Precision nephrology identified tumor necrosis factor activation variability in minimal change disease and focal segmental glomerulosclerosis.

The diagnosis of nephrotic syndrome relies on clinical presentation and descriptive patterns of injury on kidney biopsies, but not specific to underlying pathobiology. Consequently, there are variable rates of progression and response to therapy within diagnoses. Here, an unbiased transcriptomic-driven approach was used to identify molecular pathways which are shared by subgroups of patients with either minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS). Kidney tissue transcriptomic profile-based clustering identified three patient subgroups with shared molecular signatures across independent, North American, European, and African cohorts. One subgroup had significantly greater disease progression (Hazard Ratio 5.2) which persisted after adjusting for diagnosis and clinical measures (Hazard Ratio 3.8). Inclusion in this subgroup was retained even when clustering was limited to those with less than 25% interstitial fibrosis. The molecular profile of this subgroup was largely consistent with tumor necrosis factor (TNF) pathway activation. Two TNF pathway urine markers were identified, tissue inhibitor of metalloproteinases-1 (TIMP-1) and monocyte chemoattractant protein-1 (MCP-1), that could be used to predict an individual's TNF pathway activation score. Kidney organoids and single-nucleus RNA-sequencing of participant kidney biopsies, validated TNF-dependent increases in pathway activation score, transcript and protein levels of TIMP-1 and MCP-1, in resident kidney cells. Thus, molecular profiling identified a subgroup of patients with either MCD or FSGS who shared kidney TNF pathway activation and poor outcomes. A clinical trial testing targeted therapies in patients selected using urinary markers of TNF pathway activation is ongoing.

The first randomized controlled trial in pediatric nephrology: the history of the International Study of Kidney Disease in Children (ISKDC).

The International Study of Kidney Disease in Children (ISKDC), begun in 1966, conducted the first international collaborative randomized blinded controlled trial in pediatric nephrology and one of the first in either pediatrics or nephrology. For this trial, the ISKDC developed the criteria, such as those for response and relapse, used today to describe the clinical course of the nephrotic syndrome, and the trial generated the nephropathologic terminology and criteria which largely remain the current standards. Over an approximately 20-year span, the ISKDC followed the natural history and evaluated the therapeutic effectiveness of therapies in over 500 children with the nephrotic syndrome from three continents. It published 14 peer-reviewed studies and several reports and commentaries, many of which helped create current standards of practice for therapy of childhood nephrotic syndrome and consequently remain highly cited today. The ISKDC continues to be an important model for subsequent collaborative studies and was the impetus for the development of regional and national pediatric nephrology societies leading to the recognition and growth of pediatric nephrology as a separate subspecialty. A higher resolution version of the Graphical abstract is available as Supplementary information.

Effect of disease progression on the podocyte cell cycle in Alport Syndrome.

Progression of glomerulosclerosis is associated with loss of podocytes with subsequent glomerular tuft instability. It is thought that a diminished number of podocytes may be able to preserve tuft stability through cell hypertrophy associated with cell cycle reentry. At the same time, reentry into the cell cycle risks podocyte detachment if podocytes cross the G1/S checkpoint and undergo abortive cytokinesis. In order to study cell cycle dynamics during chronic kidney disease (CKD) development, we used a FUCCI model (fluorescence ubiquitination-based cell cycle indicator) of mice with X-linked Alport Syndrome. This model exhibits progressive CKD and expresses fluorescent reporters of cell cycle stage exclusively in podocytes. With the development of CKD, an increasing fraction of podocytes in vivo were found to be in G1 or later cell cycle stages. Podocytes in G1 and G2 were hypertrophic. Heterozygous female mice, with milder manifestations of CKD, showed G1 fraction numbers intermediate between wild-type and male Alport mice. Proteomic analysis of podocytes in different cell cycle phases showed differences in cytoskeleton reorganization and metabolic processes between G0 and G1 in disease. Additionally, in vitro experiments confirmed that damaged podocytes reentered the cell cycle comparable to podocytes in vivo. Importantly, we confirmed the upregulation of PDlim2, a highly expressed protein in podocytes in G1, in a patient with Alport Syndrome, confirming our proteomics data in the human setting. Thus, our data showed that in the Alport model of progressive CKD, podocyte cell cycle distribution is altered, suggesting that cell cycle manipulation approaches may have a role in the treatment of various progressive glomerular diseases characterized by podocytopenia.

