Journal Pre-proof

Antiproteinuric effect of DPP-IV inhibitors in diabetic and non-diabetic

kidney diseases

Ramona Nicotera, Alessandro Casarella, Elisa Longhitano, Davide

Bolignano, Michele Andreucci, Giovambattista De Sarro, Valeria
Cernaro, Emilio Russo, Giuseppe Coppolino

PII: S1043-6618(20)31327-X
DOI: https://doi.org/10.1016/j.phrs.2020.105019
Reference: YPHRS 105019

To appear in: Pharmacological Research

Received Date: 4 April 2020

Revised Date: 5 June 2020
Accepted Date: 10 June 2020

Please cite this article as: Nicotera R, Casarella A, Longhitano E, Bolignano D, Andreucci M,
De Sarro G, Cernaro V, Russo E, Coppolino G, Antiproteinuric effect of DPP-IV inhibitors in
diabetic and non-diabetic kidney diseases, Pharmacological Research (2020),
doi: https://doi.org/10.1016/j.phrs.2020.105019

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Antiproteinuric effect of DPP-IV inhibitors in diabetic and non-diabetic kidney diseases

Ramona Nicotera1, Alessandro Casarella2, Elisa Longhitano3, Davide Bolignano1, Michele Andreucci1,

Giovambattista De Sarro2, Valeria Cernaro3, Emilio Russo2, Giuseppe Coppolino1*

1
Renal Unit, Department of Health Sciences, "Magna Graecia" University, Catanzaro, Italy
2
Department of Health Sciences, "Magna Graecia" University, Catanzaro, Italy

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Renal Unit, Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy.

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*
Corresponding Author:

Dr. Giuseppe Coppolino, MD, PhD

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Renal Unit, Department of Health Sciences,

“Magna Graecia” University,

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Viale Europa - Germaneto

88100 Catanzaro, Italy.

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Phone +39 09613697170

Email: [email protected]
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Graphical abstract

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ABSTRACT
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Diabetes Mellitus (DM) is a chronic and severe metabolic disease, characterized by chronic

hyperglycemia due to insulin resistance and/or reduced insulin secretion. Concerning the non-insulin
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glucose-lowering therapy for diabetes, Dipeptidyl-peptidase-4 (DPP-4) inhibitors, members of the

incretin family, represent new agents, capable of a glycemic control improvement with an advantageous

safety profile, given the absence of weight gain, the low incidence of hypoglycemia and the good renal

tolerance in patients suffering from chronic renal failure. In addition to demonstrating efficacy in

glycemic control through inhibition of GLP-1 degradation, DPP-4 inhibitors (DPP-4is) seem to

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demonstrate pleiotropic effects, which also make them interesting in both diabetic and non-diabetic

nephropathies, especially for their capacity of reducing proteinuria. Several studies about diabetic

nephropathy on patients’ cohorts and murine models have demonstrated a solid direct relationship

between DPP-4 activity and urinary albumin excretion (UAE), thus confirming the capacity of DPP-4is

to reduce proteinuria; the mechanism responsible for that effect was studied to assess if it was the result

of a direct action on renal impairment or a secondary consequence of the better glycemic control related

to these agents. As a result of these more in-depth studies, DPP-4is have demonstrated an improvement

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of renal inflammation markers and consequent proteinuria reduction, regardless of glucose

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concentrations. Considering the nephroprotective effects of DPP-4is might be glycemic independent,

several studies were conducted to prove the validity of the same effects in non-diabetic nephropathies.

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Among these studies, DPP-4is demonstrated an improvement of various renal inflammatory markers on
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several models of non-diabetes dependent renal impairment, confirming their capacity to reduce

proteinuria, independently from the action on glucose metabolism. The objective of this review is to
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present and discuss the so far demonstrated antiproteinuric effect of DPP-4is and their effects on diabetic

and non-diabetic nephropathies.

