Digital technology for personalised management and therapy of hypertensive nephropathy

Hypertensive nephropathy: Transcriptomics of kidney biopsies predicts long-term outcome and identifies therapeutic targets!1. Successful RNA sequencing of archival kidney biopsies with hypertensive nephropathy; see below. Also, we have introduced kidney-on-chip technology to create a model of hypertensive nephropathy. 2. We have completed and published two introductory/pilot projects on hypertensive nephropathy connected to this main HV project, see "Research papers".Objective: Hypertensive nephropathy (HN) represents a major cause of chronic kidney disease, but it is incompletely understood why some patients show disease progression and others don’t. Our project aim is to identify potential markers of disease progression and novel treatment targets. Design and Method: Adult patients (n=50; n=36 males, n=14 females, mean age 53 years) with biopsy-verified HN were categorized as "early" (estimated glomerular filtration rate (eGFR) >45 ml/min/1.73m2) or "late" disease (eGFR ≤45 ml/min/1.73m2) at the time of biopsy. Patients were divided further into “stable” (eGFR decline <3 ml/year) or “progressive” (eGFR decline ≥3 ml/year or start of renal replacement therapy) after median follow-up of 9 years (5-24). TruSeq Exome sequencing was executed after RNA extraction (miRNeasy FFPE kit, Qiagen) at Novogene, Cambridge, UK. Quality control and data analysis was performed using R Studio (v4.2.0) and QIAGEN Ingenuity Pathway Analysis. Results: We divided and analyzed our patients in four different groups: n=12 early stable (ES), n=13 early progressive (EP), n=9 late stable (LS), and n=16 late progressive (LP) HN patients. Differentially expressed genes (DEG, log2fold change (FC) ≥1.25 and p-value < 0.05) were discovered as follows: n=345 in all stable vs. all progressive HN, n=701 in ES vs. EP, and n=883 in LS vs. LP. Principal component analysis (PCA) showed almost complete separation of ES vs. EP and LS vs. LP. K-nearest neighbour (KNN) analysis of DEG identified a 10-gene classifier in LS vs. LP (19/25 samples correctly classified), while ARHGAP5-AS1 was best as a single-gene classifier in ES vs. EP (19/25 samples). Classifier genes like RNF152, ARHGAP5-AS1, AQP1, USP2 and TMED1 as well as other DEG, such as MMP-9, SELL and HCAR3, may also represent novel treatment targets. Differentially regulated pathways were related to collagen biosynthesis in ES vs. EP, and to cell cycle in LS vs. LP. Conclusions: Transcriptomic profiling from diagnostic kidney biopsies with HN can distinguish future disease progression from non-progression and potentially detect novel therapeutic targets.

Patient-Tailored Management of Hypertensive Nephropathy through Digital TechnologiesDuring the last year we have made progress regarding the completion of a pilot study on glomerular proteomics of patients with stable or progressive hypertensive kidney injury and with respect to patient inclusion into the main project. Moreover, habe have bought the necessary equipment to establish the kidney-on-chip technology in our laboratory.Specifically, during the year of 2022, we have achieved the following: 1. Investigation completed on glomerular proteomics in a limited number of patients with hypertensive nephropathy (Work package-1; WP-1). This work has been presented at the 29th Scientific Meeting of the International Society of Hypertension (ISH) 2022 in Kyoto, Japan, and has been awarded the Best Oral Presentation Diamond Award. Subsequent publication in Hypertension Research; see summary given just below. 2. Patient identification and selection for study inclusion to perform RNA sequencing from their kidney biopsies. RNA from these biopsies has been extracted and its sequencing is currently being attempted (WP-1). 3. Patients with hypertension identified and selected from the HUNT studies in Norway for future serum analyses (WP-1). 4. Kidney-on-chip technology introduced to our laboratory (WP-3). Summary of Results: 1. Glomerular proteomic profiling of kidney biopsies with hypertensive nephropathy reveals a signature of disease progression (Hypertens Res, Epub October 14, 2022) Hypertensive nephropathy (HN) requires a kidney biopsy as diagnostic gold-standard but histological findings are unspecific and specific prognostic markers are missing. We aimed at identifying candidate prognostic markers based on glomerular protein signatures. We studied adult patients (n = 17) with eGFR >30 ml/min/1.73m2 and proteinuria <3 g/d from the Norwegian Kidney Biopsy Registry, including subjects non progressing (NP, n = 9), or progressing (P, n = 8) to end-stage renal disease (ESRD) within an average follow-up of 22 years. Glomerular cross-sections from archival kidney biopsy sections were microdissected and processed for protein extraction. Proteomic analyses were performed using Q-exactive HF mass spectrometer and relative glomerular protein abundances were compared between