Determinants of medication adherence in childhood nephrotic syndrome and associations of adherence with clinical outcomes.

BACKGROUND: Pediatric patients with nephrotic syndrome take medications long-term with significant toxicity and complex regimens, yet data on medication adherence are limited. METHODS: In a multicenter observational study of patients with nephrotic syndrome, NEPTUNE (NCT01209000), we surveyed caregivers of patients <19 years old and adolescent patients on medication adherence during longitudinal follow-up beginning in June 2015. Data extraction was in October 2020. We described the proportion of nonadherent patients at first survey. Participant social and economic factors, condition-related factors, therapy-related factors, and patient-related factors were examined for relationships with nonadherence by generalized linear mixed models using the longitudinal data. In exploratory fashion, we assessed the relationship between adherence and subsequent steroid response classification by binary logistic regression and adherence with healthcare utilization by Poisson regression. RESULTS: A total of 225 participants completed a median of 3 surveys during follow-up (IQR, 2-5), with a total of 743 surveys. Overall, 80 (36%) reported nonadherence with medications. In adjusted analysis, older age (per 1 year; OR 1.08; 95% CI, 1.03 1.12), lower maternal educational level (≥ high school vs. < high school; OR 0.47; 95% CI 0.25 to 0.89), and increased parent and self-identification of medications barriers (per 1 point; OR 1.57; 95% CI, 1.15-2.15) were significantly associated with nonadherence. No relationship between nonadherence and subsequent frequency of healthcare utilization was observed. A trend toward increased subsequent steroid resistance classification was seen with nonadherence, though not statistically significant. CONCLUSIONS: Medication nonadherence is common in pediatric nephrotic syndrome. Investigations into the use of surveys in the clinic setting to identify at-risk patients and ways to support families over time are needed. A higher resolution version of the Graphical abstract is available as Supplementary information.

APOL1 genotype-associated morphologic changes among patients with focal segmental glomerulosclerosis.

BACKGROUND: The G1 and G2 alleles of apolipoprotein L1 (APOL1) are common in the Black population and associated with increased risk of focal segmental glomerulosclerosis (FSGS). The molecular mechanisms linking APOL1 risk variants with FSGS are not clearly understood, and APOL1's natural absence in laboratory animals makes studying its pathobiology challenging. METHODS: In a cohort of 90 Black patients with either FSGS or minimal change disease (MCD) enrolled in the Nephrotic Syndrome Study Network (58% pediatric onset), we used kidney biopsy traits as an intermediate outcome to help illuminate tissue-based consequences of APOL1 risk variants and expression. We tested associations between APOL1 risk alleles or glomerular APOL1 mRNA expression and 83 light- or electron-microscopy traits measuring structural and cellular kidney changes. RESULTS: Under both recessive and dominant models in the FSGS patient subgroup (61%), APOL1 risk variants were significantly correlated (defined as FDR <0.1) with decreased global mesangial hypercellularity, decreased condensation of cytoskeleton, and increased tubular microcysts. No significant correlations were detected in MCD cohort. Independent of risk alleles, glomerular APOL1 expression in FSGS patients was not correlated with morphologic features. CONCLUSIONS: While APOL1-associated FSGS is associated with two risk alleles, both one and two risk alleles are associated with cellular/tissue changes in this study of FSGS patients. Our lack of discovery of a large group of tissue differences in FSGS and no significant difference in MCD may be due to the lack of power but also supports investigating whether machine learning methods may more sensitively detect APOL1-associated changes.

A molecular morphometric approach to diabetic kidney disease can link structure to function and outcome.

