Scientists from the University of Sheffield are part of an international collaboration to develop a new class of drugs to treat a common genetic kidney disease which is a major cause of kidney failure.
Working with Glasgow-based biotech company Mironid and colleagues in the US, the Sheffield researchers have carried out successful laboratory tests of a compound developed by Mironid to treat autosomal dominant polycystic kidney disease (ADPKD), a hereditary, progressive disease which affects over 60,000 people in the UK and around 12 million people worldwide.
ADPKD is caused by a genetic fault that disrupts the normal development of kidney cells and causes cysts to form inside the kidneys.
The cysts, which are like fluid-filled blisters, grow and eventually destroy normal kidney tissue causing the kidneys to stop working properly.
This leaves patients needing life-saving treatments, such as dialysis or a kidney transplant.
In ADPKD cells, cysts develop and grow because they express higher than normal levels of a “messenger molecule” called cyclic AMP (or cAMP).
One of the many known functions of cAMP is to tell cells when to divide and when to secrete fluid.
The new compound discovered by Mironid activates an enzyme called PDE4, whose natural role is to break down cAMP.
Researchers at Mironid Ltd describe how the novel small molecule activators of PDE4 enzymes act to suppress disease driving cAMP signaling.
Using cell lines and patient tissues derived from ADPKD patient kidneys, researchers at Sheffield and in the US confirmed that when the Mironid compound switched PDE4 on, levels of cAMP in the disease cells were reduced and the number and growth of the cysts were suppressed.
The findings are published today (18 June 2019) in the Proceedings of the National Academy of Sciences (PNAS).
Moreover, the reversible nature of the compound means that PDE4 activity may be rapidly and accurately controlled in patients, according to the dose decided by the clinician.
This suggests that a future treatment for ADPKD could be tailored to an individual patients’ needs and the severity of their disease without the side effects seen with other approaches.
In theory, the compound could also suppress cyst formation due to ADPKD in other organs apart from the kidney, especially in the liver.
Professor Albert Ong, a consultant nephrologist and professor of renal medicine who led the experiments conducted at the University of Sheffield said:
“Drug development usually focuses on looking for ways to block molecular and chemical processes, not switch them on, so this is a rare mode of action.
As a researcher, it’s very exciting to be involved in the early stage development of a completely new class of drugs.
“It’s also very heartening for me as a clinician since like most genetic diseases, ADPKD cannot be cured.
This discovery gives me hope for the many patients I see in my clinics that there could be another effective new treatment in the future to keep the disease in check for much longer, either on its own or in combination with other drugs, such as tolvaptan.
“I would love to think that ADPKD could one day be a disease that people can successfully manage throughout their natural lives, rather than one that could shorten their lives.”
Mironid’s next step is to refine the compounds in anticipation of being able to develop a new medicine to treat ADPKD patients.
Dr. David Henderson, Senior Investigator at Mironid added:
“This exciting publication highlights the innovative science behind our LoAc PDE4 activator technology.
These first-in-class small molecules have the potential to address unmet clinical need in Autosomal Dominant Polycystic Kidney Disease (ADPKD), and potentially have further utility across different therapeutic areas where unbalanced cellular signaling drives disease progression.
Looking to the future, we are excited and enthusiastic in our continued collaboration to translate this novel approach into real therapeutic benefit.”
Kidney Research UK has funded work on ADPKD at the University of Sheffield for several years, helping to develop vital knowledge on the mechanism of the disease and ways in which it might be managed.
Chief executive of the charity, Sandra Currie, said: “We are proud to have supported several ADPKD studies led by Professor Ong and his team at Sheffield over the years. Most recently, we funded Professor Ong’s investigations into how to control the ‘messenger molecule,’ cyclic AMP, which affects the rate at which cysts grow and expand in the kidneys.”
“It is really encouraging news that this collaboration with Mironid has now successfully laboratory-tested a compound which reduces levels of the cyclic AMP molecule. This could bring new hope to the 12 million people living with ADPKD worldwide—we look forward to further developments.”
Diabetes Mellitus and Diabetic Kidney Disease
Diabetes mellitus (DM) represents one of the most important health problems worldwide. Nowadays, more than 450 million people have DM and, according to recent estimations, about 690 million people will present this pathology by 2045 .
