Urinary podocyte markers of disease activity, therapeutic efficacy, and long-term outcomes in acute and chronic kidney diseases.

A critical degree of podocyte depletion causes glomerulosclerosis, and persistent podocyte loss in glomerular diseases drives the progression to end-stage kidney disease. The extent of podocyte injury at a point in time can be histologically assessed by measuring podocyte number, size, and density ("Biopsy podometrics"). However, repeated invasive renal biopsies are associated with increased risk and cost. A noninvasive method for assessing podocyte injury and depletion is required. Albuminuria and proteinuria do not always correlate with disease activity. Podocytes are located on the urinary space side of the glomerular basement membrane, and as they undergo stress or detach, their products can be identified in urine. This raises the possibility that urinary podocyte products can serve as clinically useful markers for monitoring glomerular disease activity and progression ("Urinary podometrics"). We previously reported that urinary sediment podocyte mRNA reflects disease activity in both animal models and human glomerular diseases. This includes diabetes and hypertension which together account for 60% of new-onset dialysis induction patients. Improving approaches to preventing progression is an urgent priority for the renal community. Sufficient evidence now exists to indicate that monitoring urinary podocyte markers could serve as a useful adjunctive strategy for determining the level of current disease activity and response to therapy in progressive glomerular diseases.

The Michigan O'Brien Kidney Research Center: transforming translational kidney research through systems biology.

Research on kidney diseases is being transformed by the rapid expansion and innovations in omics technologies. The analysis, integration, and interpretation of big data, however, have been an impediment to the growing interest in applying these technologies to understand kidney function and failure. Targeting this urgent need, the University of Michigan O'Brien Kidney Translational Core Center (MKTC) and its Administrative Core established the Applied Systems Biology Core. The Core provides need-based support for the global kidney community centered on enabling incorporation of systems biology approaches by creating web-based, user-friendly analytic and visualization tools, like Nephroseq and Nephrocell, guiding with experimental design, and processing, analysis, and integration of large data sets. The enrichment core supports systems biology education and dissemination through workshops, seminars, and individualized training sessions. Meanwhile, the Pilot and Feasibility Program of the MKTC provides pilot funding to both early-career and established investigators new to the field, to integrate a systems biology approach into their research projects. The relevance and value of the portfolio of training and services offered by MKTC are reflected in the expanding community of young investigators, collaborators, and users accessing resources and engaging in systems biology-based kidney research, thereby motivating MKTC to persevere in its mission to serve the kidney research community by enabling access to state-of-the-art data sets, tools, technologies, expertise, and learning opportunities for transformative basic, translational, and clinical studies that will usher in solutions to improve the lives of people impacted by kidney disease.

Quantitative morphometrics reveals glomerular changes in patients with infrequent segmentally sclerosed glomeruli.

AIMS: Detection of one segmentally sclerosed glomerulus (SSG) identifies patients with focal segmental glomerulosclerosis (FSGS) but rare SSGs may be missed in kidney biopsies. It is unknown whether alterations of unaffected glomeruli in patients with infrequent SSG can be detected by quantitative morphometrics. METHODS: We determined SSG frequency and obtained quantitative morphometrics in glomeruli without a pathologic phenotype in large kidney sections of non-involved kidney tissue from 137 patients undergoing total nephrectomy. We used multivariate modelling to identify morphometrics independently associated with increasing frequency of SSG and Receiver Operator Curve (ROC) analysis to determine the ability of quantitative morphometrics to identify patients with FSGS. We used the geometric distribution to estimate the sensitivity and specificity of a needle biopsy to identify patients with FSGS. RESULTS: In seventy-one patients (51.8%), at least one SSG was observed, and of those, 39 (54.9%) had an SSG lesion in less than 2% of all glomeruli (mean of 249 glomeruli per specimen). Increasing percent of SSG was independently associated with decreasing podocyte density and increasing mesangial index in multivariate modelling. For infrequent SSG lesions (<1% of glomeruli), kidney biopsy could miss FSGS diagnosis more than 74% of the time, and podocyte density had an area under the curve (AUC) of 0.77, and mesangial index, an AUC of 0.79 to identify patients with FSGS. CONCLUSIONS: More than half of patients had FSGS, although 30% had infrequent SSG. Quantitative morphometrics in glomeruli without pathology, such as podocyte density and mesangial index, identified patients with infrequent SSG and may serve as clinical markers to identify patients with FSGS.