Diabetic kidney disease is the leading cause of kidney failure. However, studies of molecular mechanisms of early kidney damage are lacking. Here we examined for possible linkage between transcriptional regulation and quantitative structural damage in early diabetic kidney disease in Pima Indians with type 2 diabetes. Tissue obtained from protocol kidney biopsies underwent genome-wide compartment-specific gene expression profiling and quantitative morphometric analysis. The ultrastructural lesion most strongly associated with transcriptional regulation was cortical interstitial fractional volume (VvInt), an index of tubule-interstitial damage. Transcriptional co-expression network analysis identified 1843 transcripts that correlated significantly with VvInt. These transcripts were enriched for pathways associated with mitochondrial dysfunction, inflammation, migratory mechanisms, and tubular metabolic functions. Pathway network analysis identified IL-1ß as a key upstream regulator of the inflammatory response and five transcription factors cooperating with p53 to regulate metabolic functions. VvInt-associated transcripts showed significant correlation with the urine albumin to creatinine ratio and measured glomerular filtration rate 10 years after biopsy, establishing a link between the early molecular events and long-term disease progression. Thus, molecular mechanisms active early in diabetic kidney disease were revealed by correlating intrarenal transcripts with quantitative morphometry and long-term outcomes. This provides a starting point for identification of urgently needed therapeutic targets and non-invasive biomarkers of early diabetic kidney disease.

Urine metabolites are associated with glomerular lesions in type 2 diabetes.

INTRODUCTION: Little is known about the association of urine metabolites with structural lesions in persons with diabetes. OBJECTIVES: We examined the relationship between 12 urine metabolites and kidney structure in American Indians with type 2 diabetes. METHODS: Data were from a 6-year clinical trial that assessed renoprotective efficacy of losartan, and included a kidney biopsy at the end of the treatment period. Metabolites were measured in urine samples collected within a median of 6.5 months before the research biopsy. Associations of the creatinine-adjusted urine metabolites with kidney structural variables were examined by Pearson's correlations and multivariable linear regression after adjustment for age, sex, diabetes duration, hemoglobin A1c, mean arterial pressure, glomerular filtration rate (iothalamate), and losartan treatment. RESULTS: Participants (n = 62, mean age 45 ± 10 years) had mean ± standard deviation glomerular filtration rate of 137 ± 50 ml/min and median (interquartile range) urine albumin:creatinine ratio of 34 (14-85) mg/g near the time of the biopsy. Urine aconitic and glycolic acids correlated positively with glomerular filtration surface density (partial r = 0.29, P = 0.030 and r = 0.50, P < 0.001) and total filtration surface per glomerulus (partial r = 0.32, P = 0.019 and r = 0.43, P = 0.001). 2-ethyl 3-OH propionate correlated positively with the percentage of fenestrated endothelium (partial r = 0.32, P = 0.019). Citric acid correlated negatively with mesangial fractional volume (partial r=-0.36, P = 0.007), and homovanillic acid correlated negatively with podocyte foot process width (partial r=-0.31, P = 0.022). CONCLUSIONS: Alterations of urine metabolites may associate with early glomerular lesions in diabetic kidney disease.

Interstitial fibrosis scored on whole-slide digital imaging of kidney biopsies is a predictor of outcome in proteinuric glomerulopathies.

Background: Interstitial fibrosis (IF), tubular atrophy (TA) and interstitial inflammation (II) are known determinants of progression of renal disease. Standardized quantification of these features could add value to current classification of glomerulopathies. Methods: We studied 315 participants in the Nephrotic Syndrome Study Network (NEPTUNE) study, including biopsy-proven minimal change disease (MCD = 98), focal segmental glomerulosclerosis (FSGS = 121), membranous nephropathy (MN = 59) and IgA nephropathy (IgAN = 37). Cortical IF, TA and II were quantified (%) on digitized whole-slide biopsy images, by five pathologists with high inter-reader agreement (intra-class correlation coefficient >0.8). Tubulointerstitial messenger RNA expression was measured in a subset of patients. Multivariable Cox proportional hazards models were fit to assess association of IF with the composite of 40% decline in estimated glomerular filtration rate (eGFR) and end-stage renal disease (ESRD) and separately as well, and with complete remission (CR) of proteinuria. Results: IF was highly correlated with TA (P < 0.001) and II (P < 0.001). Median IF varied by diagnosis: FSGS 17, IgAN 21, MN 7, MCD 1 (P < 0.001). IF was strongly correlated with baseline eGFR (P < 0.001) and proteinuria (P = 0.002). After adjusting for clinical pathologic diagnosis, age, race, global glomerulosclerosis, baseline proteinuria, eGFR and medications, each 10% increase in IF was associated with a hazard ratio of 1.29 (P < 0.03) for ESRD/40% eGFR decline, but was not significantly associated with CR. A total of 981 genes were significantly correlated with IF (|r| > 0.4, false discovery rate (FDR) < 0.01), including upstream regulators such as tumor necrosis factor, interferon gamma (IFN-gamma), and transforming growth factor beta 1 (TGF-B1), and signaling pathways for antigen presentation and hepatic fibrosis. Conclusions: The degree of IF is associated with risk of eGFR decline across different types of proteinuric glomerulopathy, correlates with inflammatory and fibrotic gene expression, and may have predictive value in assessing risk of progression.

