Alexander, Mariam P.

Key Points Increased protein expression of tissue inhibitor of metalloproteinase-3, glypican 6, synuclein γ , apolipoprotein A-4, and Ecto-5′-nucleotidase was observed in old adults compared with young adults. In old adults, overexpressed proteins were enriched in mitochondrial processes while under expressed proteins enriched in RNA metabolism. Validated differentially expressed proteins were not significantly correlated with kidney pathology after adjusting for age. Background Kidney aging is characterized by a loss of glomeruli, predominantly in the superficial cortex, with a resultant decline in GFR and an increased risk of various kidney-related diseases. The early molecular alterations in glomeruli associated with the aging process are not well studied. Methods We combined laser capture microdissection and mass spectrometry–based unbiased proteomic analysis of nonsclerosed, nonischemic glomeruli in the superficial cortex from young and old adults who underwent a radical nephrectomy for a tumor to understand the age-related molecular changes in glomeruli. Twenty-four young and 30 old adults were used for the discovery dataset, and the significant differentially expressed proteins were further validated using an independent set comprising six young and eight old adults. Results Kidneys from old adults had lower eGFR, less kidney parenchyma on computed tomography imaging, and more glomerulosclerosis and arteriosclerosis on histology. Quantitative proteomic analysis of nonsclerosed, nonischemic glomeruli identified increased expression of tissue inhibitor of metalloproteinase-3, glypican 6, synuclein γ , apolipoprotein A-4, and Ecto-5′-nucleotidase in old adults that were further validated in an independent set. Pathway analysis indicated that proteins with increased expression in old adults were enriched in mitochondrial translational processes, aerobic respiration, and TCA cycle, whereas proteins with decreased expression in old adults were enriched in mRNA splicing, mRNA processing, and nonsense mediated decay. Furthermore, validated differentially expressed proteins did not show any significant correlation with the kidney pathology independent of age group. Conclusions Overall, this study identified proteins that are specifically associated with the aging process in otherwise normal-appearing glomeruli.

Significance Statement Glomerular size differs by cortex depth. Larger nephrons are prognostic of progressive kidney disease, but it is unknown whether this risk differs by cortex depth or by glomeruli versus proximal or distal tubule size. We studied the average minor axis diameter in oval proximal and distal tubules separately and by cortex depth in patients who had radical nephrectomy to remove a tumor from 2019 to 2020. In adjusted analyses, larger glomerular volume in the middle and deep cortex predicted progressive kidney disease. Wider proximal tubular diameter did not predict progressive kidney disease independent of glomerular volume. Wider distal tubular diameter showed a gradient of strength of prediction of progressive kidney disease in the more superficial cortex than in the deep cortex. Background Larger nephrons are prognostic of progressive kidney disease, but whether this risk differs by nephron segments or by depth in the cortex is unclear. Methods We studied patients who underwent radical nephrectomy for a tumor between 2000 and 2019. Large wedge kidney sections were scanned into digital images. We estimated the diameters of proximal and distal tubules by the minor axis of oval tubular profiles and estimated glomerular volume with the Weibel–Gomez stereological model. Analyses were performed separately in the superficial, middle, and deep cortex. Cox proportional hazard models assessed the risk of progressive CKD (dialysis, kidney transplantation, sustained eGFR <10 ml/min per 1.73 m 2 , or a sustained 40% decline from the postnephrectomy baseline eGFR) with glomerular volume or tubule diameters. At each cortical depth, models were unadjusted, adjusted for glomerular volume or tubular diameter, and further adjusted for clinical characteristics (age, sex, body mass index, hypertension, diabetes, postnephrectomy baseline eGFR, and proteinuria). Results Among 1367 patients were 62 progressive CKD events during a median follow-up of 4.5 years. Glomerular volume predicted CKD outcomes at all depths, but only in the middle and deep cortex after adjusted analyses. Proximal tubular diameter also predicted progressive CKD at any depth but not after adjusted analyses. Distal tubular diameter showed a gradient of more strongly predicting progressive CKD in the superficial than deep cortex, even in adjusted analysis. Conclusions Larger glomeruli are independent predictors of progressive CKD in the deeper cortex, whereas in the superficial cortex, wider distal tubular diameters are an independent predictor of progressive CKD.

