Novel Strategies in Nephrology: What To Expect From The Future?

ABSTRACT Chronic kidney disease (CKD) will become the fifth global case of death by 2040. Its largest impact is on premature mortality but the number of persons with kidney failure requiring renal replacement therapy (RRT) is also increasing dramatically. Current RRT is suboptimal due to the shortage of kidney donors and dismal outcomes associated with both hemodialysis and peritoneal dialysis. Kidney care needs a revolution. In this review, we provide an update on emerging knowledge and technologies that will allow an earlier diagnosis of CKD, addressing the current so-called blind spot (e.g. imaging and biomarkers), and improve renal replacement therapies (wearable artificial kidneys, xenotransplantation, stem cell-derived therapies, bioengineered and bio-artificial kidneys).

Keywords: artificial kidney, bioengineering, chronic kidney disease, induced pluripotent stem cells, xenotransplantation

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INTRODUCTION

There are currently 850 million persons in the world with chronic kidney disease (CKD), and CKD is predicted to become the fifth global cause of death by 2040 and the second cause of death in counties with long life expectancy by 2100 [1]. Moreover, CKD is the leading chronic condition with increased incidence, prevalence, and overall health impact. The terminal stage of CKD referred as an end-stage renal disease (ESRD) or kidney failure, is defined by an estimated glomerular filtration rate (eGFR) below 15 mL/min/1.73 m2 by the Kidney Disease: Improving Global Outcomes (KDIGO), affecting approximately 800 000 patients in the USA (71% on dialysis and 29% with a kidney transplant) [2], whereas, in Europe, the estimated ESRD population is over 1 million people, with considerable variations across individual countries [3, 4]. 

Kidney transplantation was first performed in 1954 by Dr Joseph Murray and is the current gold standard for treatment. However, there are still fewer donors than the relentlessly increasing waiting list [5]. Chronic hemodialysis was introduced in 1960 by Dr Belding Scribner, and despite being the major form of renal replacement therapy (RRT) it is associated with numerous short- and long-term complications. Importantly, the life expectancy of patients in dialysis in their twenties is 40 years shorter than of the general population [6]. Peritoneal dialysis is less frequently utilized, and besides specific complications (peritonitis, cellulitis, metabolic disturbances) it is limited by almost inexorable membrane failure [7].shorter than for the general population [6]. Peritoneal dialysis is less frequently utilized, and besides specific complications (peritonitis, cellulitis, metabolic disturbances) it is limited by almost inexorable membrane failure [7].

Figure 1: General overview of the novel strategies in nephrology. (Diagnostics will be further elaborated in detail in Fig. 2.)

Thus, overall, the treatment of ESRD patients remains sub-optimal, due to a shortage of kidney donors and multiple significant complications associated with both dialysis modalities, although multiple alternatives have been considered over the years. Some of the recent developments have the potential to revolutionize the field of nephrology in the upcoming decades. In addition, a large proportion of patients with CKD, especially those with stage 2 and stage 3 CKD, are older individuals who will never require any form of renal replacement therapy mainly since these patients die earlier of cardiovascular diseases. Thus, the real increase in the need of RRT is much less. The main benefits of new drugs such as sodium–glucose cotransporter 2 (SGLT2) inhibitors and mineralocorticoid receptor antagonists (MRAs) are in this group of kidney disease patients.

In this review, we aim to describe the novel diagnostic methods in nephrology such as the advancements in kidney imaging and modalities utilized to estimate renal function as well as the novel therapeutic approaches in kidney disease including wearable artificial kidneys, xenotransplantation, stem cell-derived therapies, bioengineering models and medications on the rise (Fig. 1).