White blood cell fractions correlate with lesions of diabetic kidney disease and predict loss of kidney function in Type 2 diabetes.

Background: Inflammation linked to diabetic kidney disease (DKD) may affect white blood cell (WBC) counts and differentials. We examined the cross-sectional associations of total WBC count and WBC fractions with structural lesions of DKD in 108 Pima Indians with Type 2 diabetes who underwent research kidney biopsies. We also examined the longitudinal association of these WBC variables with renal function loss (RFL) in 941 Europeans with Type 2 diabetes from the SURDIAGENE study. Methods: Associations of WBC variables with morphometric parameters were assessed by linear regression. RFL was defined as≥40% loss of estimated glomerular filtration rate from baseline. Associations with RFL were evaluated by Cox regression. Hazard ratios (HRs) were reported per standard deviation increment of each WBC variable. Results: After multivariable adjustment, lymphocyte (r  = -0.20, P = 0.043) and eosinophil (r = 0.21, P = 0.032) fractions in the Pima Indians correlated with glomerular basement membrane width. Eosinophil fraction also correlated with glomerular filtration surface density (r  = -0.21, P = 0.031). Lymphocyte fraction (r = 0.25, P = 0.013), neutrophil fraction (r  = -0.23, P = 0.021) and the neutrophil:lymphocyte ratio (r  = -0.22, P = 0.024) correlated with percentage of normally fenestrated endothelial cells. During median follow-up of 4.5 years, 321 SURDIAGENE participants developed RFL. Lower lymphocyte fraction [HR = 0.67, 95% confidence interval (95% CI) 0.60-0.76] and higher neutrophil fraction (HR = 1.35, 95% CI 1.20-1.52), total WBC count (HR = 1.20, 95% CI 1.08-1.35) and neutrophil:lymphocyte ratio (HR = 1.44, 95% CI 1.28-1.62) each predicted RFL in this cohort. Conclusions: WBC fractions associate with morphometric lesions of DKD and predict RFL in individuals with Type 2 diabetes.

Mechanical challenges to the glomerulus and podocyte loss: evolution of a paradigm.

In this article, I shall outline some of the most important aspects of the evidentiary basis of the so-called Kriz model for the development of glomerular sclerosis, a model that we continue to modify to this day. In my mind, the most important findings include the fact that podocytes are generally post-mitotic cells, so that loss of a significant number for any cause leads to podocyte insufficiency. Another pivotal finding is that in many experimental models and in human disease, podocytes detach from the GBM as living cells. These facts, together with biomechanical deduction, have led to the ongoing evolution of the original Heidelberg model.

Combined use of electron microscopy and intravital imaging captures morphological and functional features of podocyte detachment.

The development of podocyte injury and albuminuria in various glomerular pathologies is still incompletely understood due to technical limitations in studying the glomerular filtration barrier (GFB) in real-time. We aimed to directly visualize the early morphological and functional changes of the GFB during the development of focal segmental glomerulosclerosis (FSGS) using a combination of transmission electron microscopy (TEM) and in vivo multiphoton microscopy (MPM) in the rat puromycin aminonucleoside (PAN) model. We hypothesized that this combined TEM + MPM experimental approach would provide a major technical improvement that would benefit our mechanistic understanding of podocyte detachment. Male Sprague-Dawley (for TEM) or Munich-Wistar-Frömter (for MPM) rats were given a single dose of 100-150 mg/kg body weight PAN i.p. and were either sacrificed and the kidneys processed for TEM or surgically instrumented for in vivo MPM imaging at various times 2-14 days after PAN administration. Both techniques demonstrated hypertrophy and cystic dilatations of the subpodocyte space that developed as early as 2-3 days after PAN. Adhesions of the visceral epithelium to the parietal Bowman's capsule (synechiae) appeared at days 8-10. TEM provided unmatched resolution of podocyte foot process remodeling, while MPM revealed the rapid dynamics of pseudocyst filling, emptying, and rupture, as well as endothelial and podocyte injury, misdirected filtration, and podocyte shedding. Due to the complementary advantages of TEM and MPM, this combined approach can provide an unusally comprehensive and dynamic portrayal of the alterations in podocyte morphology and function during FSGS development. The results advance our understanding of the role and importance of the various cell types, hemodynamics, and mechanical forces in the development of glomerular pathology.