Glomerular endothelial cell-podocyte stresses and crosstalk in structurally normal kidney transplants.

Increased podocyte detachment begins immediately after kidney transplantation and is associated with long-term allograft failure. We hypothesized that cell-specific transcriptional changes in podocytes and glomerular endothelial cells after transplantation would offer mechanistic insights into the podocyte detachment process. To test this, we evaluated cell-specific transcriptional profiles of glomerular endothelial cells and podocytes from 14 patients of their first-year surveillance biopsies with normal histology from low immune risk recipients with no post-transplant complications and compared these to biopsies of 20 healthy living donor controls. Glomerular endothelial cells from these surveillance biopsies were enriched for genes related to fluid shear stress, angiogenesis, and interferon signaling. In podocytes, pathways were enriched for genes in response to growth factor signaling and actin cytoskeletal reorganization but also showed evidence of podocyte stress as indicated by reduced nephrin (adhesion protein) gene expression. In parallel, transcripts coding for proteins required to maintain podocyte adherence to the underlying glomerular basement membrane were downregulated, including the major glomerular podocyte integrin α3 and the actin cytoskeleton-related gene synaptopodin. The reduction in integrin α3 protein expression in surveillance biopsies was confirmed by immunoperoxidase staining. The combined growth and stress response of patient allografts post-transplantation paralleled similar changes in a rodent model of nephrectomy-induced glomerular hypertrophic stress that progress to develop proteinuria and glomerulosclerosis with shortened kidney life span. Thus, even among patients with apparently healthy allografts with no detectable histologic abnormality including alloimmune injury, transcriptomic changes reflecting cell stresses are already set in motion that could drive hypertrophy-associated glomerular disease progression.

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.

Urinary podocyte mRNAs precede microalbuminuria as a progression risk marker in human type 2 diabetic nephropathy.

Earlier detection of progression risk in diabetic nephropathy will allow earlier intervention to reduce progression. The hypothesis that urinary pellet podocyte mRNA is a more sensitive progression risk marker than microalbuminuria was tested. A cross sectional cohort of 165 type 2 diabetics and 41 age and sex-matched controls were enrolled. Podocyte stress (Urinary pellet podocin:nephrin mRNA ratio), podocyte detachment (Urinary pellet podocin mRNA:creatinine ratio: UPPod:CR) and a tubular marker (Urinary pellet aquaporin 2:creatinine ratio) were measured in macro-albuminuric, micro-albuminuric and norm-albuminuric groups. eGFR was reassessed after 4 years in 124 available diabetic subjects. Urinary pellet podocyte and tubular mRNA markers were increased in all diabetic groups in cross-sectional analysis. After 4 years of follow-up univariable and multivariate model analysis showed that the only urinary markers significantly related to eGFR slope were UPPod:CR (P < 0.01) and albuminuria (P < 0.01). AUC analysis using K-fold cross validation to predict eGFR loss of ≥ 3 ml/min/1.73m2/year showed that UPPod:CR and albuminuria each improved the AUC similarly such that combined with clinical variables they gave an AUC = 0.70. Podocyte markers and albuminuria had overlapping AUC contributions, as expected if podocyte depletion causes albuminuria. In the norm-albuminuria cohort (n = 75) baseline UPPod:CR was associated with development of albuminuria (P = 0.007) and, in the tertile with both normal kidney function (eGFR 84 ± 11.7 ml/min/1.73m2) and norm-albuminuria at baseline, UPPod:CR was associated with eGFR loss rate (P = 0.003). In type 2 diabetics with micro- or macro-albuminuria UPPod:CR and albuminuria were equally good at predicting eGFR loss. For norm-albuminuric type 2 diabetics UPPod:CR predicted both albuminuria and eGFR 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.

Hypertension induces glomerulosclerosis in phospholipase C-ε1 deficiency.