NOVEL DIAGNOSTIC METHODS 

Novel diagnostic methods that would allow an earlier diagnosis of CKD are an unmet clinical need. Reliance on the current eGFR threshold to diagnose CKD means that by the time CKD is diagnosed, over 50% of the functional kidney mass has been lost and the combined risk of CKD progression and premature death is already increased by around 2- to 7-fold, while current interventions decrease the risk of adverse outcomes by 20%–40% [8, 9]; in other words, diagnosis is too late. While CKD may be diagnosed earlier based on high albuminuria values, most patients progress to CKD category G3 while having physiological albuminuria, as evidenced by epidemiological data that show that G3 is the most common category of CKD [10], i.e. albuminuria did not allow an earlier diagnosis (i.e. G1 or G2) for most patients that progressed to G3. This subclinical stage of CKD progression, potentially lasting decades, as evidenced by those forms of CKD in which we have a tool that allows an earlier diagnosis (e.g. sonography for autosomal dominant polycystic kidney disease) is in fact the blind spot for CKD diagnosis [11–13]. Several approaches are under study to address the blind spot in CKD, mainly using imaging and assessment of biomarkers in biological fluids (Fig. 2).

Imaging: assessing nephron number as a determinant of kidney disease and kidney fibrosis

Imaging techniques have the advantage of being non-invasive and, thus, may be safely repeated to evaluate changes, providing information for both kidneys and potentially combining functional with morphological information. The most interesting advances relate to the estimation of nephron number, overall kidney functions, fibrosis, and new functional magnetic resonance imaging (MRI), as well as ultrasound techniques such as diffusion-weighted MRI (DWI or DW-MRI), blood oxygenation level–dependent MRI (BOLD-MRI), perfusion MRI, hyperpolarized (HP) carbon 13 MRI (13C MRI) and contrast-enhanced ultrasound (CEUS).

Functional nephron number is considered an important determinant of kidney health and disease susceptibility throughout life [14, 15]. In humans, nephron number varies widely and a low nephron endowment at birth and/or a loss of nephrons throughout life is strongly associated with kidney disease [15]. Novel technologies to measure nephron number are under development, and functional nephron number has the potential to be used as a clinical biomarker [14]. Nephron number, when used as a biomarker, could provide important information regarding the progression of kidney disease and provide early detection of CKD onset or assessment of recovery after acute kidney injury, improve the evaluation and assessment of donor organs, predict graft survival times, predict the risk of drug-induced nephrotoxicity, and help develop strategies for dosing and toxicity testing for a wide range of therapeutic drugs [14].

New methods have been suggested to measure nephron number ex vivo in the intact kidney: cationized ferritin-enhanced MRI (CFE-MRI) [16], light sheet microscopy after optical clearing [17], and computed tomography (CT) [18]. However, the utilization of these tools in vivo and in the clinics necessitates them being non-destructive and relatively non-invasive, and thus far only CFE-MRI has been used in vivo [14].

CFE-MRI uses ferritin filled with iron oxide [14]. Following intravenous injection, the ferritin is cationized and bound to the glomerular basement membrane. The accumulation of ferritin in the glomeruli allows its detection, mapping of the entire kidney in vivo, and co-localization of glomeruli with other structures such as the microvasculature. To support the use of nephron number as a clinical parameter, the cationized ferritin molecule of CFE-MRI has been modified to form radiolabeled cationic ferritin (RadioCF), a radiotracer used in positron emission tomography (PET) to map functioning glomeruli in vivo in the kidney [19]. RadioCF-PET accurately quantifies nephron mass in animals and had the potential for clinical translation [19]. Radio-CF is formed by the integration of a radioisotope, Cu-64, to the cationic ferritin (CF) and has been shown to bind functional glomeruli when given intravenously [19]. RadioCFPET can map and measure the areas of functional nephron loss making it a diagnostic tool that can also predict CKD progression [14].