Potential relevance of shear stress for slit diaphragm and podocyte function.

Filtrate flow through the glomerular barrier produces shear stresses that tend to disconnect podocytes from the glomerular basement membrane. Forces are highest within the filtration slits. The slit diaphragm mechanically balances the lateral components of the shear stresses on opposing foot processes, preventing widening of the slit.

Mechanical challenges to the glomerular filtration barrier: adaptations and pathway to sclerosis.

Podocytes are lost as viable cells by detachment from the glomerular basement membrane (GBM), possibly due to factors such as pressure and filtrate flow. Distension of glomerular capillaries in response to increased pressure is limited by the elastic resistance of the GBM. The endothelium and podocytes adapt to changes in GBM area. The slit diaphragm (SD) seems to adjust by shuttling SD components between the SD and the adjacent foot processes (FPs), resulting in changes in SD area that parallel those in perfusion pressure.Filtrate flow tends to drag podocytes towards the urinary orifice by shear forces, which are highest within the filtration slits. The SD represents an atypical adherens junction, mechanically interconnecting the cytoskeleton of opposing FPs and tending to balance the shear forces.If under pathological conditions, increased filtrate flows locally overtax the attachment of FPs, the SDs are replaced by occluding junctions that seal the slits and the attachment of podocytes to the GBM is reinforced by FP effacement. Failure of these temporary adaptive mechanisms results in a steady process of podocyte detachment due to uncontrolled filtrate flows through bare areas of the GBM and, subsequently, the labyrinthine subpodocyte spaces, presenting as pseudocysts. In our view, shear stress due to filtrate flow-not capillary hydrostatic pressure-is the major challenge to the attachment of podocytes to the GBM.

Integrative Genomics Identifies Novel Associations with APOL1 Risk Genotypes in Black NEPTUNE Subjects.

APOL1 variants have been associated with renal phenotypes in blacks. To refine clinical outcomes and discover mechanisms of APOL1-associated kidney injury, we analyzed clinical and genomic datasets derived from 90 black subjects in the Nephrotic Syndrome Study Network (NEPTUNE), stratified by APOL1 risk genotype. Ninety subjects with proteinuria ≥0.5 g/d were enrolled at first biopsy for primary nephrotic syndrome and followed. Clinical outcomes were determined, and renal histomorphometry and sequencing of Mendelian nephrotic syndrome genes were performed. APOL1 variants were genotyped, and glomerular and tubulointerstitial transcriptomes from protocol renal biopsy cores were analyzed for differential and correlative gene expression. Analyses were performed under the recessive model (high-risk genotype defined by two risk alleles). APOL1 high-risk genotype was significantly associated with a 17 ml/min per 1.73 m(2) lower eGFR and a 69% reduction in the probability of complete remission at any time, independent of histologic diagnosis. Neither APOL1 risk group was enriched for Mendelian mutations. On renal biopsy, high-risk genotype was associated with increased fractional interstitial area, interstitial fibrosis, and tubular atrophy. Risk genotype was not associated with intrarenal APOL1 mRNA expression levels. Differential expression analysis demonstrated an increased steady-state level of five genes associated with the high-risk genotype (CXCL9, CXCL11, and UBD in glomerulus; SNOR14B and MUC13 in tubulointerstitium). APOL1 tubulointerstitial coexpression analysis showed coexpression of APOL1 mRNA levels with a group of intrarenal transcripts that together were associated with increased interstitial fibrosis and tubular atrophy. These data indicate the high-risk APOL1 genotype confers renal risk across histopathologic diagnoses.

Fetal Serum ß2-Microglobulin and Postnatal Renal Function in Lower Urinary Tract Obstruction Treated with Vesicoamniotic Shunt.