Subsequently, target organ complications secondary to DM, especially micro- and macro-vascular complications, will constitute one of the most important medical concerns in the near future.Diabetic kidney disease (DKD) is a relevant complication of DM, constituting the single most common cause of end-stage renal disease (ESRD) in the western world .
In addition, the presence of any stage of DKD is strongly associated with the development of cardiovascular disease, and therefore renal involvement is a major cause of morbidity and mortality in the diabetic population.
The pathogenesis of DKD includes mesangial expansion, impairment of endothelial cell function and loss of podocytes in the glomerulus, and interstitial fibrosis in the tubular compartment.
The most important clinical manifestation is proteinuria, together with a progressive decline in renal function .
Regarding the treatment of DKD, current practice guidelines are focused on halting or delaying the progression of the disease by adequate metabolic regulation and control of the blood pressure (BP), with blockade of the renin-angiotensin aldosterone system (RAAS) as a cornerstone therapy .
RAAS blockers such as angiotensin converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) are effective in slowing progression of the disease, but this approximation does not generally halt the progression to ESRD.
Therefore, there is a need to evaluate new strategies to improve kidney function, delay the progression of the disease, and eventually improve kidney survival.
These new approaches become even more necessary if we consider that recent trials designed to find effective renoprotection in DM patients have failed [9,10] or were prematurely stopped because of safety concerns [8,11,12].
Recent studies with new antidiabetic drugs (sodium-glucose co-transporter-2 inhibitors and glucagon-like peptide-1 receptor agonists) have shown beneficial renal effects, and thus, these new drugs have emerged as promising drug classes for treating DKDOld Drug Repositioning
De novo drug discovery is a very costly and time-consuming process.
The discovery of one drug takes more than 10 years, with more than one billion USD as the overall estimate of costs and with less than 10% chances of success due to several reasons, including failure rates, high cost, poor safety, poor bioavailability, and limited efficacy.
Thus, new strategies for drug discovery have been needed. One of these new approaches is based on the process of finding new uses for existing drugs outside the scope of the original indication; i.e., drug repositioning.
This concept evolved in the early 1990s from the fact that different diseases share common molecular pathways and targets in the cell.
It gives an extended life for marketed drugs via new indications .
Thalidomide was used in pregnant women to prevent morning sickness, but it was withdrawn after cases of phocomelia in newborn babies had been reported.
It was repositioned for the treatment of multiple myeloma.
Minoxidil, which was initially approved for the treatment of hypertension, was repositioned for the treatment of male pattern baldness, based on the finding that it promotes facial hair growth.
Sildenafil is a phosphodiesterase-5 inhibitor that was initially used for the treatment of angina, but it was switched to the treatment of erectile dysfunction. In this context, pentoxifylline (PTX) could be a potential candidate for repositioning based on its beneficial effects in the treatment of DKD.
Table 1. Examples of successfully repositioned drugs.
|Bromocriptine||Parkinson’s disease||Diabetes mellitus|
|Finasteride||Benign prostatic hyperplasia||Male pattern baldness|
|Methotrexate||Cancer||Psoriasis, rheumatoid arthritis|
|Minoxidil||Hypertension||Male pattern baldness|
|Sildenafil||Angina||Erectile dysfunction, pulmonary hypertension|
|Thalidomide||Morning sickness||Erythema nodosum leprosum|
RAAS, Renin-Angiotensin Aldosterone System; ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker; CAN, chronic allograft nephropathy; CKD, chronic kidney disease; DM, diabetes mellitus; GN, glomerulonephritis; GFR, glomerular filtration rate; hsCRP, high sensitivity C reactive protein; MCP1, monocyte chemoattractant protein 1; PTX, pentoxifylline; TNFα, tumor necrosis factor α; UAE, urinary albumin excretion.
PTX (3,7-dimethyl-1-(5-oxohexyl)-3,7-dihydro-1H-purine-2,6-dione) is a methyl-xanthine derivative that was approved by the United States Food and Drug Administration for the treatment of intermittent claudication more than 30 years ago .
The primary hemorheological effects of PTX are due to increased red blood cell deformability and decreased blood viscosity, although it affects almost all factors responsible for blood viscosity and can be considered as an almost complete rheological drug . However, PTX also has important effects as a modulator of inflammation , which supports its use as a renoprotective drug in DKD.
