Avi Z. Rosenberg

Background: Traditional immunohistochemistry (IHC) with chromogen detection has limited multiplex capacity, detecting at most 4 protein markers per tissue section simultaneously, thereby restricting comprehensive spatial analysis of valuable human biopsies. We developed and validated a robust serial IHC (sIHC) staining method to detect multiple antigens on a single kidney biopsy slide, maximizing data yield for diagnosing and studying complex kidney diseases. Methods: Formalin-fixed, paraffin-embedded kidney biopsy sections were subjected to repeated IHC/imaging cycles with antibody removal using an optimized sodium dodecyl sulfate-glycerol buffer stripping protocol. Images were then co-registered, and analysis was performed using a variety of methodologies, including color deconvolution, cell segmentation, and spatial clustering. Results: This optimized sIHC method successfully detected up to 20 antigens on a single slide. Combining image analysis and artificial intelligence software, for example with HALO (Indica Labs), the assay assembles high-dimensional images and enables quantitative histology and single-cell spatial analysis. Using this advanced method, we were able to identify rare cell populations, such as double-negative T cells, that are challenging to detect conventionally. Conclusion: We have developed a validated, high-capacity sIHC protocol that uses standard IHC procedures with commercially available, clinically validated off-the-shelf antibodies. This method is a valuable, cost-effective tool for obtaining extensive, high-dimensional single-cell-resolved spatial data from limited pathology samples, such as a human kidney biopsy.

BACKGROUND: Acute interstitial nephritis (AIN) is a common cause of acute kidney injury (AKI), but the diagnosis may be missed as kidney biopsies are rarely obtained when acute tubular injury (ATI) is suspected. METHODS: The Kidney Precision Medicine Project is a cohort study that obtains kidney biopsies from individuals with AKI, which undergo pathologic and molecular interrogation. We compared ATI and AIN cases among the first 60 AKI participants. RESULTS: On clinicopathologic adjudication, 30 patients (50%) had a primary adjudicated diagnosis of ATI, 13 (22%) patients had AIN, 9 (15%) had diabetic nephropathy, and 3 (5%) had other conditions. There were increased interstitial white blood cells and tubulitis ( P < 0.05 for both) in AIN compared with ATI. Prior to biopsy, the treating clinician suspected ATI in 83% of the cases with adjudicated ATI, while the treating clinician suspected AIN in 54% of the cases with AIN. Tissue transcriptomic signatures showed enrichment of proinflammatory signaling and increased expression of CXCL9 , a chemokine induced by IFN-γ, in myeloid cells of participants with AIN. CXCL9 localized to inflammatory infiltration in spatial transcriptomic data. CONCLUSION: Adjudication of kidney biopsies revealed distinct pathologic and molecular profiles between ATI and AIN. Kidney biopsy should be considered more frequently in AKI, as AIN is clinically underrecognized. TRIAL REGISTRATION: ClinicalTrials.gov NCT04334707. FUNDING: National Institute of Diabetes and Digestive and Kidney Diseases grants U01DK133081, U01DK133091, U01DK133092, U01DK133093, U01DK133095, U01DK133097, U01DK114866, U01DK114908, U01DK133090, U01DK133113, U01DK133766, U01DK133768, U01DK114907, U01DK114920, U01DK114923, U01DK114933, U24DK114886, UH3DK114926, UH3DK114861, UH3DK114915, and UH3DK114937.

Early diagnosis of cystinosis is critical to limit disease progression. YKL-40, a protein in the chitinase family, released by inflammatory cells, may be a useful biomarker for cystinosis. In a case-control study of 10 children with cystinosis and 20 without cystinosis, matched by age and baseline eGFR, we measured urine YKL-40, NGAL, and EGF. A lateral flow device (LFD) for YKL-40 was also developed and tested. Urine YKL-40 was over 200-fold higher in children with cystinosis (64.6 ng/mL [IQR: 23.4, 83.8]) compared with controls (0.3 [IQR: 0.3, 0.79]; P = 0.0001) with excellent diagnostic discrimination (AUC = 0.99) that was superior to other biomarkers. LFD measurements for YKL-40 showed similar results (AUC = 0.93). YKL-40 results were verified in 5 cystinosis patients, and YKL-40 staining was markedly higher in kidney biopsies from cystinosis patients than in healthy controls. Urine YKL-40 has excellent diagnostic potential for cystinosis, and point-of-care technologies may facilitate early screening and management of this disease.