Loss-of-function mutations in phospholipase C-ε1 (PLCE1) have been detected in patients with nephrotic syndrome, but other family members with the same mutation were asymptomatic, suggesting additional stressor are required to cause the full phenotype. Consistent with these observations, we determined that global Plce1-deficient mice have histologically normal glomeruli and no albuminuria at baseline. Angiotensin II (ANG II) is known to induce glomerular damage in genetically susceptible individuals. Therefore, we tested whether ANG II enhances glomerular damage in Plce1-deficient mice. ANG II increased blood pressure equally in Plce1-deficient and wild-type littermates. Additionally, it led to 20-fold increased albuminuria and significantly more sclerotic glomeruli in Plce1-deficient mice compared with wild-type littermates. Furthermore, Plce1-deficient mice demonstrated diffuse mesangial expansion, podocyte loss, and focal podocyte foot process effacement. To determine whether these effects are mediated by hypertension and hyperfiltration, rather than directly through ANG II, we raised blood pressure to a similar level using DOCA + salt + uninephrectomy and norepinephrine. This caused a fivefold increase in albuminuria in Plce1-deficient mice and a significant increase in the number of sclerotic glomeruli. Consistent with previous findings in mice, we detected strong PLCE1 transcript expression in podocytes using single cell sequencing of human kidney tissue. In hemagglutinin-tagged Plce1 transgenic mice, Plce1 was detected in podocytes and also in glomerular arterioles using immunohistochemistry. Our data demonstrate that Plce1 deficiency in mice predisposes to glomerular damage secondary to hypertensive insults.

Podocyte hypertrophic stress and detachment precedes hyperglycemia or albuminuria in a rat model of obesity and type2 diabetes-associated nephropathy.

Type2 diabetes-associated nephropathy is the commonest cause of renal failure. Mechanisms responsible are controversial. Leptin-deficient hyperphagic Zucker (fa/fa) rats were modeled to test the hypothesis that glomerular enlargement drives podocyte hypertrophic stress leading to accelerated podocyte detachment, podocyte depletion, albuminuria and progression. By 6weeks, prior to development of either hyperglycemia or albuminuria, fa/fa rats were hyperinsulinemic with high urinary IGF1/2 excretion, gaining weight rapidly, and had 1.6-fold greater glomerular volume than controls (P < 0.01). At this time the podocyte number per glomerulus was not yet reduced although podocytes were already hypertrophically stressed as shown by high podocyte phosphor-ribosomal S6 (a marker of mTORC1 activation), high urinary pellet podocin:nephrin mRNA ratio and accelerated podocyte detachment (high urinary pellet podocin:aquaporin2 mRNA ratio). Subsequently, fa/fa rats became both hyperglycemic and albuminuric. 24 hr urine albumin excretion correlated highly with decreasing podocyte density (R2 = 0.86), as a consequence of both increasing glomerular volume (R2 = 0.70) and decreasing podocyte number (R2 = 0.63). Glomerular podocyte loss rate was quantitatively related to podocyte detachment rate measured by urine pellet mRNAs. Glomerulosclerosis occurred when podocyte density reached <50/106um3. Reducing food intake by 40% to slow growth reduced podocyte hypertrophic stress and "froze" all elements of the progression process in place, but had small effect on hyperglycemia. Glomerular enlargement caused by high growth factor milieu starting in pre-diabetic kidneys appears to be a primary driver of albuminuria in fa/fa rats and thereby an under-recognized target for progression prevention. Progression risk could be identified prior to onset of hyperglycemia or albuminuria, and monitored non-invasively by urinary pellet podocyte mRNA markers.

Organoid single cell profiling identifies a transcriptional signature of glomerular disease.