Significance Statement Nephrosclerosis (glomerulosclerosis, interstitial fibrosis, and tubular atrophy) is the defining pathology of both kidney aging and CKD. Optimal thresholds for nephrosclerosis that identify persons with a progressive disease are unknown. This study determined a young-age threshold (18–29 years) and age-based 95th percentile thresholds for nephrosclerosis on the basis of morphometry of kidney biopsy sections from normotensive living kidney donors. These thresholds were 7.1-fold to 36-fold higher in older (70 years or older) versus younger (aged 18–29 years) normotensive donors. Age-based thresholds, but not young-age threshold, were prognostic for determining risk of progressive CKD among patients who underwent a radical nephrectomy or a for-cause native kidney biopsy, suggesting that age-based thresholds are more useful than a single young-age threshold for identifying CKD on biopsy. Background Nephrosclerosis, defined by globally sclerotic glomeruli (GSG) and interstitial fibrosis and tubular atrophy (IFTA), is a pathology of both kidney aging and CKD. A comparison of risk of progressive CKD using aged-based thresholds for nephrosclerosis versus a single young-adult threshold is needed. Methods We conducted morphometric analyses of kidney biopsy images for %GSG, %IFTA, and IFTA foci density among 3020 living kidney donors, 1363 patients with kidney tumor, and 314 patients with native kidney disease. Using normotensive donors, we defined young-age thresholds (18–29 years) and age-based (roughly by decade) 95th percentile thresholds. We compared age-adjusted risk of progressive CKD (kidney failure or 40% decline in eGFR) between nephrosclerosis that was “normal compared with young,” “normal for age but abnormal compared with young,” and “abnormal for age” in patients with tumor and patients with kidney disease. Results The 95th percentiles in the youngest group (18–29 years) to the oldest group (70 years or older) ranged from 1.7% to 16% for %GSG, 0.18% to 6.5% for %IFTA, and 8.2 to 59.3 per cm 2 for IFTA foci density. Risk of progressive CKD did not differ between persons with nephrosclerosis “normal compared with young” versus “normal for age but abnormal compared with young.” Risk of progressive CKD was significantly higher with %GSG, %IFTA, or IFTA foci density that was abnormal versus normal for age in both cohorts. Conclusions Given that increased risk of progressive CKD occurs only when nephrosclerosis is abnormal for age, age-based thresholds for nephrosclerosis seem to be better than a single young-age threshold for identifying clinically relevant CKD.

Significance Statement Nephron number currently can be estimated only from glomerular density on a kidney biopsy combined with cortical volume from kidney imaging. Because of measurement biases, refinement of this approach and validation across different patient populations have been needed. The prognostic importance of nephron number also has been unclear. The authors present an improved method of estimating nephron number that corrects for several biases, resulting in a 27% higher nephron number estimate for donor kidneys compared with a prior method. After accounting for comorbidities, the new nephron number estimate does not differ between kidney donors and kidney patients with tumor and shows consistent associations with clinical characteristics across these two populations. The findings also indicate that low nephron number predicts CKD independent of biopsy and clinical characteristics in both populations. Background Nephron number can be estimated from glomerular density and cortical volume. However, because of measurement biases, this approach needs refinement, comparison between disparate populations, and evaluation as a predictor of CKD outcomes. Methods We studied 3020 living kidney donors and 1354 patients who underwent radical nephrectomy for tumor. We determined cortex volume of the retained kidney from presurgical imaging and glomerular density by morphometric analysis of needle core biopsy of the donated kidney and wedge sections of the removed kidney. Glomerular density was corrected for missing glomerular tufts, absence of the kidney capsule, and then tissue shrinkage on the basis of analysis of 30 autopsy kidneys. We used logistic regression (in donors) and Cox proportional hazard models (in patients with tumor) to assess the risk of CKD outcomes associated with nephron number. Results Donors had 1.17 million nephrons per kidney; patients with tumor had 0.99 million nephrons per kidney. A lower nephron number was associated with older age, female sex, shorter height, hypertension, family history of ESKD, lower GFR, and proteinuria. After adjusting for these characteristics, nephron number did not differ between donors and patients with tumor. Low nephron number (defined by <5th or <10th percentile by age and sex in a healthy subset) in both populations predicted future risk of CKD outcomes independent of biopsy and clinical characteristics. Conclusions Compared with an older method for estimating nephron number, a new method that addresses several sources of bias results in nephron number estimates that are 27% higher in donors and 1% higher in patients with tumor and shows consistency between two populations. Low nephron number independently predicts CKD in both populations. Podcast This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_06_22_JASN0000000000000124.mp3