Introduction: Heart failure with preserved ejection fraction (HFpEF) is strongly associated with cardiometabolic comorbidities, including obesity, diabetes, hypertension, chronic kidney disease and aging, yet the mechanistic contribution of cellular senescence to HFpEF pathogenesis remains poorly defined. Methods and Results: To model clinically relevant HFpEF, we subjected p16-3MR mice to a novel chronic “four-hit” cardiovascular-kidney-metabolic stress regimen (10 months of a high-fat diet, low-dose streptozotocin, L-NAME, and aging). These mice developed a robust HFpEF phenotype characterized by left ventricular hypertrophy, impaired diastolic function (reduced E′/A′ and elevated E/E′), preserved ejection fraction, reduced −dP/dt, exercise intolerance, pulmonary congestion, and increased cardiac CD68⁺ macrophage infiltration. Cardiac proteomics identified 821 proteins significantly altered by four-hit stress. Selective genetic ablation of p16⁺ senescent cells using ganciclovir ameliorated HFpEF phenotypes, reduced cardiac p16 expression and inflammation, and normalized proteomic remodeling, without affecting body weight or glycemic status. Comparative network analysis of mouse and human HFpEF cardiac proteomes revealed highly concordant upstream regulatory networks, prominently involving cell-cycle control, DNA damage responses, and inflammatory signaling. Immunohistochemical analysis of human HFpEF cardiac biopsies confirmed increased p16, γH2AX, STING, IRF3, NF-κB p65, and CD68⁺ macrophages, mirroring the murine findings. The 4-Hit mice also developed chronic diabetic kidney disease with increased kidney inflammation, both of which were attenuated by Senolytic therapy. Mechanistically, the cGAS-STING (cyclic GMP-AMP synthase – stimulator of interferon genes) is activated in response to damaged DNA, which in turn activates the downstream immune responses, including NF-κB and interferons. Cross-species validation further demonstrated that combined metabolic stress impaired cardiac function and nephrocyte function in Drosophila. Cardiac and nephrocyte dysfunctions were independently rescued by cardiomyocyte-specific and nephrocyte-specific inhibition of the cGAS-STING pathway, respectively. In human iPSC-derived cardiomyocytes, irradiation and palmitate induced senescence, DNA damage sensing via ZBP1, and activation of the cGAS-STING-IRF3 signaling axis; ZBP1 knockdown or senolytic treatment suppressed this inflammatory axis. Conclusions: Across mouse, human, fly, and human iPSC models, our findings identify DNA damage–driven senescence and ZBP1–cGAS–STING signaling as conserved, causal mechanisms linking cardiovascular-kidney-metabolic comorbidities to HFpEF, highlighting senescence and innate immune pathways as promising therapeutic targets.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are two interconnected clinical conditions, both defined by degree of functional impairment, but with heterogeneous clinical trajectories. Using new transcriptomic technologies, recent studies have described the cellular diversity in the healthy and injured kidney at the single cell level. Here, we used single nucleus transcriptomics to investigate the molecular diversity and commonalities in kidney biopsies from over 150 participants with AKI and CKD enrolled within the Kidney Precision Medicine Project (KPMP) and did so at the patient participant level. Using an unsupervised approach, we identified two multi-cellular programs associated with clinical and histopathological features of acute injury and chronic damage, respectively. We found that these programs are expressed across patients with AKI and CKD, supporting shared, rather than distinct, underlying molecular mechanisms. These programs capture tissue-level compositional changes towards adaptive and failed-repair states in tubular epithelial cells, as well as intra-cellular molecular changes characteristic of stress in all cell types. We identified subunits of the NFkB and AP-1 complexes, as well as members of the STAT family, as putative upstream regulators of the acute and chronic programs. We were able to map these continuous molecular measures of acute injury and chronic damage to urine and plasma protein profiles obtained at time of biopsy. These non-invasive protein signatures were predictive of renal outcomes in an independent cohort of 44 thousand participants from the UK biobank. In summary, unbiased identification of cellular programs in kidney disease biopsies defined molecular programs of injury cutting across conventional disease categorization and established a non-invasive molecular link to long term patient outcomes. Graphical