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Abbreviations:
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CKD, chronic kidney disease; DKD, diabetic kidney disease; DM, diabetes mellitus; DN, diabetic

nephropathy; DPP-4, dipeptidyl-peptidase-4; DPP-4is, dipeptidyl-peptidase-4 inhibitors; GLP-1,

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glucagon-like peptide-1; UAE, urinary albumin excretion; ESRD, end-stage renal disease; SGLT2,

sodium glucose cotransporter 2; GFR, glomerular filtration rate; eGFR, estimated glomerular filtration

rate; ROS, reactive oxygen species; PKC, protein kinase C; AGE, advanced glycation end-products;

RAAS, renin-angiotensin-aldosterone system, gastric inhibitory polypeptide/glucose-dependent

insulinotropic polypeptide; sDPP-4, soluble form of dipeptidyl-peptidase-4; NHE3, sodium/proton

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exchanger isoform 3; Ang II, angiotensin 2; CYP, cytochrome P450; RCT, randomized control trial;

UACR, albumin/creatinine ratio; HbA1c, glycated hemoglobin; BMI, body mass index; ORG, obesity-

related glomerulopathy;

Chemical compounds studied in this article:

Glucagon-like peptide-1 (PubChem CID:16135499)

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Sitagliptin (PubChem CID: 4369359)

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Vildagliptin (PubChem CID: 6918537)

Saxagliptin (PubChem CID: 11243969)

Linagliptin (PubChem CID: 10096344)

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Alogliptin (PubChem CID: 11450633)
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Keywords
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DPP-4; DPP-4 inhibitors; diabetic nephropathy; antiproteinuric effect; proteinuria; Kidney function;

1. Introduction
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Diabetes Mellitus (DM) is a severe metabolic disease characterized by reduced insulin action and/or
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secretion, resulting in chronic hyperglycemia. DM is recognized as one of the most common health

problems of the 21st century. Significant morbidity and mortality in diabetic patients depend on micro-

and macro-vascular complications. Diabetic nephropathy (DN) is one of the most common and important

microvascular DM complications1, the proportion of diabetic patients with chronic kidney disease (CKD)

is relatively stable at 25-30%. Currently, DM is among the leading causes of end-stage renal disease

(ESRD) and renal replacement therapy worldwide2. Besides, diabetic patients with CKD of stages 4 and

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5 have a higher prevalence of anemia than non-diabetic patients with comparable glomerular filtration

rate with important clinical and economical consequences3, 4. The pathogenesis of DM is multifactorial.

Hyperglycemia induces afferent arteriolar dilatation by releasing vasoactive mediators. The high filtered

load of glucose up-regulates sodium-glucose cotransporter 2 (SGLT2) in the renal proximal tubules; as

a result, tubular reabsorption of both glucose and sodium chloride is increased leading to glomerular and

tubular hypertrophy and, eventually, to glomerulosclerosis and tubule atrophy5. The consequent decrease

in the sodium chloride delivery to the macula densa, causes afferent arteriole dilatation, due to tubule-

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glomerular feedback, and concomitant activation of the renin-angiotensin system. As a consequence of

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inappropriate activation of the renin-angiotensin system, a concomitant constriction of efferent arteriole

induces changes in autoregulation, glomerular hypertension and increased glomerular filtration rate

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(GFR)6, 7. Furthermore, high blood glucose levels, insulin resistance and compensatory hyperinsulinemia
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independently determine endothelial dysfunction through different intracellular mechanisms, including

the production of reactive oxygen species (ROS), the activation of protein kinase C (PKCs), the
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stimulation of advanced glycation end-products (AGEs)-induced inflammation and the expression of

profibrotic genes. These pathophysiological processes are responsible for oxidative stress and chronic
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inflammation in renal parenchyma, ending up with the worsening of kidney function8-12. These

hemodynamic and metabolic abnormalities also contribute to podocyte damage, leading to proteinuria
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and glomerulosclerosis. Proteinuria and hypertension are the main progression factors of CKD of all

causes13, 14. Low GFR values, associated with proteinuria, are expected to decline more rapidly over
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time15, 16. This is the reason why the latest Kidney Disease: Improving Global Outcomes (KDIGO)

classification defines as CKD stages of greater severity, those associated with macroalbuminuria (6

categories of estimated GFR [eGFR] [G], and 3 categories of albuminuria [A])17.

Therefore, reducing proteinuria, as much as possible, is the main therapeutic aim for any cause of CKD.

Undoubtedly, the drugs acting on the renin-angiotensin-aldosterone system (RAAS) should be the basis

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of this approach7, 10, 18-21. In recent years, the guidelines on DM treatment have been enriched with drugs

that, in addition to demonstrating efficacy in primary metabolic compensation targets, seem to have

pleiotropic effects that also make them interesting in non-diabetic nephropathy; among these new drugs,

dipeptidyl peptidase-4 (DPP-4) inhibitors and inhibitors of sodium-glucose cotransport in the renal

proximal tubule appear to reduce albuminuria22, 23. This brief review aims to present the first results of

the antiproteinuric effect of DPP-4is in diabetic and non-diabetic kidney diseases.