Podocyte injury is central to many forms of kidney disease, but transcriptional signatures reflecting podocyte injury and compensation mechanisms are challenging to analyze in vivo. Human kidney organoids derived from pluripotent stem cells (PSCs), a potentially new model for disease and regeneration, present an opportunity to explore the transcriptional plasticity of podocytes. Here, transcriptional profiling of more than 12,000 single cells from human PSC-derived kidney organoid cultures was used to identify robust and reproducible cell lineage gene expression signatures shared with developing human kidneys based on trajectory analysis. Surprisingly, the gene expression signature characteristic of developing glomerular epithelial cells was also observed in glomerular tissue from a kidney disease cohort. This signature correlated with proteinuria and inverse eGFR, and it was confirmed in an independent podocytopathy cohort. Three genes in particular were further characterized as potentially novel components of the glomerular disease signature. We conclude that cells in human PSC-derived kidney organoids reliably recapitulate the developmental transcriptional program of podocytes and other cell lineages in the human kidney and that transcriptional profiles seen in developing podocytes are reactivated in glomerular disease. Our findings demonstrate an approach to identifying potentially novel molecular programs involved in the pathogenesis of glomerulopathies.

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.

Urinary podocyte and TGF-ß1 mRNA as markers for disease activity and progression in anti-glomerular basement membrane nephritis.

BACKGROUND: Podocyte depletion causes glomerulosclerosis, with persistent podocyte loss being a major factor driving disease progression. Urinary podocyte mRNA is potentially useful for monitoring disease progression in both animal models and in humans. To determine whether the same principles apply to crescentic glomerular injury, a rat model of anti-glomerular basement membrane (anti-GBM) nephritis was studied in parallel with a patient with anti-GBM nephritis. METHODS: Podocyte loss was measured by Wilms' Tumor 1-positive podocyte nuclear counting and density, glomerular epithelial protein 1 or synaptopodin-positive podocyte tuft area and urinary podocyte mRNA excretion rate. Glomerulosclerosis was evaluated by Azan staining and urinary transforming growth factor (TGF)-ß1 mRNA excretion rate. RESULTS: In the rat model, sequential kidney biopsies revealed that after a threshold of 30% podocyte loss, the degree of glomerulosclerosis was linearly associated with the degree of podocyte depletion, compatible with podocyte depletion driving the sclerotic process. Urinary podocyte mRNA correlated with the rate of glomerular podocyte loss. In treatment studies, steroids prevented glomerulosclerosis in the anti-GBM model in contrast to angiotensin II inhibition, which lacked a protective effect, and urinary podocyte and TGF-ß1 mRNA markers more accurately reflected both the amount of podocyte depletion and the degree of glomerulosclerosis compared with proteinuria under both scenarios. In a patient successfully treated for anti-GBM nephritis, urinary podocyte and TGB-ß1 mRNA reflected treatment efficacy. CONCLUSION: These results emphasize the role of podocyte depletion in anti-GBM nephritis and suggest that urinary podocyte and TGF-ß1 mRNA could serve as markers of disease progression and treatment efficacy.

Two-Photon Intravital Fluorescence Lifetime Imaging of the Kidney Reveals Cell-Type Specific Metabolic Signatures.

In the live animal, tissue autofluorescence arises from a number of biologically important metabolites, such as the reduced form of nicotinamide adenine dinucleotide. Because autofluorescence changes with metabolic state, it can be harnessed as a label-free imaging tool with which to study metabolism in vivo Here, we used the combination of intravital two-photon microscopy and frequency-domain fluorescence lifetime imaging microscopy (FLIM) to map cell-specific metabolic signatures in the kidneys of live animals. The FLIM images are analyzed using the phasor approach, which requires no prior knowledge of metabolite species and can provide unbiased metabolic fingerprints for each pixel of the lifetime image. Intravital FLIM revealed the metabolic signatures of S1 and S2 proximal tubules to be distinct and resolvable at the subcellular level. Notably, S1 and distal tubules exhibited similar metabolic profiles despite apparent differences in morphology and autofluorescence emission with traditional two-photon microscopy. Time-lapse imaging revealed dynamic changes in the metabolic profiles of the interstitium, urinary lumen, and glomerulus-areas that are not resolved by traditional intensity-based two-photon microscopy. Finally, using a model of endotoxemia, we present examples of the way in which intravital FLIM can be applied to study kidney diseases and metabolism. In conclusion, intravital FLIM of intrinsic metabolites is a bias-free approach with which to characterize and monitor metabolism in vivo, and offers the unique opportunity to uncover dynamic metabolic changes in living animals with subcellular resolution.

Podocyte number and density changes during early human life.