Key Points: Kidney biopsy remains the gold standard for diagnosis of kidney disease. Prebiopsy and postbiopsy diagnosis concordance is low for acute interstitial nephritis and acute tubular injury. Background: Percutaneous kidney biopsy remains the gold standard for evaluation of AKI, given that clinicians' prebiopsy clinical impression of AKI, on the basis of history, examination, and noninvasive testing may not lead to a unifying diagnosis. In this study, we evaluated the concordance of prebiopsy clinical diagnosis with postbiopsy final diagnosis among patients with clinical AKI. Methods: We leveraged data collected prospectively through the Novel Approaches in the Investigation of Kidney Disease Study between 2020 and 2023, in which adult patients admitted to the Johns Hopkins Hospital and scheduled for clinical kidney biopsies consented to provide biosamples paired with data collection. Up to three prebiopsy clinical diagnoses were recorded for each patient, along with up to three postbiopsy diagnoses, adjudicated by a study nephrologist postbiopsy. We investigated the concordance of prebiopsy and postbiopsy diagnoses among patients with suspected acute interstitial nephritis (AIN) or acute tubular injury (ATI). Results: Among 164 total participants, 29 patients had a suspected clinical diagnosis of AIN and 47 had a suspected clinical diagnosis of ATI prebiopsy. Among the participants with suspected AIN, only seven (24%) had AIN confirmed on biopsy. Of the 22 biopsies without AIN present, alternative diagnoses noted on histology included FSGS, ATI, and various glomerular diseases. Of 47 participants with suspected ATI, ATI was confirmed on biopsy in 27 (57%) participants. In the 20 biopsies without ATI present, alternative histological findings also included glomerular diseases, diabetic nephropathy, and FSGS predominantly. Conclusions: Patients with suspected ATI or AIN who undergo percutaneous kidney biopsy are often found to have alternative, significant findings present on histology. Among patients without relative or absolute contraindications, kidney biopsy remains an essential part of clinical evaluation, while future research should focus on the development of noninvasive approaches for kidney disease diagnosis.

Key Points: Used multiplex protein imaging to elucidate the intratubular cast components and the associated tubular alterations. Identified (Prominin-1/CD133), a dedifferentiation marker, as a major constituent of intratubular casts. Protein components within casts were associated with the injury of the surrounding tubular epithelium. Background: Kidney intratubular casts are frequently observed in the distal nephron segments of the kidney and have long been regarded as a sign of kidney disease. However, the composition and pathologic significance of intratubular casts have remained understudied. Methods: We leveraged Hematoxylin and Eosin (H&E) staining to identify intratubular casts along with concurrent codetection by indexing multiplexed spatial protein imaging on human kidney biopsy sections from the Kidney Precision Medicine Project. We also conducted immunoblotting of Prominin-1 (PROM1/CD133) in urine and assessed its levels from publicly available urinary proteomics datasets of the Kidney Precision Medicine Project consortium. Results: We analyzed 493 intratubular casts across 42 individuals with kidney disease or healthy controls. We identified PROM1 and insulin-like growth factor binding protein 7 as major constituents of casts (positive staining in 89.0% and 39.1%, respectively). Staining for uromodulin, an established cast component, was present in 86.6%. These components showed variable patterns across disease states. Intratubular casts were predominantly detected in the distal nephron segments, and their presence was associated with a marked loss of sodium-chloride cotransporter and aquaporin-2 expression in the cast-containing tubular epithelium, suggesting underlying injury. The loss of these transporters correlated with protein components within casts, and the presence of intracast PROM1 showed the strongest association, with an odds ratio of 26.7 (95% confidence interval, 13.1 to 54.7). Urinary PROM1 secretion was confirmed by immunoblotting and was greater in patients with AKI compared with healthy controls ( P = 0.01). Conclusions: We identified PROM1, a dedifferentiation and injury marker expressed in epithelial cells, as a novel major constituent of intratubular casts. Our studies suggest that protein composition signature within casts varies with disease state and is associated with tubular injury in distal nephron segments. Our study also suggests that urinary PROM1 may have potential as a biomarker for AKI. Podcast: This article contains a podcast at https://dts.podtrac.com/redirect.mp3//www.asn-online.org/media/podcast/JASN/2026_10_22_KTS_October2026.mp3

Mitochondrial dysfunction is a hallmark of aging and a key contributor to age-related diseases including cardiovascular disease. However, molecular pathways that safeguard mitochondrial homeostasis in the aging heart remain poorly understood. Here, we identify MTFR1L as a regulator of mitophagy that binds p-S65-Ub, a key signal amplifying the PINK1/Parkin axis. We find that MTFR1L is enriched in metabolically active tissues, particularly in the heart, where it regulates Parkin signaling. Genetic deletion of Mtfr1l in mice impairs stress-induced mitophagy and Parkin activation, leading to accumulation of damaged mitochondria, increased inflammation and senescence, and accelerated age-related cardiac dysfunction. Strikingly, cardiac expression of MTFR1L progressively decreases along with aging in mice, primates, and humans, coinciding with cardiomyocyte senescence and lipofuscin accumulation. Together, these findings uncover a role for MTFR1L in regulation of the p-S65-Ub/Parkin mitophagy axis and maintenance of mitochondrial homeostasis during cardiac aging and suggest that age-associated loss of MTFR1L may contribute to age-related cardiac dysfunction. Based on these findings, we propose a therapeutic paradigm for the prevention of heart aging by restoring MTFR1L function.