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1.1 Literature search strategy

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PubMed database was searched for English-language articles, without time restriction up to 15 January

2020. The following search terms were combined through Boolean search method and used to locate
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relevant articles: “renal disease”, “chronic kidney disease” or “albuminuria” or “serum creatinine” or

“proteinuria” or “nephroprotective” with the terms “DPP-4 inhibitor”, “sitagliptin”, “vildagliptin”,

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“saxagliptin”, “alogliptin”, “linagliptin”. Variations of these terms were included, and other reference

were extracted from relevant articles. We included in the paper some relevant randomized controlled trial
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(RCT), pilot, observational, prospective or retrospective studies including diabetic patients with any

degree of renal impairment treated with DPP-4is, with or without other comedication, and animal models
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resembling diabetic nephropathy or other kinds of renal damage. Outcomes considered for the analysis

were: 1) CKD progression or changes in eGFR or in serum creatinine; 2) albuminuria or proteinuria or

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changes in urine albumin/creatinine ratio (UACR); 3) Changes in cytological/histological structure of

human or animal kidneys.

2. DPP-4 enzyme: physiological function.

DPP-4 is the enzyme responsible for the degradation of incretins, which are hormones released by the

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enteroendocrine cells in response to food consumption. Glucagon-like peptide-1 (GLP-1) and gastric

inhibitory polypeptide/glucose-dependent insulinotropic polypeptide (GIP), the two most important

incretins, increase insulin secretion from pancreatic beta-cells with a mechanism dependent on glucose

level; moreover, they inhibit glucagon secretion from alpha-cells and make gastric emptying slower.

GLP-1 has a half-life of about 2 minutes, since it is promptly degraded by the enzyme DPP-4. DPP-4is

suppress DPP-4 activity, thus reducing the clearance of both GLP-1 and GIP. The increase in their

concentrations stimulates the release of insulin from pancreatic beta-cells24-26, maintaining their glycemic

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regulation functions27. DPP-4 is an ubiquitously expressed glycoprotein that belongs to the family of

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serine proteases. DPP-4 enzyme exists in two forms: a soluble form circulating in the blood (sDPP-4)

and a membrane-bound one that is present in several cell types28. Membrane-bound DPP-4, a protein of

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110 kDa, is a type II transmembrane protein of 766 amino acids (AAs) and mainly consists of four
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domains: a short cytoplasmic domain (1–6 AA), a transmembrane domain (7–28 AA), a flexible stalk

segment (29–39 AA), and an extracellular domain (40–766 AA), responsible for ligand binding and DPP-
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4 activity, which can be further separated in a highly glycosylated region involved in binding to anti-

CD26 antibodies, adenosine deaminase and caveolin (101-350 AA), a cysteine-rich region for collagen
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and fibronectin-binding (55-100 AA, 351-497 AA), and a catalytic region (506-766 AA) 29, 30. sDPP-4 is

a protein of 727 AAs containing the DPP-4 membrane-bound residues 39-766; it lacks the cytoplasmic
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domain, the transmembrane region, and the flexible stalk31. Both DPP-4 forms exert catalytic activity: in

particular, they selectively remove the N-terminal dipeptide from peptides with proline or alanine in the
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second position, inactivating them or generating new mediators with different actions. The enzyme has

a greater affinity for proline than for alanine31. DPP-4 is expressed in a variety of tissues and organs

including the kidney 29, 32. Within the immune system, DPP-4 is better known as CD26 30. In the kidneys,

DPP-4 is present in 98 portions of nephron; it has been localized in the brush border membrane of the

renal proximal tubules (segments S1-S3), but also in the loops of Henle, distal tubules and collecting

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ducts, where it has unclear functions33-35. In the renal proximal tubule, however, it exerts more recognized

actions. Firstly, its role is related to the sodium/proton exchanger isoform 3 (NHE3) activity in response

to Angiotensin II (Ang II)33. Both Ang II inhibition and DPP-4 blockade result in reduced sodium

absorption in the renal proximal tubule33. Moreover, thanks to the gradient generated by the current of

H+ ions, DPP-4 allows the reabsorption of oligopeptides containing proline in the same portion of the
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renal tubule . It has also been observed, in vitro and in vivo, that inhibition of DPP-4 results in a

restoration of megaline levels, reduced by the action of Ang II on the renal proximal tubule: this would

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determine an increased uptake of albumin and other low-molecular-weight proteins 25, 38-40.

