Drug Discovery for Remission of Chronic Kidney Disease

Repeated doses of Cisplatin (CP) causes Chronic Kidney Disease (CKD). Renalase (RNLS) activates kinases linked to survival and attenuates acute ischemic and CP-induced kidney injury. We now seek to target delivery of RNLS specifically to kidney to prevent CP-induced CKD.

Severe CKD was induced in RNLS knockout (KO) mice by 2 doses of CP 15 mg/kg 2 weeks apart. The RNLS agonist peptide RP81 was encapsulated in mesoscale nanoparticles (MNP) that target the kidney. Its cytoprotective activity was tested in vitro in TKPTS cells and in vivo using RNLS KO mice. RP81MNP or empty MNP was administered weekly for 4 weeks. CKD was evaluated by serum creatinine (Cr) and immunohistochemistry as well as single cell RNA sequencing (scRNAseq) of whole kidney. MNP were retained intracellularly by TKPTS cells and were localized to proximal tubules in vivo. RP81MNP enhanced cell viability significantly in vitro (cell viability was enhanced 3.5-fold, n=6, p<.05, vs empty MNP) and in vivo (sCr RP81MMP:0.095± 0.006 vs 0.177± 0.019) and KIM-1 (227±28.36 pg/ml vs. 124.3±15.12). RP81MMP significantly reduced plasma cytokines IL-1β, IL-2, IL-6, KC, and TNFα and inhibited regulated necrosis. RP81MNP preserved tubule and vasculature cell mass and decreased infiltrated immune cells caused by CP.

RP81MNP reduced CP-induced CKD, diminishing cell death pathways activated by CP. RP81MNP may be an effective therapeutic agent to prevent CKD in patients treated with repeated doses of cisplatin.​

BACKGROUND: Donor-derived, cell free DNA (ddcfDNA) levels correlate with allograft injury with clinical validity and utility for quiescence and active acute rejection (AR) in kidney transplant (KT) recipients. We analyzed dd-cfDNA level trends immediately preceding and during the CoVid-19 pandemic with "shelter in place" and implemented tele-health strategy with remote phlebotomy tolimit viral exposures.

METHODS: We surveyed weekly U.S. and Metropolitan New York area (06 January 2020 – 25 May 2020) metrics for dd-cfDNA corresponding to "low risk" for AR (dd-cfDNA <0.5%) and cohorts with "indeterminate levels" of 0.5-1.0% and >1.0%. Over 11,000 patient samples (67%) from 150 KT centers, were transitioned from facility-based to remote phlebotomy.

RESULTS: The proportion in 21 weekly aggregated cohorts for each risk-stratification category, were unchanged during CoVid-19 escalation in U.S. Linearized slopes for total U.S. and NYC for indeterminate risk AR cohorts of >1.0% and 0.5-1.0%, were -0.31, -4.97 and -0.12, -4.13, respectively; an indication that incidence decreased during the Covid-19 era. Approximately 73% of samples corresponded to low risk AR category (dd-cfDNA <0.5%), while 15% of samples had dd-cfDNA ≤ 1.0%.

CONCLUSIONS: A combination of remote phlebotomy including ddcfDNA and tele-health, offer a new paradigm for monitoring the status of KT health during the CoVid-19 pandemic. Prospective multi-center studies with robust outcomes data are warranted​

Rates of kidney disease are increasing worldwide, but our ability to detect kidney damage and treat patients early has remained low. New in vivo imaging tools are emerging to provide information about kidney structure and function at the level of the individual nephron. These tools could guide a new generation of precision therapies and enable sensitive monitoring in preclinical models, transplant organs, and patients at or at risk of chronic kidney disease. This talk will provide an overview of these new tools and their current and future applications in both drug development and in patient care.

Background: Bone marrow chimera has been experimented for clinical kidney transplant tolerance in human kidney transplant recipients. However, these approaches require aggressive recipient conditioning, and the long-term risk of graft-versus-host disease remains formidable. The use of donor apoptotic cells is an emerging alternative therapy for inducing transplantation tolerance. In this presentation, I will discuss current understanding of mechanisms of this approach, as well as crucial aspects necessary for successful translation of this approach to clinical kidney transplantation.

Recent findings: Transplantation tolerance to kidney allografts by donor apoptotic cells is mediated by their homeostatic interaction with recipient phagocytes, and subsequent expansion of suppressor cell populations as well as inhibition of effector T cells via deletion and anergy. To ensure their tolerogenicity, it is critical to procure non-stressed donor cells, and to induce and arrest their apoptosis at the appropriate stage prior to their administration. Equally important is the monitoring of dynamics of kidney recipient immunological status, and its influences on tolerance efficacy and longevity. Emerging concepts and technologies may significantly streamline tolerogen manufacture and delivery of this approach, and smooth its transition to clinical application.

Summary: Hijacking homeostatic clearance of donor apoptotic cells is a promising strategy for immune tolerance for kidney transplantation. Timing is now mature for concerted efforts for transitioning this strategy to clinical kidney transplantation.

Kidneys cannot naturally regenerate lost tissue, and few preventive medications exist. This limits treatment options to dialysis or transplant, which are temporary salves with substantial side effects. We have developed a simple method to differentiate human pluripotent stem cells into intricately patterned, multi-segment organoids that resemble kidney tissues. These organoids form via a developmental pathway that induces the nephron progenitor cell, which gives rise to podocytes, proximal tubules, and distal tubules along a proximal-to-distal axis. While beautiful, how to translate organoids into innovative therapies for organs as complex as human kidneys remains a critical question. To address this challenge, we have applied CRISPR gene editing to reveal disease mechanisms in organoids and test therapeutic interventions. Mutations associated with polycystic kidney disease or cilia cause organoid tubules to swell thousands of times in size, producing large, fluid-filled cysts of centimeter diameters. In contrast, mutations associated with podocytes, the filtering cells of the kidney, do not affect tubules but cause junctional deformities that explain urinary defects in vivo. Harnessing the power of automation, scRNA-seq analysis of organoids reveals sixteen different cell types, and identifies novel gene signatures of glomerular disease that appears in human patients.

To improve organoid function and seek therapies, thousands of organoids can be manufactured simultaneously in high throughput screening formats, and analyzed for multi-dimensional phenotypes of differentiation, toxicity, and disease. Screening reveals treatments that dramatically increase the vascular endothelium, and a surprising role for non-muscle myosin in cystogenesis, which can be targeted pharmacologically. Organoids with live fluorescence reporters and in microfluidic kidney-on-a-chip formats provide next-generation platforms for phenotypic screening and illumination of intracellular mechanisms at the tissue scale. Implantation in vivo of organoids from kidney disease patients produces more vascularized, glomerulus-like structures, supporting the prospect of autologous grafts that could be generated on-demand.

Collectively, our findings delineate key strategies and focus areas for advancement of kidney therapeutics using human organoids as surrogates for drug discovery, gene therapy, and regeneration.