Figure 2: Novel diagnostic methods in nephrology. CFE-MRI: cationized ferritin-enhanced MRI; RadioCF-PET: radiolabeled cationic ferritin-positron emission tomography; CKD: chronic kidney disease; AKI: acute kidney injury; GBM: glomerular basement membrane; KRIS: Kidney Risk Inflammatory Signature; THSD7A: thrombospondin type-1 domain–containing 7A antibodies; NAD: nicotinamide adenine dinucleotide; FAT1: glomerular antigen-protocadherin FAT1; ESRD: end-stage renal disease; DKD: diabetic kidney disease; HST: hematopoietic stem cell transplantation; PCX: podocalyxin; KIM-1: kidney injury molecule 1; TNF-α: tumor necrosis factor- α; 8-OHdG: 8-oxo-7,8-dihydro- 2 -deoxyguanosine; L-FABP: liver-type fatty acid binding protein; IGFBP-7: insulin-like growth factor-binding protein-7; TIMP-2: tissue inhibitor of metalloprotease-2; DIKI: drug-induced kidney injury; CNA35-CT: collagen-binding adhesion protein-35 CT; ESMA-based MRI: elastin-specific contrast agent MRI; DW-MRI: diffusion-weighted MRI; BOLD-MRI: blood oxygen level–dependent MRI; HP-13C MRI: hyperpolarized carbon 13 MRI; plus sign: advantages; minus sign: disadvantages/limitations (indicated in red color).

MRI has been used to assess the functional status of both kidneys. Multiparametric MRI may evaluate diverse aspects of kidney function and vascularization [20]. A novel imaging modality is sodium MRI. Compared with “normal MRI,” which is a map of hydrogen atoms in the body, sodium MRI is a map of sodium atoms in the body. The kidney has a baseline gradient of sodium concentrations from the cortex to the medulla (corticomedullary sodium gradient) [21, 22]. The first study of sodium MRI on human kidneys demonstrated that the sodium gradient increases linearly from the cortex to the medulla which is followed by a linear decrease until the renal pelvis [23, 24]. Although alterations in the sodium gradient have been demonstrated in multiple clinical and pre-clinical trials, the sodium MRI technique is far from being integrated into clinical practice [25–27] mainly due to the cost and technical factors involved in MRI recalibration and operation. An additional major setback of this technique, for now, is the lack of adequate characterization of findings in different CKD stages and in various kidney diseases.

Specific techniques have been designed to assess kidney fibrosis and its dynamics in both kidneys simultaneously in vivo [28]. These include fluorescent CNA35 CT, which takes advantage of a collagen-binding peptide, and elastin-specific contrast agent MRI (ESMA)-based molecular MRI of elastin [28]. For instance, collagen-binding adhesion protein (CNA35) which has a high affinity for type I and III collagen molecules has been utilized as a tool to visualize collagen deposition in mouse subjects and has the potential to be utilized in human subjects as a non-invasive method of fibrosis assessment [29, 30]. On the other hand, ESMA-based MRI, which is a small peptide specific to elastin, a component of the extracellular matrix, has been shown to demonstrate kidney fibrosis in mouse models as well as the efficiency of anti-fibrotic therapies [31]. These techniques will eventually also test the potential impact of anti-fibrotic therapies, a current unmet need.

In addition, novel MRI techniques allow to generate imaging biomarkers that can improve the management of kidney disease [20]. MRI enables to the measurement of kidney volumes, blood flow in renal arteries, renal water content, and tissue oxygenation [20]. DWI, or DW-MRI, can detect the displacement of water molecules within the tissue architecture [20]. This technique can inform about any changes in the renal microstructure such as renal fibrosis, cellular infiltration (inflammatory or tumorous) or edema as well as changes in renal perfusion and in the water handling in the tubular compartment [20]. BOLD-MRI, which measures renal tissue deoxyhemoglobin levels voxel by voxel, is a promising technique to monitor renal tissue oxygenation in humans [32]. It allows the monitoring of changes in renal oxygenation or changes in the capillary bed microstructure. In addition, T2 is altered by several other factors such as hydration status, dietary sodium, and susceptibility effects [20]. In addition, HP 13C MRI is a potential tool, not currently used in the clinic, for the non-invasive assessment of oxidative stress and mitochondrial pyruvate dehydrogenase activity following renal ischemia–reperfusion injury [33].