Significance Statement Glomerular volume, ischemic glomeruli, and global glomerulosclerosis are not consistently assessed on kidney transplant biopsies. The authors evaluated morphometric measures of glomerular volume, the percentage of global glomerulosclerosis, and the percentage of ischemic glomeruli and assessed changes in these measures over time to determine whether such changes predict late allograft failure. All three features increased from transplant to five-year biopsy. Kidneys with smaller glomeruli at 5 years had more global glomerulosclerosis and a higher percentage of ischemic-appearing glomeruli. Smaller glomeruli and increasing percentages of global glomerulosclerosis and ischemic glomeruli at 5 years predicted allograft failure. Only increased percentage of ischemic glomeruli predicted allograft failure at 5 years independent of all Banff scores. Glomerular changes reflect pathologic processes that predicted allograft loss; measuring them quantitatively might enhance the current Banff system and provide biomarkers for intervention trials. Background Histology can provide insight into the biology of renal allograft loss. However, studies are lacking that use quantitative morphometry to simultaneously assess changes in mean glomerular volume and in the percentages of globally sclerosed glomeruli (GSG) and ischemic-appearing glomeruli in surveillance biopsies over time to determine whether such changes are correlated with late graft failure. Methods We used digital scans of surveillance biopsies (at implantation and at 1 and 5 years after transplantation) to morphometrically quantify glomerular volume and the percentages of GSG and ischemic-appearing glomeruli in a cohort of 835 kidney transplants. Cox proportional hazards models assessed the risk of allograft failure with these three glomerular features. Results From implantation to 5 years, mean glomerular volume increased by nearly 30% (from 2.8×10 6 to 3.6×10 6 µm 3 ), mean percentage of GSG increased from 3.2% to 13.2%, and mean percentage of ischemic-appearing glomeruli increased from 0.8% to 9.5%. Higher percentages of GSG and ischemic-appearing glomeruli at 5-year biopsy predicted allograft loss. The three glomerular features at 5-year biopsy were related; the percentage of GSG and the percentage of ischemic glomeruli were positively correlated, and both were inversely correlated to glomerular volume. At 5 years, only 5.3% of biopsies had ≥40% ischemic glomeruli, but 45% of these grafts failed (versus 11.6% for <40% ischemic glomeruli). Higher Banff scores were more common with increasing percentages of GSG and ischemia, but at 5 years, only the percentage of ischemic glomeruli added to predictive models adjusted for Banff scores. Conclusions Glomerular changes reflect important pathologic processes that predict graft loss. Measuring glomerular changes quantitatively on surveillance biopsies, especially the proportion of ischemic-appearing glomeruli, may enhance the current Banff system and be a useful surrogate end point for clinical intervention trials. Podcast This article contains a podcast at