INTRODUCTION: Although mortality has decreased for fetuses with lower urinary tract obstruction treated with vesicoamniotic shunt (VAS) placement, survivors remain at risk for long-term renal impairment. We tested the association of fetal serum ß2-microglobulin (fsß2M) with postnatal renal function in these patients, hypothesizing that fsß2M may predict such renal impairment. MATERIALS AND METHODS: fsß2M was obtained in patients undergoing VAS placement. The primary outcome was renal function at 3-12 months of life, as assessed by a pediatric nephrologist using medical records. Patients were divided into two groups: (1) 'stable renal function' - probable stable long-term renal function and reasonable growth - and (2) 'loss of renal function' - early loss of renal function and failure to thrive. RESULTS: Nineteen patients with preoperative fsß2M received a VAS. Of the 14 survivors, those with fsß2M ≤5.6 mg/l tended to have stable renal function compared to those with fsß2M >5.6 mg/l [5/6 (83.3%) vs. 2/8 (25.0%), OR = 15.00, 95% CI 0.70-709.89; p = 0.1026]. Eight of 9 patients followed for >12 months of age had outcomes consistent with the initial renal assessments. DISCUSSION: Patients with initial fsß2M >5.6 mg/l and treated with VAS tended to have poor renal outcomes.

Glomerular pathology and the progression of chronic kidney disease.

Structural studies of the glomerulus, largely undertaken in animal models, have informed our understanding of the progression of chronic kidney disease (CKD) for decades. A fundamental tenet of that understanding is that a loss of podocytes underlies progression in many or most cases of progressive CKD. Recent attempts have been made to reconcile earlier findings from glomerular physiology (the primacy of glomerular capillary hypertension in causation of secondary glomerular sclerosis) with structural findings and have suggested a more detailed model of the mechanisms underlying podocyte detachment as viable cells. A new appreciation of the main locus of mechanical challenges to the podocyte (in the filtration slit) may both explain the renoprotective action of some current therapies and help to suggest novel therapeutic strategies.

Tumor necrosis factor receptors 1 and 2 are associated with early glomerular lesions in type 2 diabetes.

Elevated serum tumor necrosis factor receptor 1 (TNFR1) and 2 (TNFR2) concentrations are strongly associated with increased risk of end-stage renal disease in type 2 diabetes. However, little is known about the early glomerular structural lesions that develop in patients when these markers are elevated. Here, we examined the relationships between TNFRs and glomerular structure in 83 American Indians with type 2 diabetes. Serum TNFRs and glomerular filtration rate (GFR, iothalamate) were measured during a research exam performed within a median of 0.9 months from a percutaneous kidney biopsy. Associations of TNFRs with glomerular structural variables were quantified by Spearman's correlations and by multivariable linear regression after adjustment for age, gender, diabetes duration, hemoglobin A1c, body mass index, and mean arterial pressure. The baseline mean age was 46 years, median GFR 130 ml/min, median albumin/creatinine ratio 26 mg/g, median TNFR1 1500 pg/ml, and median TNFR2 3284 pg/ml. After multivariable adjustment, TNFR1 and TNFR2 significantly correlated inversely with the percentage of endothelial cell fenestration and the total filtration surface per glomerulus. There were significant positive correlations with mesangial fractional volume, glomerular basement membrane width, podocyte foot process width, and percentage of global glomerular sclerosis. Thus, TNFRs may be involved in the pathogenesis of early glomerular lesions in diabetic nephropathy.

A potential role for mechanical forces in the detachment of podocytes and the progression of CKD.

Loss of podocytes underlies progression of CKD. Detachment of podocytes from the glomerular basement membrane (GBM) rather than apoptosis or necrosis seems to be the major mechanism of podocyte loss. Such detachment of viable podocytes may be caused by increased mechanical distending and shear forces and/or impaired adhesion to the GBM. This review considers the mechanical challenges that may lead to podocyte loss by detachment from the GBM under physiologic and pathophysiologic conditions, including glomerular hypertension, hyperfiltration, hypertrophy, and outflow of filtrate from subpodocyte spaces. Furthermore, we detail the cellular mechanisms by which podocytes respond to these challenges, discuss the protective effects of angiotensin blockade, and note the questions that must be addressed to better understand the relationship between podocyte detachment and progression of CKD.