Inflammation in Diabetic Kidney Disease
The understanding of the pathophysiologic processes leading to DKD has evolved tremendously in recent years.
Renal injury was previously explained by metabolic and hemodynamic alterations, which increase systemic and intraglomerular pressure, and by the modification of molecules under hyperglycemic conditions.
Nowadays, it is recognized that both the chronic low-grade inflammation and the activation of the innate immune system occurring in DM are related to diabetic complications, becoming key pathophysiological mechanisms involved in the development and progression of DKD.
Proinflammatory cytokines can lead to the development of microvascular diabetic complications, including nephropathy.
Recent studies have shown that the concentrations of these substances increase as nephropathy progresses [21,22] being independently related to clinical markers of glomerular and tubulointerstitial damage, including urinary albumin excretion (UAE), the clinical hallmark of DKD [22,23].
Moreover, accumulation of inflammatory cells in the kidney is closely associated with DKD, and, indeed, inhibition of inflammatory cell recruitment into the kidney has been related to protective effects in experimental models of DKD [24,25,26].
Proinflammatory cytokines synthesized and secreted by these cells in the local microenvironment directly damage kidney architecture and subsequently trigger the epithelial-to-mesenchymal transition process , resulting in extracellular matrix accumulation.
Furthermore, the expression of chemoattractant cytokines and adhesion molecules is upregulated in kidney cells of diabetic patients.
These molecules are key mediators of renal injury by virtue of their ability to attract circulating white blood cells (monocytes, neutrophils, and lymphocytes) and facilitate their transmigration into the renal tissue.
These infiltrating cells become a source of new cytokines and other mediators that contribute to the development and progression of renal injury feeding back the process and amplifying the inflammatory reaction.
Serum and urinary levels of interleukin (IL) 18, a potent proinflammatory cytokine, have been reported to be higher in patients with DKD than in control subjects, showing significant positive correlations with UAE .
Many cells produce this cytokine, such as infiltrating monocytes, macrophages, and T cells, and, importantly, tubular renal cells also show increased expression levels of IL18 in patients with DKD .
This enhanced expression has been related to the triggering of the mitogen-activated protein kinase (MAPK) pathways secondary to the action of TGF-β .
In addition, diverse renal cells (endothelial, epithelial, mesangial, and tubular cells) are also capable of synthesizing other proinflammatory cytokines such as tumor necrosis factor (TNF)α, IL1, and IL6, and therefore these cytokines, acting in a paracrine or autocrine manner, may induce a variety of effects on different renal structures [33,34] playing a significant role in pathophysiology of DKD.
Many clinical studies have reported that the serum and urinary concentrations of TNFα are elevated in patients with DKD as compared with nondiabetic individuals or with diabetic subjects without renal involvement and that these concentrations increase concomitantly with the progression of renal damage .
This cytokine is cytotoxic to glomerular, mesangial, and epithelial cells and may induce significant renal damage .
The direct harmful effect of TNFα on the protein permeability barrier of the glomerulus is independent from alterations in hemodynamic factors or effects of recruited inflammatory cells .
In DKD, it is particularly relevant that urinary TNFα has been suggested as a critical factor contributing to sodium retention and renal hypertrophy, important renal alterations that occur during the initial stage of this disease .
Moreover, it has been demonstrated that increased urinary as well as renal interstitial concentrations of TNFα precede the rise in albuminuria .
Similarly, IL6 levels are also higher in patients with DKD in comparison with diabetic patients without nephropathy .
Human renal samples also present increased expression levels of mRNA encoding IL6 in cells infiltrating the mesangium, the interstitium, and the tubules, with a positive correlation with the severity of mesangial expansion .
Other functional and structural abnormalities related to DKD and the progression of renal damage have been associated with IL6, including abnormalities in the permeability of glomerular endothelium, the expansion of mesangial cells and enhanced expression of fibronectin , the increase in the thickness of the glomerular basement membrane [43,44], and renal hypertrophy .
The glomerulus has been the focus of research into DKD, although tubulointerstitial injury is also a major feature, with pathological changes in the tubulointerstitium being closely correlated with renal dysfunction .