Furthermore, CEUS has been a promising imaging modality for renal lesions. CEUS lacks nephrotoxicity, ionizing radiation, and has the ability to evaluate the enhancement pattern of renal lesions quickly and in real-time [34]. Some of the well-defined applications of CEUS are the differentiation of solid tumors, pseudo lesions, and complex cysts; the characterization of complex cysts with different malignant potentials; and the evaluation of tumor ablation [34]. Microbubble contrast agents are safe with rare adverse reactions [34].

These approaches are promising tools to assess nephron number, diverse kidney functions and kidney fibrosis and may be transitioned to clinical use if their safety, efficacy, and regulatory requirements are established.

Biological fluid biomarkers 

Proteomics and metabolomics have recently been tested in the field of nephrology with intriguing and promising results. In some cases, proteomic or metabolomic signatures themselves are used as biomarkers. In others, they are tools that are used to identify individual biomarkers that are then assessed using more conventional techniques. Additionally, both RNA and DNA in biological fluids may serve as biomarkers. These biomarkers should correlate well with kidney disease, histopathology, progression, outcomes or early disease, and allow for rapid, noninvasive, and specific measurements with high sensitivity and specificity.

Proteomic and metabolomic analysis for the detection of biomarkers 

Pontillo and Mischak identified 273 urinary peptides that differ between patients with CKD and healthy subjects, namely the marker CKD273 which includes fragments of collagen and of proteins involved in inflammation and tissue repair, by using capillary electrophoresis-mass spectroscopy (CE-MS) on 230 CKD patients and 379 control subjects [35, 36]. Further studies have implicated the potential superiority of CKD73 in the prediction and diagnosis of CKD over traditional markers leading to a letter of support of the US Food and Drug Administration (FDA). According to a cross-sectional study conducted on 1990 participants, CKD273 performed better than traditional markers and correlated better than albuminuria with eGFR and better predicted rapid CKD progression [37]. Similar findings were observed in another study conducted on 2087 participants, in which CKD273 was added to the prediction of CKD G3 after accounting for baseline eGFR, albuminuria, and covariables [38]. In a randomized controlled trial in diabetic patients without albuminuria, CKD273 predicted the development of albuminuria [39]. Furthermore, the same CE-MS analysis of a single urine sample may be used to derive other peptidomics markers that predict the rapid loss of eGFR better than albuminuria in patients who do not fulfill current eGFR criteria for CKD (i.e. may allow an earlier diagnosis of CKD than albuminuria) [40], or correlate with kidney fibrosis as detected in kidney biopsy [41] or provide information on the underlying cause of CKD and its prognosis [42, 43].

The Kidney Risk Inflammatory Signature (KRIS) includes 17 proteins directly involved in inflammation and correlated with the 10-year risk of ESRD in diabetic kidney disease [44]. In this study, 194 circulating inflammatory proteins have been evaluated in three different cohorts comprised of type 1 and 2 diabetic patients, revealing that 17 novel proteins enriched for TNF Receptor Superfamily members are linked to early and late renal function decline leading to ESRD in diabetic subjects. Even though the major source, hypothesized to be white blood cells, of those KRIS proteins is yet to be determined, their appearance years prior to the onset of ESRD appears to be related to overproduction rather that disrupted renal clearance. In addition to their predictive role in ESRD, KRIS proteins provide a potential therapeutic intervention point as evidenced by a clinical trial conducted in one of those three cohorts demonstrating a decline in albuminuria consistent with the decline in KRIS proteins in response to a 24-week trial with 4 mg of baricitinib, a JAK-1/2 inhibitor [45]. Additionally, certain metabolites of nicotinamide adenine dinucleotide (NAD) have recently been correlated with acute kidney injury (AKI) risk [46].