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Figure 1. Mechanism of action of DPP-4is on glucose metabolism. Created by Biorender.com

2.1 Pleiotropic action of DPP-4 enzyme

Despite its clinical importance resides in glycemic modulation due to incretin degradation, we cannot

ignore the fact that DPP-4 can interact with at least 35 different substrates, and directly or indirectly

influence multiple metabolic pathways. As an example, a study highlighted that DPP-4 is also responsible

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for neuropeptide Y1-36 and peptide YY1-36 metabolism; thus, DPP-4is are capable of inducing

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augmentation of both peptides, causing preglomerular vascular smooth muscle and mesangial cells

proliferation and collagen production in the kidneys of Spontaneous Hypertensive rats. The same effects

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were not replicable in normotensive Wistar Kyoto rats , maybe because the pro-growth effect of this

mechanism was counterbalanced by reduction of glucagon secretion, exerted by DPP-4is41. As a matter

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of fact, glucagon may stimulate glomerular mesangial cell proliferation, and obviously its inhibition
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produces the opposite effect42. This evidence puts attention on the possible role of DPP-4 in a large

extension of metabolic pathways.

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The sole principal therapeutic target of DPP-4is is considered GLP-1 and its half-life prolongation,

however, this interpretation may be excessively narrowing, considering evidence from a study that
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proved how GLP-1 gene expression and peptide synthesis were unmodified in animal models of Type 1

and Type 2 Diabetes (T2DM). On the contrary, DPP-4 expression in kidney, liver, and intestine has been
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found augmented by chronic hyperglycemia, in animal models resembling human T2DM. Due to its

multifunctional properties and multiple pathways in which it is involved, DPP-4 may be accountable for

many common findings of T2DM. For example, we know that DPP-4 is involved in immune response

regulation, specifically in T cell activation, signal transduction, proliferation and cytokines, and

chemokines metabolism, which excessive production was demonstrated in T2DM. Despite its therapeutic

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relevance, few studies have been conducted to clarify a possible link between Beta cell damage and DPP-

IV overexpression. Considering this evidence, DPP-4is may exert nephroprotective effects from diabetic

nephropathy, also acting on diabetes through an independent GLP-1 pathway43.

Regarding its capacity to regulate cytokines and chemokines metabolism, the role of DPP-4 in acute

kidney ischemia-reperfusion damage, was investigated by using DPP knockout rats. In the study, DPP-4

deficient rats showed reduced levels of circulating creatinine and blood urea nitrogen (BUN), with

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reduced creatine ratio, proteinuria, and anatomical/histopathological renal damage in response to acute

kidney ischemia-reperfusion injury; which involves oxidative stress, mitochondrial damage, apoptosis,

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complement cascade activation and inflammation. Regarding this mechanism of injury, the exact role of

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DPP-4 was not investigated, especially relating to GLP-1, which augmentation seems to be directly

responsible for anti-inflammatory effects. These findings highlight potential therapeutic effects of DPP-
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4is on acute kidney ischemic-reperfusion injury, in a dependent manner from GLP-1 but independent

from its glycemic regulation capacities44.

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Given the fact that DPP-4 under-expression leads to nephroprotective effects from many kinds of renal
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injuries, it seems reasonable to speculate about the potential pathogenic role of its overexpression in

kidneys. As part of a study conducted on db/db mice, an animal model of T2DM, demonstrating the
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nephroprotective effect of DPP-4i linagliptin, DPP-4 immunoreactivity was also detected in the

glomeruli of patients with diabetic nephropathy, but not in healthy controls. This result could indicate
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that the enzyme upregulation may be in part responsible for albuminuria, renal impairment, and

glomerulosclerosis in diabetic nephropathy45.

Another study has provided evidence about the possible link between DPP-4 expression in the urine and

the onset and progression of diabetic kidney disease (DKD). The study aimed to prove the possibility to

consider urinary microvescicle-bound DPP-4 as a DKD biomarker, for screening and diagnosis purposes.