Accumulating evidence indicates that increased UAE is not simply an aftermath of glomerular injury but is also involved in the development and progression of this complication, since it has been identified as a mechanism associated with the induction of tubulointerstitial inflammation .
Pentoxifylline: Renoprotection and Targeting Inflammation in Diabetic Kidney Disease
PTX is clinically used to treat intermittent claudication resulting from peripheral vascular disease .
This drug exerts hemorheological actions, since it reduces blood viscosity, erythrocyte aggregation, erythrocyte rigidity, and platelet aggregation.
In fact, data derived from clinical studies and animal models support the use of PTX as an antiproteinuric agent. Interestingly, this antiproteinuric property has been related with its, more recently described, anti-inflammatory effect [52,53,54,55,56,57,58].
Likewise, it has a considerable capacity to modulate other pro-inflammatory cytokines, including IL1, IL6, interferon γ [60,61,62], and other molecules like the intercellular adhesion molecule 1 (ICAM1), the vascular cell adhesion molecule 1 (VCAM1), and the reactive C protein (CRP) [63,64].
Since DKD is a proinflammatory state  with increased glomerular permeability to proteins , the anti-inflammatory effect of PTX could result in a reduction of proteinuria. Therefore, PTX might represent a novel therapeutic approximation to the treatment of DKD.To date, a number of clinical trials evaluating PTX in renal patients, most of them diabetic subjects, have been conducted (Table 2).
The first clinical evidence of the renal protective effects of PTX was reported in 1982 by Blagosklonnaia et al. .
In that work, administration of 300 mg/day of PTX for three weeks to diabetic patients improved glomerular filtration rate (GFR) and decreased proteinuria.
However, it was not until almost the turn of the century that the interest in the anti-proteinuric effects of PTX was renewed.
In 1999, Navarro et al.  reported, in a group of diabetic patients with advanced renal failure, a decrease both in serum TNFα and proteinuria after treatment with PTX (400 mg/day) for 6 months.
In 2005, Aminorroaya et al.  and Rodríguez-Morán et al.  observed that the administration of 400 mg PTX three times daily to non-hypertensive patients with type 2 diabetes displayed anti-proteinuric effects comparable to those achieved with ACEI treatment.
In the same year, Navarro et al. , in a randomized, open-label trial, found that an add-on therapy of PTX at a dose of 1200 mg/day for 4 months in DM patients with a background of ARB additively decreased proteinuria.
Importantly, this extra antiproteinuric effect of PTX was associated with significant reductions in serum and urinary levels of TNFα, although only variations in urinary TNFα correlated with the change of albuminuria.
In a later study in 2006, Rodríguez-Morán et al.  also found a reduction in the levels of both high and low molecular weight urinary protein excretion in DM patients with microalbuminuria.
The anti-proteinuric effect of PTX has also been found in non-diabetic subjects. In 2006, Chen et al.  reported that the treatment with PTX (800 mg/day for 6 months) decreased proteinuria in 17 patients with primary glomerulonephritis.
The reduction of proteinuria was associated with a decline in urinary monocyte chemoattractant protein (MCP) 1 excretion, which allowed the authors to propose a mechanistic basis for PTX in non-diabetic patients with proteinuria.
One year later, Shu et al.  reported a reduction of proteinuria in non-diabetic patients with chronic allograft nephropathy and microalbuminuria.
The Thl/Th2 intracytoplasmic cytokine pattern analysis of peripheral blood CD4+ cells showed a significant decrease of cells bearing TNFα and IL10.
Moreover, the graft function was stabilized in more than a half of the patients by the end of the study, pointing to a renal protection exerted by PTX.
The potential renoprotective effect of PTX was posteriorly evaluated in diverse clinical trials, some of which also examined the anti-inflammatory effect of this drug. However, these trials include a variety of study designs, drug dosages, and follow-up periods, which results in inconclusive results.
In 2007, Diskin et al. , in an open-label, controlled trial including DM patients with nephrotic proteinuria, did not find any additive anti-proteinuric or renoprotective effects of PTX treatment on the background of ACEI and ARB therapy after 1 year of follow-up.
Another randomized clinical trial published by Badri et al. in 2013  with add-on PTX therapy to background RAAS blockade in a small group of non-diabetic patients showed a reduction of proteinuria in the PTX group without affecting eGFR.