P and NP patients. Immunohistochemistry (IHC) was used to validate selected data. Amongst 1870 quality filtered proteins, 58 were differentially expressed in P and NP patients' glomeruli, with absolute fold changes (FC) ≥1.5, p ≤ 0.05. Supervised classifier analysis (K nearest neighbor) identified a set of five proteins, including Gamma-butyrobetaine dioxygenase (BBOX1, O75936) and Cadherin 16 (CDH16, O75309), overexpressed in P, and Eosinophil peroxidase (EPX, P11678), DnaJ homolog subfamily B member 1 (DNAJB1, P25685) and Alpha-1-syntrophin (SNTA1, Q13424), overexpressed in NP glomeruli, correctly classifying 16/17 kidney biopsy samples. Geneset Enrichment Analysis (GSEA), showed that metabolic pathways were generally enriched in P, and structural cell pathways in NP. Pathway analysis identified Epithelial Adherens Junction Signaling as most affected canonical pathway. IHC analysis confirmed overexpression of BBOX1 and Cadherin 16 in glomeruli from P patients. In conclusion, glomerular proteomic profiling can be used to discriminate P from NP HN patients.

PATIENT-TAILORED MANAGEMENT OF HYPERTENSIVE NEPHROPATHY THROUGH DIGITAL TECHNOLOGIESMain Results and Milestones 1. Pilot study: Glomerular proteomic profiling of kidney biopsies with hypertensive nephropathy reveals a signature of disease progression. 2. Personnel: We have appointed Øystein Eikrem and Lea Landolt, who share a postdoctoral position. 3. Main Project: 3a) Identification of patients. 3b) Establishing new technology.Summary 1. Pilot Study: Glomerular proteomic profiling of kidney biopsies with hypertensive nephropathy reveals a signature of disease progression. We plan to resubmit this study for publication during spring of 2022. Background: We aimed at identifying prognostic candidate markers for hypertensive nephropathy based on glomerular protein signatures. Methods: We studied adult patients (n=17) with an estimated glomerular filtration rate (eGFR) >30 ml/min/1.73m2 and proteinuria <3g/d from the Norwegian Kidney Biopsy Registry, including stable non-progressive patients (n=9) and patients progressing (n=8) to end-stage renal disease within 20 years. Glomerular cross-sections from archival kidney biopsy sections were microdissected and processed for protein extraction and proteomic analyses. Results: Amongst 1870 quality filtered proteins, 58 were differentially expressed in progressive and non-progressive glomerular samples, with absolute fold changes >1.5, p>0.05. By using 17 glomerular proteins with absolute fold changes >2 and p<0.05, hierarchical clustering and principal component analysis effectively separated progressors and non-progressors. Supervised classifier analysis (K nearest neighbour) identified a set of five proteins, including Gamma-butyrobetaine dioxygenase (BBOX1, O75936) and Cadherin 16 (CDH16, O75309), overexpressed in progressors, and Eosinophil peroxidase (EPX, P11678), DnaJ homolog subfamily B member 1 (DNAJB1, P25685) and Alpha-1-syntrophin (SNTA1, Q13424), overexpressed in non-progressive glomeruli, correctly classifying 16/17 samples. Geneset Enrichment Analysis showed that metabolic pathways were enriched in progressors, and structural cell pathways in non-progressors. Pathway analysis identified Epithelial Adherens Junction Signaling as most affected canonical pathway. Conclusion: Glomerular proteomic profiling can be used to discriminate progressive from non-progressive patients with HN. 2. Personnell We have advertised and hired Øystein Eikrem, PhD, and Lea Landolt, PhD, from our institution in spring 2021. They share the 100% postdoctoral position equally (50% each). Furthermore, we have also appointed Marius Altern Øvrehus in a 30% postdoctoral position at St. Olav Sykehus/NTNU in Trondheim. 3a. Identification of patients The correct identification of patients with their respective kidney biopsies represents one of the crucial steps of this research proposals. We are based on data from the Norwegian Renal Registry, including its Norwegian KidneyBiopsy registry (NBKR), and from the electronic patient charts (DIPS). The definition of patients with stable/advanced and stable/progressive hypertensive nephropathy (n=60) and the respective control groups (n=30) over a prolonged follow-up period of 10 years or longer requires precise, intense work by the postdoctoral fellows and is very time-consuming. We hope to have this work accomplished in summer 2022. 3b. Establishing kidney-on-chip technology in our laboratory During the fall of 2021 we have acquired the proposed kidney-on-chip equipment from Emulate AS, Boston, Ma, USA. Ole Patter Nordbø, PhD scholar, and Jessica Furriol, PhD, are in the process of fully establishing this technology. Unfortunately, this process has been slowed down due to the current corona pandemic regarding shipment of replacement parts and solutions as well of visits/tutorials from the vendor to help with installations. We hope to have this technology up and running in summer of 2022.