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The study compared urine and serum DPP-4 analysis of 127 diabetic patients (divided into 3 groups

sorted for normal, macro, and microalbuminuria) with that of 34 healthy patients. Results showed no

significant difference in DPP-4 activity between T2DM patients’ serum and healthy patients’ serum, but

an increment of urine micro vesicle-bound DPP-4 in diabetic patients in respect of healthy patients, with

a clear correlation with its augmentation and the worsening of albuminuria46.

Many other studies have been conducted to assess the correlation between serum/urine DPP-4 activity

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and renal pathologies, aiming to check the value of this enzyme as a diagnostic biomarker of kidney

injury. In a group of various enzymes, DPP-4 activity was determined in serum, lymphocytes, and urine

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of 178 individuals (grouped for renal disease in: IgA nephropathy, membranous nephropathy, diabetic

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nephropathy, lupus nephritis, and healthy control subjects). Urine DPP-4 activity was significantly higher

in diabetic nephropathy, resulted from Type 1 diabetes and T2DM with microalbuminuria, than the
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control group. Creatine clearance correlated significantly with urinary DPP-4, in IgA nephropathy.

Moreover, serum DPP-4 activity was significantly higher than the control level in membranous
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nephropathy and IgA nephropathy47, 48

. Furthermore, serum DPP-4 activity was found negatively

associated with eGFR of patients affected by CKD, in a cohort of patients49. To better comprehend the
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relationship between renal function and DPP-4, another study investigated whether serum DPP-4 activity

is related to urine albumin excretion (UAE) in 113 patients with type 1 diabetes and GFR within normal
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range. The results showed that DPP-4 activity correlated with systolic blood pressure, HbA1c, and UAE.
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Higher waist circumference and worse lipid profile were reported in patients showing the highest DPP-

4 activity. Furthermore, patients with the lowest UAE showed the lowest DPP-4 activity. In the linear

regression analysis, the association remained significant even after adjusting DPP-4 activity for gender,

age, HbA1c, disease duration, waist circumference, treatment with angiotensin-converting enzyme

inhibitors, and hypolipemic drugs50.

This evidence underlines the possible diagnostic and prognostic significance of DPP-4, raising questions

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about its possible direct pathogenetic role, and underlining its importance in multiple metabolic

pathways.

2.3 DPP-4 inhibitors: pharmacokinetics and pharmacodynamics

DPP-4is are a class of drugs belonging to the family of incretins, new oral hypoglycemic agents used in

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the treatment of T2DM, with high tolerance and good safety profile 22, 51. Together with GLP-1 agonists,

DPP-4is provide a reduction in glucose levels with a low risk of hypoglycemia compared to more

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conventional therapies52, 53. Sitagliptin, saxagliptin, linagliptin, alogliptin, and vildagliptin are orally

taken and they are absorbed rapidly, regardless of simultaneous patients’ food intake; the peak of their

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plasma concentrations (Tmax) occurs between 1 and 3 hours after administration. Oral bioavailability is
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generally high, with lower values with linagliptin, whose absorption is inhibited by P-glycoprotein. In

plasma, only a low percentage of circulating drug forms reversible bonds with proteins; the only
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exception is linagliptin, and this is precisely the underlying mechanism explaining the different drug

excretion54. Indeed, all gliptins are predominantly excreted in the urine, with 60–85% of each dose
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eliminated as the unchanged parent compound. In contrast, linagliptin undergoes enterohepatic cycling

and is mainly excreted in the feces (~90%).

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Therefore, according to the guidelines of the Food and Drug Administration (FDA) and the European
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Medicines Agency (EMA), dose modifications for alogliptin, sitagliptin, saxagliptin and vildagliptin are

recommended in the presence of moderate or severe renal impairment; conversely, dosage adjustments

are not necessary for linagliptin under these circumstances55. DPP-4is are divided into peptidomimetics

and not peptidomimetics. All molecules are designed to exert a reversible competitive inhibitory action,

although there are differences in the type of chemical bond established with the target enzyme.

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 Sitagliptin, alogliptin, and linagliptin form a non-covalent bond with the enzyme in the catalytic site,

while vildagliptin and saxagliptin act in two phases: reversible covalent binding and dissociation from

the enzyme, with a slow balance between active and inactive enzyme forms. These chemical and binding

differences are at the basis of the different half-life and maximum dosage, however, these differences do

not seem to be significant in terms of the relationship between inhibition of sDPP-4 and acute glucose

decrease in mice56. In general, the half-life of the compounds varies between 1.5 and 40 hours (Table 1).