There are multiple ongoing clinical trials (NCT01550393, NCT02743273, NCT00690586, NCT04851145) investigating the role of proteomics and metabolomics in the field of nephrology. However, these trials should demonstrate that proteomics or metabolomics biomarkers offer additional information over conventional biomarkers that are clinically relevant and may change therapeutic decision-making in a cost-effective manner. It is important to emphasize that studies investigating novel biomarkers need not only a detection cohort but also an independent (second) validation cohort.

There are multiple ongoing clinical trials (NCT01550393, NCT02743273, NCT00690586, NCT04851145) investigating the role of proteomics and metabolomics in the field of nephrology. However, these trials should demonstrate that proteomics or metabolomics biomarkers offer additional information over conventional biomarkers that are clinically relevant and may change therapeutic decision-making in a cost-effective manner. It is important to emphasize that studies investigating novel biomarkers need not only a detection cohort but also an independent (second) validation cohort.

In addition, multiple novel biomarkers have been identified recently, sometimes using proteomics, although they are in diverse stages of translation to the clinic [47]. More than 40 potential biomarkers have emerged in recent years and most can be sorted based on their association with features such as glomerular injury [podocalyxin (PCX)], tubular injury [kidney injury molecule 1 (KIM-1)], inflammation [tumor necrosis factor-α (TNF-α), TNF receptors-1 and -2) [48]] and oxidative stress [8-oxo- 7,8-dihydro- 2 -deoxyguanosine (8-OHdG)] [47]. CKD biomarkers can be identified by a variety of conventional methods such as solid-phase fluorescent immunoassay, liquid chromatography–mass spectrometry, liquid chromatography–mass spectrometry, high-performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA) [47]. Additionally, microfluidics allows for short sample processing times along with a small footprint, automated operation and a high degree of flexibility, and may potentially offer a robust, cost-effective, and simple-to-operate instrument for early diagnosis of CKD and other pathological events [47].

Membranous nephropathy is the most common cause of nephrotic syndrome in adults and in hematopoietic stem cell transplant (HST) patients. A flurry of autoantigens has been identified recently, following the description of anti-phospholipase A2 receptor (PLA2R) antibodies, a biomarker that according to guidelines can now replace kidney biopsy in patients with membranous nephropathy [49]. Anti-protocadherin FAT1 antibodies are found in over 80% of cases of membranous nephropathy following HST [50]. Patients with anti-PLA2R antibody–negative primary membranous nephropathy may have an anti-thrombospondin type-1 domain–containing 7A antibodies [51, 52]. Additionally, anti-nephrin antibodies have been detected via serological and immunohistochemical studies in a subset of biopsy-proven minimal change disease patients [53].

The field of biomarkers of AKI is also very active; urinary insulin-like growth factor-binding protein-7 (IGFBP-7) and tissue inhibitor of metalloprotease-2 (TIMP-2) testing are now in clinical use under the brand name Nephrocheck [47, 54]. Kidney tubular injury results in the release of blood and urine biomarkers [55]. Urinary KIM-1, liver-type fatty acid binding protein (L-FABP), IGFBP-7 and TIMP-2 are released from the proximal tubule, whereas uromodulin (UMOD) is secreted from the loop of Henle and neutrophil gelatinase-associated lipocalin (NGAL) originates from distal tubules [55]. These biomarkers could potentially localize specific segments of injured tubules. Biomarkers involved in inflammation, repair, and fibrosis [55] could also predict the transition from AKI to CKD, help distinguish between kidney dysfunction and injury, guide AKI management, and improve the diagnosis of diseases such as acute interstitial nephritis [55]. Furthermore, some biomarkers are ready for use in clinical trials of AKI and could guide management in certain clinical settings. The Kidney Precision Medicine Project is an ongoing effort to build a kidney tissue atlas and increase the use of biomarkers to evaluate nephron health [55].

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