In contrast with these results, Perkins et al.  observed in 2009 an amelioration of renal function decline in 40 diabetic patients with chronic kidney disease (CKD) stages 3 and 4 after 1 year of add-on PTX to RAAS blockade.
However, the authors did not observe a decrease in proteinuria levels and proposed that this parameter does not always constitute an optimal surrogate outcome in these studies.
this parameter does not always constitute an optimal surrogate outcome in these studies.
In 2012, Goicoechea et al.  reported a stabilization of renal function and a reduction of several markers of inflammation (TNFα, fibrinogen, and high sensitivity CRP) in CKD patients with stage 3 or higher.
Again, PTX therapy did not reduce proteinuria in this group, but the high percentages of dropped-out and incomplete follow-up patients should be noted.
Similarly, in 2008, Lin et al. found  in CKD stage 3 patients with macroalbuminuria that 1 year of treatment with add-on PTX to ARB background therapy reduced proteinuria and urinary levels of TNFα and MCP1 as compared with the ARB monotherapy group.
Moreover, further analysis revealed a significant decrease of eGFR in the ARB group but not in the add-on PTX group after 1 year of follow-up.
A few larger studies comprising a higher number of participants have been more conclusive, clearly showing the anti-proteinuric ability of PTX and pointing to the therapeutic benefits of the clinical use of PTX in renal disease.
In 2015, Navarro et al.  published the PREDIAN trial, to date the largest randomized controlled study to evaluate the renoprotective effects of PTX.
The study comprised 169 type 2 diabetic subjects with CKD stages 3 and 4 and residual albuminuria despite RAAS blockade, who were randomized to a control group or an active group.
Patients in the active group received PTX (1200 mg/day) on top of RAAS blockers. After 2 years of follow-up, the rate of progression of renal disease was reduced in the PTX group, which was accompanied by a decrease in proteinuria and a reduction in the urinary excretion of TNFα.
The smaller decrease of eGFR in the PTX group with respect to the control group showed a trend at 6 months and reached statistical significance after 1 year, suggesting that a longer duration of treatment with PTX is necessary to observe a therapeutic benefit on renal function.
Also in 2015, Chen et al.  published a retrospective analysis of 661 patients with CKD stages 3–5 treated with PTX. As in the PREDIAN trial, this study also explored the renoprotective effects of add-on PTX therapy to RAAS blockade.
The authors observed that PTX provided nephroprotection in the subset of patients with higher levels of proteinuria, suggesting that proteinuria may be a predictor of response to PTX. Finally, Wu et al.  reported the results of an analysis of a nationwide administrative dataset of advanced CKD patients identifying two propensity score-matched cohorts: PTX users and nonusers.
The authors found that the PTX group was protected from ESRD.
This is the first evidence of the ability of PTX in reducing the risk of developing ESRD even in patients with advanced CKD.
Mechanisms Underlying the Renoprotective Effects of PTX
A meta-analysis published in 2008 pointed to the capacity of PTX to reduce the production of proinflammatory cytokines as the most likely explanation for its antiproteinuric action in patients with DKD .
A subsequent meta-analysis by Tian et al.  concluded that PTX therapy was also able to additively reduce proteinuria and urinary TNFα in patients with DKD under RAAS blockade. Importantly, this antiproteinuric action is observed without metabolic or hemodynamic changes .
PTX is a drug with several effects including the inhibition of phosphodiesterases (PDEs).
The activity of PDEs modulates intracellular second messenger cyclic nucleotides levels in mammalian cells by controlling the degradation of cyclic adenosine-3,5-monophosphate (cAMP) as a feedback mechanism to return to basal levels [84,85].
The inhibition of PDEs by PTX prevents the inactivation of cAMP, resulting in increased levels of cAMP which in turn activate protein kinase A (PKA) , leading to a reduced synthesis of the pro-inflammatory cytokines IL1, IL6, and TNFα (Figure 1) [85,86].
Therefore, the inhibitory effects on PDEs exerted by PTX result in a reduction of inflammation.
PDEs are constituted by 11 gene-related families of isozymes (PDE1–PDE11) with more than 60 isoforms, and PTX is able to suppress the activity of several isoforms.