All drugs are administered once daily, except vildagliptin that requires twice-daily administration. They

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reach the steady-state in 3-6 days 57, 58. Metabolism of the different DPP-4is is variable, but, despite their

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variability, no DPP-4i has demonstrated to be a potential inhibitor or inducer of Cytochrome P450 (CYP)

isoforms. Accordingly, the risk of drug interactions is very low. However, a potential risk with

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saxagliptin should be considered when it is administered with pharmacological agents that interfere with
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CYP 3A4/5 isoforms, such as ketoconazole (a potent inhibitor) or rifampicin (a potent inducer). In these

cases, dose adjustment is necessary59-61. Moreover, linagliptin possesses a weak inhibitory enzyme action
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Table 1. Pharmacokinetic properties of approved DPP-4 Inhibitors

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Sitagliptin Saxagliptin Linagliptin Alogliptin Vildagliptin

Oral bioavailability (%) 87 75 30 70 85

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Distribution Volume (L) 198 151 368-918 300 71

Half Life (h) 8-14 2.2-3.8 120-184 12.4-21.4 2-3

Fraction Bound to protein (%) 38 <10 70 20 9.3

Bile excretion (%) 13 22 85 13 4.5

Renal excretion (%) 87 75 5 76 85

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Proportion excreted 79 24 ̴90 95 23

unchanged (%)

Substrate for CYP3A4/5 Low Yes No No No

Drugs interactions Unknown CYP3A4/5 inducers Unknown Unknown Unknown

and inhibitors

DPP, dipeptidyl peptidase; CYP, cytochrome P450

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Table reference:

- (adapted from: Capuano et Al. Dipeptidyl peptidase-4 inhibitors in type 2 diabetes therapy – focus on alogliptin,

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201362) * Small differences in pharmacokinetic parameters can be found from different sources.

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2.4 DPP-4 Inhibitors: clinical aspects

DPP-4is are generally well tolerated. Several meta-analyses have documented a good safety profile, also
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in elderly patients63. From the cardiological point of view, they seem to guarantee an appreciable

protection, even so an increased risk of hospitalization with saxagliptin (27%) has been reported64,
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determining need for careful monitoring of administration to cardiopathic patients. In particular, the

SAVOR-TIMI trial evaluated a composite outcome of cardiovascular death, myocardial infarction, and
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stroke, as the primary endpoint to assess the cardiovascular effects of saxagliptin. There was no

difference in documented pathological events, however, hospitalization among patients treated with
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saxagliptin was greater . Improvements of glycemic profile and organ protection are, of course, the

main advantage of DPP-4is. Indeed, several studies have found a reduction in proteinuria, that was

independent from the glycemic control, the blood pressure, and the body weight; this opens insights about

an independent activity from inhibition of GLP-1 degradation in the kidneys24-26.

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 Four large placebo-controlled randomized controlled trials (SAVOR-TIMI, EXAMINE, TECOS, and

CARMELINA studies) demonstrated the safety of DPP-4is in patients with renal disease (Table 2)65.

Table 2. Clinical aspects of approved DPP-4 Inhibitors

Sitagliptin Saxagliptin Linagliptin Alogliptin Vildagliptin

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Clinical Use Treatment of type 2 Treatment of type 2 Treatment of type 2 Treatment of type 2 Treatment of type 2

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diabetes in diabetes diabetes diabetes in combination diabetes mellitus in

combination with with combination

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metformin or a other therapies with other antidiabetic

thiazolidinedione drugs

Dose in normal renal 100 mg/die 5 mg/die 5 mg/die 25 mg/die 2 x 50 mg/die

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function With a sulphonylurea:

50 mg in the morning
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Dose in renal

impairment GFR

(mL/min)
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30–50 50 mg/die 20–50 2.5 mg/die 20–50 Dose as in 30–50 12.5 mg/die 20–50 50 mg/die

normal renal function.

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<30 25 mg/die 10–20 2.5 mg/die 10–20 Dose as in 10–30 6.25 mg/die 10–20 50 mg/die

normal renal function.

<10 2.5 mg/die <10 Dose as in normal <10 6.25 mg/die <10 50 mg/die
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renal function

DPP, dipeptidyl peptidase; CYP, cytochrome P450

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3. DPP-4 inhibitors in diabetic nephropathy

The effects of DPP-4is on diabetic nephropathy has not been fully comprehended, despite the extensive

number of results published.