PATIENT-TAILORED MANAGEMENT OF HYPERTENSIVE NEPHROPATHY THROUGH DIGITAL TECHNOLOGIESWe have almost completed two studies on RNA sequencing and proteomics of kidney biopsies with hypertensive nephropathy as a preparation for the main project proposal. Results are reported below. With regard to the main project, we have started to select patients from the Norwegian Kidney Biopsy Registry and to send out informed consent letters.1. PROTEOMIC PROFILING OF GLOMERULI FROM KIDNEYS WITH HYPERTENSIVE NEPHROPATHY REVEALS SIGNATURE OF DISEASE PROGRESSION. Introduction: Kidney biopsy-based findings of hypertensive nephropathy (HN) are not specific, and prognosis is difficult to determine. We aimed at defining protein markers for disease progression. Methods: We included adult HN patients (n=17) with eGFR >30 ml/min/1.73m2 and proteinuria <3g/d from the Norwegian Kidney Biopsy Registry, divided into patients with stable HN (n=9) and with progressive HN (n=8) leading to end-stage renal disease within 20 years. Glomerular cross-sections were microdissected from 10 μm whole archival kidney biopsy sections for proteomic analyses using a Q-exactive HF mass spectrometer. Results: Amongst 1870 quality filtered proteins, we identified 58 proteins with absolute fold change (FC) >1.5, p<0.05, including 17 proteins with absolute FC >2, indicative of HN progression. Hierarchical cluster and principal component analysis (PCA) with these 17 proteins showed a clear sample separation into two clusters. Regarding prognostic biomarkers, a set of five proteins (incl. cadherin 16) performed best and separated the two groups in PCA. This classifier classified 16 of 17 samples correctly (AUC 0.993). Applying Geneset Enrichment Analysis, metabolic pathways were up-regulated in progressors, and structural cell pathways up-regulated in non-progressors. Ingenuity Pathway Analysis identified Epithelial Adherens Junction Signaling as the most affected canonical pathway; five of six member proteins were down-regulated in progressors. Conclusion: Glomerular proteomic profiling differentiates HN progressors from non-progressors. Publication: Manuscript in preparation. 2. PARTIAL EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) AND T CELL-MEDIATED INFLAMMATION IN HYPERTENSIVE AND DIABETIC NEPHROPATHY Objective: We wanted to investigate to what degree patients with hypertensive or diabetic nephropathy show evidence of concurrent inflammation and/or partial epithelial-to-mesenchymal transition (EMT) in their renal biopsies. Methods: Formalin-fixed and paraffin-embedded kidney biopsies from adult patients were selected from the Norwegian Kidney Biopsy Registry. The following three groups (n=6 biopsies per group) were chosen: hypertensive nephropathy (HN), type 2 diabetic nephropathy (T2DN) and normal-appearing renal biopsy tissue as controls. Total RNA was extracted from 10 μm whole kidney biopsy sections and processed for RNA sequencing, while immunohistochemistry (IHC) was performed on 3-μm tissue sections. Results: Principal component analysis separated diseased and normal tissue, with overlap between HN and T2DN. Gene expression profiling revealed activation of T-helper type 1 (TH1) cells and CD8+ cytotoxic T-cells, as well as the upregulation of the complement system in both diseases. IHC confirmed presence of CD8+ and CD4+ cells as part of T-cell–mediated inflammation as well as complement factors, such as C3 and C5b-9. EMT-related genes were upregulated in both diseases with confirmation of AXL and vimentin by IHC. All shared transcripts between HN and T2DN were regulated in the same direction. Conclusion: HN and T2DN show increased mesenchymal and inflammatory phenotype related to TH1, CD8+ cytotoxic inflammation and to the complement system. HN and T2DN are very homogenous regarding its underlying transcriptomic profile. Publication: Manuscript submitted for publication.