as an Adaptive Response to Growth-Induced Podocyte Stress.

Glomerular sclerotic lesions develop when the glomerular filtration surface area exceeds the availability of podocyte foot process coverage, but the mechanisms involved are incompletely characterized. We evaluated potential mechanisms using a transgenic (podocin promoter-AA-4E-BP1) rat in which podocyte capacity for hypertrophy in response to growth factor/nutrient signaling is impaired. FSGS lesions resembling human FSGS developed spontaneously by 7 months of age, and could be induced earlier by accelerating kidney hypertrophy by nephrectomy. Early segmental glomerular lesions occurred in the absence of a detectable reduction in average podocyte number per glomerulus and resulted from the loss of podocytes in individual glomerular capillary loops. Parietal epithelial cell division, accumulation on Bowman's capsule, and tuft invasion occurred at these sites. Three different interventions that prevented kidney growth and glomerular enlargement (calorie intake reduction, inhibition of mammalian target of rapamycin complex, and inhibition of angiotensin-converting enzyme) protected against FSGS lesion development, even when initiated late in the process. Ki67 nuclear staining and unbiased transcriptomic analysis identified increased glomerular (but not podocyte) cell cycling as necessary for FSGS lesion development. The rat FSGS-associated transcriptomic signature correlated with human glomerular transcriptomes associated with disease progression, compatible with similar processes occurring in man. We conclude that FSGS lesion development resulted from glomerular growth that exceeded the capacity of podocytes to adapt and adequately cover some parts of the filtration surface. Modest modulation of the growth side of this equation significantly ameliorated FSGS progression, suggesting that glomerular growth is an underappreciated therapeutic target for preservation of renal function.

Podocyte Depletion in Thin GBM and Alport Syndrome.

The proximate genetic cause of both Thin GBM and Alport Syndrome (AS) is abnormal α3, 4 and 5 collagen IV chains resulting in abnormal glomerular basement membrane (GBM) structure/function. We previously reported that podocyte detachment rate measured in urine is increased in AS, suggesting that podocyte depletion could play a role in causing progressive loss of kidney function. To test this hypothesis podometric parameters were measured in 26 kidney biopsies from 21 patients aged 2-17 years with a clinic-pathologic diagnosis including both classic Alport Syndrome with thin and thick GBM segments and lamellated lamina densa [n = 15] and Thin GBM cases [n = 6]. Protocol biopsies from deceased donor kidneys were used as age-matched controls. Podocyte depletion was present in AS biopsies prior to detectable histologic abnormalities. No abnormality was detected by light microscopy at <30% podocyte depletion, minor pathologic changes (mesangial expansion and adhesions to Bowman's capsule) were present at 30-50% podocyte depletion, and FSGS was progressively present above 50% podocyte depletion. eGFR did not change measurably until >70% podocyte depletion. Low level proteinuria was an early event at about 25% podocyte depletion and increased in proportion to podocyte depletion. These quantitative data parallel those from model systems where podocyte depletion is the causative event. This result supports a hypothesis that in AS podocyte adherence to the GBM is defective resulting in accelerated podocyte detachment causing progressive podocyte depletion leading to FSGS-like pathologic changes and eventual End Stage Kidney Disease. Early intervention to reduce podocyte depletion is projected to prolong kidney survival in AS.

Glomerular Aging and Focal Global Glomerulosclerosis: A Podometric Perspective.

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) µm(3)), but by 70-80 years of age, average podocyte nuclear density decreased to, <100 per 10(6) µm(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.

The two kidney to one kidney transition and transplant glomerulopathy: a podocyte perspective.

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.

Urine podocyte mRNAs, proteinuria, and progression in human glomerular diseases.

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 minimal-change 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.

Urine podocin:nephrin mRNA ratio (PNR) as a podocyte stress biomarker.

BACKGROUND: Proteinuria and/or albuminuria are widely used for noninvasive assessment of kidney diseases. However, proteinuria is a nonspecific marker of diverse forms of kidney injury, physiologic processes and filtration of small proteins of monoclonal and other pathologic processes. The opportunity to develop new glomerular disease biomarkers follows the realization that the degree of podocyte depletion determines the degree of glomerulosclerosis, and if persistent, determines the progression to end-stage kidney disease (ESKD). Podocyte cell lineage-specific mRNAs can be recovered in urine pellets of model systems and in humans. In model systems, progressive glomerular disease is associated with decreased nephrin mRNA steady-state levels compared with podocin mRNA. Thus, the urine podocin:nephrin mRNA ratio (PNR) could serve as a useful progression biomarker. The use of podocyte-specific transcript ratios also circumvents many problems inherent to urine assays. METHODS: To test this hypothesis, the human diphtheria toxin receptor (hDTR) rat model of progression was used to evaluate potentially useful urine mRNA biomarkers. We compared histologic progression parameters (glomerulosclerosis score, interstitial fibrosis score and percent of podocyte depletion) with clinical biomarkers [serum creatinine, systolic blood pressure (BP), 24-h urine volume, 24-h urine protein excretion and the urine protein:creatinine ratio(PCR)] and with the novel urine mRNA biomarkers. RESULTS: The PNR correlated with histologic outcome as well or better than routine clinical biomarkers and other urine mRNA biomarkers in the model system with high specificity and sensitivity, and a low coefficient of assay variation. CONCLUSIONS: We concluded that the PNR, used in combination with proteinuria, will be worth testing for its clinical diagnostic and decision-making utility.

Growth-dependent podocyte failure causes glomerulosclerosis.

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 × 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.

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

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.