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Multiple animal models showed clearer evidence of the possible nephroprotective effect of DPP-4is,

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even in a glycemia independent manner.

Pleiotropic action of DPP-4is on inflammation was explored in a mouse model of cultured renal proximal

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tubule cells. Linagliptin and high concentrations of sitagliptin, vildagliptin, and alogliptin significantly
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improved tubulointerstitial damage, inflammation, fibrosis, and apoptosis induced by proteinuria,

regardless of glucose concentrations66.

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The nephroprotective effect of DPP-4is has also been demonstrated in other murine experimental models

in which it became evident that saxagliptin limits glomerular hypertrophy, interstitial fibrosis and
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macrophage infiltration mediated by NF-kBp65, and that the effects of gemigliptin on oxidative stress-

related podocytopathy in DM are significant in terms of harm reduction, related to the decrease in AGEs
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and ROS, regardless of hyperglycemia, in 12 weeks of treatment67-69. Similarly, DPP-4 deficiency could

have beneficial effects in an experimental model of diabetic nephropathy, partially due to suppressing
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the AGE-AGE receptor (RAGE) axis, implied in oxidative stress generation and fibrotic and

inflammatory reactions causing kidney alterations in diabetes. This conclusion was provided comparing

two models of streptozotocin-induced diabetes in wild type rats and DPP-4 deficient rats, that resulted in

the significant suppression of renal AGE accumulation, albuminuria, and oxidative stress markers, in the

latter group. The mechanism responsible for the effect was not completely clear, however, it is unlikely

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related to glycemia, since there was no difference for this parameter between the two groups 70. Further

evidence was provided by studying DPP-4is effects on diabetic Sprague-Dawley rats and comparing

them with non-diabetic and diabetic rats. DPP-4is treatment reduced Urine Albumin-to-Creatinine Ratio

(UACR) and histological changes of diabetic nephropathy, moreover, prevented macrophage infiltration

and inflammatory reaction in glomeruli, through a direct anti-inflammatory action rather than glycemic

control71; notably, the DPP-4i used (PKF275-055) in these experiments did not seem to have any effect

on glycemia indicating that this effect may be independent. Similarly, linagliptin demonstrated the

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capacity to delay the progression of diabetic nephropathy damage in db/db mice, an animal model of

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T2DM, in a glucose independent manner45.

A pilot study investigated the possible effect of vildagliptin on LDL heterogeneity and albuminuria, in a

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limited number of T2DM patients with diabetic nephropathy. Albuminuria was measured as UACR and
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LDL heterogeneity was evaluated on the presence of small dense LDLs (sdLDL), potent atherosclerosis

inducers, and their proportion in respect of the total serum LDL cholesterol. After 8 weeks of treatment,
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total serum LDL cholesterol resulted not significantly changed, otherwise serum sdLDL decreased by

8.8%, and triglyceride metabolism-related markers (i.e. triglyceride, remnant-like particle cholesterol,
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apolipoproteins B, C-2, and C-3) significantly decreased. Moreover, UACR decreased by 44.6%, despite

eGFR remained unchanged. Variation in sdLDL proportion occurred independently from HbA1c, and
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multivariate regression analysis of data revealed that variation of their proportion was a predictor of

UACR variation, independent from HbA1c72.

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Another pilot study investigated the anti-inflammatory effects of Linagliptin in T2DM patients,

evaluating any variation of oxidative stress markers, C-Reactive Protein (CRP), UACR, eGFR and

urinary liver fatty acid-binding protein (L-FABP) as tubulointerstitial injury marker. After 3 months

treatment period, L-FABP and oxidative stress markers significantly decrease, independently from

HbA1c, while CRP, UACR, and eGFR remained almost unchanged73.