BACKGROUND: Podocyte depletion, which drives progressive glomerulosclerosis in glomerular diseases, is caused by a reduction in podocyte number, size or function in the context of increasing glomerular volume. METHODS: Kidneys obtained at autopsy from premature and mature infants who died in the first year of life (n = 24) were used to measure podometric parameters for comparison with previously reported data from older kidneys. RESULTS: Glomerular volume increased 4.6-fold from 0.13 ± 0.07 µm3 x106 in the pre-capillary loop stage, through 0.35 µm3 x106 at the capillary loop, to 0.60 µm3 x106 at the mature glomerular stage. Podocyte number per glomerulus increased from 326 ± 154 per glomerulus at the pre-capillary loop stage to 584 ± 131 per glomerulus at the capillary loop stage of glomerular development to reach a value of 589 ± 166 per glomerulus in mature glomeruli. Thus, the major podocyte number increase occurs in the early stages of glomerular development, in contradistinction to glomerular volume increase, which continues after birth in association with body growth. CONCLUSIONS: As glomeruli continue to enlarge, podocyte density (number per volume) rapidly decreases, requiring a parallel rapid increase in podocyte size that allows podocyte foot processes to maintain complete coverage of the filtration surface area. Hypertrophic stresses on the glomerulus and podocyte during development and early rapid growth periods of life are therefore likely to play significant roles in determining how and when defects in podocyte structure and function due to genetic variants become clinically manifest. Therapeutic strategies aimed at minimizing mismatch between these factors may prove clinically useful.

Quantitative podocyte parameters predict human native kidney and allograft half-lives.

BACKGROUND: Kidney function decreases with age. A potential mechanistic explanation for kidney and allograft half-life has evolved through the realization that linear reduction in glomerular podocyte density could drive progressive glomerulosclerosis to impact both native kidney and allograft half-lives. METHODS: Predictions from podometrics (quantitation of podocyte parameters) were tested using independent pathologic, functional, and outcome data for native kidneys and allografts derived from published reports and large registries. RESULTS: With age, native kidneys exponentially develop glomerulosclerosis, reduced renal function, and end-stage kidney disease, projecting a finite average kidney life span. The slope of allograft failure rate versus age parallels that of reduction in podocyte density versus age. Quantitative modeling projects allograft half-life at any donor age, and rate of podocyte detachment parallels the observed allograft loss rate. CONCLUSION: Native kidneys are designed to have a limited average life span of about 100-140 years. Allografts undergo an accelerated aging-like process that accounts for their unexpectedly short half-life (about 15 years), the observation that older donor age is associated with shorter allograft half-life, and the fact that long-term allograft survival has not substantially improved. Podometrics provides potential readouts for these processes, thereby offering new approaches for monitoring and intervention. FUNDING: National Institutes of Health.

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.

Proximal Tubules Have the Capacity to Regulate Uptake of Albumin.

Evidence from multiple studies supports the concept that both glomerular filtration and proximal tubule (PT) reclamation affect urinary albumin excretion rate. To better understand these roles of glomerular filtration and PT uptake, we investigated these processes in two distinct animal models. In a rat model of acute exogenous albumin overload, we quantified glomerular sieving coefficients (GSC) and PT uptake of Texas Red-labeled rat serum albumin using two-photon intravital microscopy. No change in GSC was observed, but a significant decrease in PT albumin uptake was quantified. In a second model, loss of endogenous albumin was induced in rats by podocyte-specific transgenic expression of diphtheria toxin receptor. In these albumin-deficient rats, exposure to diphtheria toxin induced an increase in albumin GSC and albumin filtration, resulting in increased exposure of the PTs to endogenous albumin. In this case, PT albumin reabsorption was markedly increased. Analysis of known albumin receptors and assessment of cortical protein expression in the albumin overload model, conducted to identify potential proteins and pathways affected by acute protein overload, revealed changes in the expression levels of calreticulin, disabled homolog 2, NRF2, angiopoietin-2, and proteins involved in ATP synthesis. Taken together, these results suggest that a regulated PT cell albumin uptake system can respond rapidly to different physiologic conditions to minimize alterations in serum albumin level.