Background Membranous nephropathy (MN) is a common cause of proteinuria in patients receiving a hematopoietic stem cell transplant (HSCT). The target antigen in HSCT-associated MN is unknown. Methods We performed laser microdissection and tandem mass spectrometry (MS/MS) of glomeruli from 250 patients with PLA2R-negative MN to detect novel antigens in MN. This was followed by immunohistochemical (IHC)/immunofluorescence (IF) microscopy studies to localize the novel antigen. Western blot analyses using serum and IgG eluted from frozen biopsy specimen to detect binding of IgG to new 'antigen'. Results MS/MS detected a novel protein, protocadherin FAT1 (FAT1), in nine patients with PLA2R-negative MN. In all nine patients, MN developed after allogeneic HSCT (Mayo Clinic discovery cohort). Next, we performed MS/MS in five patients known to have allogeneic HSCT-associated MN (Cedar Sinai validation cohort). FAT1 was detected in all five patients by MS/MS. The total spectral counts for FAT1 ranged from 8 to 39 (mean±SD, 20.9±10.1). All 14 patients were negative for known antigens of MN, including PLA2R, THSD7A, NELL1, PCDH7, NCAM1, SEMA3B, and HTRA1. Kidney biopsy specimens showed IgG (2 to 3+) with mild C3 (0 to 1+) along the GBM; IgG4 was the dominant IgG subclass. IHC after protease digestion and confocal IF confirmed granular FAT1 deposits along the GBM. Lastly, Western blot analyses detected anti-FAT1 IgG and IgG4 in the eluate obtained from pooled frozen kidney biopsy tissue and in the serum of those with FAT1-asssociated MN, but not from those with PLA2R-associated MN. Conclusions FAT1-associated MN appears to be a unique type of MN associated with HSCT. FAT1-associated MN represents a majority of MN associated with HSCT.

Significance Statement Although corticosteroids are an effective first-line therapy for minimal change disease, relapse, steroid dependence, and intolerance are common in this podocytopathy of unknown etiology. The efficacy of B cell–targeted therapies in some patients suggests an autoantibody-mediated etiology. This study describes the novel discovery in both adults and children with minimal change disease of autoantibodies targeting nephrin, a critical component of the podocyte slit diaphragm that ensures integrity of the glomerular filtration barrier. This observation aligns with the established proteinuric effect of antinephrin antibodies demonstrated in animal models. These findings identify an important autoimmune mechanism in a subset of patients with minimal change disease and provide a framework for the application and development of precision medicine strategies in this condition. Background Failure of the glomerular filtration barrier, primarily by loss of slit diaphragm architecture, underlies nephrotic syndrome in minimal change disease. The etiology remains unknown. The efficacy of B cell–targeted therapies in some patients, together with the known proteinuric effect of anti-nephrin antibodies in rodent models, prompted us to hypothesize that nephrin autoantibodies may be present in patients with minimal change disease. Methods We evaluated sera from patients with minimal change disease, enrolled in the Nephrotic Syndrome Study Network (NEPTUNE) cohort and from our own institutions, for circulating nephrin autoantibodies by indirect ELISA and by immunoprecipitation of full-length nephrin from human glomerular extract or a recombinant purified extracellular domain of human nephrin. We also evaluated renal biopsies from our institutions for podocyte-associated punctate IgG colocalizing with nephrin by immunofluorescence. Results In two independent patient cohorts, we identified circulating nephrin autoantibodies during active disease that were significantly reduced or absent during treatment response in a subset of patients with minimal change disease. We correlated the presence of these autoantibodies with podocyte-associated punctate IgG in renal biopsies from our institutions. We also identified a patient with steroid-dependent childhood minimal change disease that progressed to end stage kidney disease; she developed a massive post-transplant recurrence of proteinuria that was associated with high pretransplant circulating nephrin autoantibodies. Conclusions Our discovery of nephrin autoantibodies in a subset of adults and children with minimal change disease aligns with published animal studies and provides further support for an autoimmune etiology. We propose a new molecular classification of nephrin autoantibody minimal change disease to serve as a framework for instigation of precision therapeutics for these patients.