Importantly, PDE3 and PDE4 are mainly present in inflammatory cells .
The increase of cAMP secondary to the inhibition of PDE3 and PDE4 by PTX in turn activates PKA. In several models of renal disease, PTX is able to attenuate proteinuria via the modulation of signaling pathways or components triggered by inflammatory cytokines .
In a streptozotocin-induced diabetic rat model, PTX ameliorated sodium retention and renal hypertrophy together with a reduction in renal TNFα, IL1, and IL6 expression .
Similarly, in an alloxan-induced diabetic rat model, PTX also exerted anti-inflammatory effects via decreasing the levels of TNF α and IL6 .
Another favorable effect of PTX in the prevention or therapy of DKD could come from the impact on other factors directly related with kidney health.
One of these factors is Klotho, a type I single-pass transmembrane protein predominantly expressed in the kidneys.
Interestingly, Klotho is also found in blood and urine as a soluble form, which can be generated by cleavage of the transmembrane form or by secretion to the extracellular space of a shorter form derived from an alternative spliced transcript .
Klotho has been demonstrated to have beneficial biological effects including antiaging and nephroprotective functions.
Some studies have reported diminished soluble Klotho levels in patients with type 2 DM [94,95], whereas a reduced renal Klotho expression has been observed in biopsies from patients with early stages of DKD , suggesting that soluble Klotho may be an early biomarker for predicting renal impairment in type 2 diabetic patients .
One of the most novel aspects regarding Klotho is the relationship with inflammation.
The proinflammatory cytokines TNFα and TWEAK (tumor necrosis factor–like weak inducer of apoptosis) are able to produce a decrease in renal Klotho expression mediated by NF-κB, both in vitro and in vivo [98,99].
On the other hand, in renal cells and in human umbilical vein endothelial cells (HUVECs), the addition of Klotho inhibits the production of proinflammatory cytokines  and the expression of TNFα-induced adhesion molecules .
A recent post-hoc analysis of the PREDIAN trial by Navarro-Gonzalez et al.  has reported that the administration of PTX to type 2 diabetic patients with CKD stages 3 and 4 drives a reduction in serum and urinary TNFα as well as a significant increase in serum and urinary Klotho concentrations.
Although the intimate mechanisms need to be further investigated, it may be suggested that PTX positively regulates Klotho based on its anti-inflammatory properties since changes in urinary TNFα were negatively and independently associated with the variation in serum and urinary Klotho concentrations.
Moreover, in vitro experiments showed that PTX prevented the TWEAK and TNFα-induced Klotho downregulation in cultured renal tubular cells.
In addition, the reduction of albuminuria in patients treated with PTX could contribute to this effect on Klotho expression, since albuminuria causes tubular inflammation and renal injury  and directly reduces Klotho levels in tubular cells in vivo and in culture .
Conclusions and Future Perspectives
Given the burden of diabetes worldwide, promising and relatively inexpensive therapeutic options for DKD derived from old drugs should not be overlooked.
In this context, based on its anti-inflammatory effects, PTX may constitute a further therapeutic intervention in DKD patients to background RAAS blockade.
Furthermore, PTX may have a beneficial impact on novel factors directly related with kidney health, such as Klotho.
Although the intimate mechanisms need additional investigation, PTX may positively regulate Klotho on the basis of its anti-inflammatory properties.
In addition to its potential repositioning for the treatment of DKD, PTX may also contribute to the design of novel therapeutic strategies aimed at preserving renal Klotho expression and therefore improving kidney and survival outcomes in CKD.
Finally, despite the potential beneficial effects of PTX commented on in this review, it is necessary to recognize that at the present time there are no definitive data regarding renal outcomes.
Therefore, large-scale multicenter trials, which are adequately powered, prospective, placebo-controlled, and with definitive endpoints on efficacy and safety, are necessary to demonstrate the renoprotective properties of PTF in the diabetic population with the maximum grade of evidence.
More information: Faisa Omar et al. Small-molecule allosteric activators of PDE4 long form cyclic AMP phosphodiesterases, Proceedings of the National Academy of Sciences (2019). DOI: 10.1073/pnas.1822113116
Journal information: Proceedings of the National Academy of Sciences
Provided by University of Sheffield