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In a retrospective observational study on a cohort of patients with T2DM, the nephroprotective effect of

some DPP-4is (sitagliptin, linagliptin, saxagliptin, vildagliptin, and gemigliptin), in coadministration

with other antidiabetic drugs (especially metformin and sulfonylureas), was investigated, evaluating

albuminuria and eGFR. Changes in the UACR, eGFR, and metabolic parameters were compared before

and after treatment. After a year, UACR was reduced by an average of 45 mg/g. Patients with

macroalbuminuria had a significant decrease in proteinuria (p < 0.05), whereas the effects on

microalbuminuria were not statistically significant. Although eGFR did not change one year after DPP-

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4 inhibitor treatment, reductions in eGFR were slowed in patients with microalbuminuria and reversed

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in the macroalbuminuric or normoalbuminuric groups, four years after treatment74, 75. These studies

support the evidence of a relevant DPP-4 influence on albuminuria in diabetic nephropathy, with

limitations relative to the absence of a control group.

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Aiming to certainly assess the antialbuminuric properties of DPP-4is, MARLINA-T2D trial was designed

as phase IIIb, multicenter, randomized, double-blind, placebo-controlled study to evaluate the glycemic
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and renal efficacy of Linagliptin in T2DM patients with micro or macroalbuminuria. Linagliptin 5mg

administered once daily for 24 weeks has determined significative improvements in glycemic control,
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however, albuminuria reduction was not significant over the short period of treatment. Even so, post hoc

analysis of data derived from the trial, has suggested that linagliptin may be responsible for significant
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albuminuria reduction (>20% based on authors judgement) in some patients. Albuminuria may result

from several different mechanisms as endothelial disfunction, podocyte damage, and mesangial
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proliferation, thus, responding patients may have one of these pathways activated and then inhibited by

linagliptin. This study cannot be adequate to completely exclude nephroprotective effects of DPP-4, due

to the short time of treatment and the limited number of enrolled individuals affected by advanced CKD76.

Long term effects of linagliptin were investigated through CARMELINA study.

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CARMELINA study was a large, multicenter, randomized, placebo-controlled trial that involved T2DM

patients at high risk of cardiovascular events and with a high prevalence of CKD. The study aimed to

assess cardioprotective and nephroprotective effects of linagliptin, added to usual antidiabetic therapy.

The primary endpoint of the study was defined as the instance of cardiovascular death, non-fatal

myocardial infarction, non-fatal myocardial stroke (3 points major adverse cardiovascular event -

MACE); the secondary endpoint was defined as the instance of ending stage renal disease (ESRD), death

by renal failure, or a prolonged decrease of at least 40% of eGFR, in respect of baseline. For both drug

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and placebo groups, the median duration of treatment was 1.9 years and the median duration of

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observation was 2.2 years. Regarding both primary and secondary endpoints, this study did not

demonstrate any superiority or inferiority of linagliptin group in respect of the placebo group. Despite

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this occurrence, exploratory kidney and microvascular analyses found a less frequent worsening of
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albuminuria category (as an example, change from norm to micro-albuminuria, and from micro to

macroalbuminuria) in the linagliptin group, giving support to the hypothesis that linagliptin and other
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DPP-4is might exert nephroprotective effects, for which although glycemic control might have been in

part responsible77.
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Avogaro and colleagues reviewed the results of studies conducted from 1980 to 2014 on the positive

effects of DPP-4is on microvascular complications of diabetes. It appears that DPP-4 inhibitors improve
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inflammation, endothelial function, blood pressure control, lipid metabolism, and bone marrow function;

however, the Authors failed to demonstrate that these effects are not the consequence of a better glycemic
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control, due to lack of further evidence 78. Moreover, a systematic review of literature and metanalysis

of randomized placebo-controlled trials found that DPP-4is were not associated with additional

albuminuria reduction, compared with other conventional antidiabetic therapies or placebo, in contrast

with GLP-1 receptor agonists which resulted exerting this effect79.

19
Comparative analysis of different DPP-4is effects gave insights about the possible glucose independent

action of these drugs. Alogliptin and vildagliptin effects on glucose metabolism, renal function, and lipid

metabolism of T2DM patients, were compared in a 24 weeks prospective randomized open-label study,

divided into two phases. During the first phase, DPP-4is naïve patients were assigned to alogliptin

25mg/day or vildagliptin 50mg/bid groups. HbA1c levels, considered as the primary endpoint, decreased

comparably between both groups (-0.5% for alogliptin and -0.7% for vildagliptin) while UACR,

considered as a secondary endpoint, was better improved by vildagliptin.

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In the second phase, patients in treatment with sitagliptin switched to either alogliptin or vildagliptin, at

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the same dose regimens of the first phase. Switching between different DPP-4is-based therapy did not

exert any significant change on HbA1c levels at 24 weeks, although vildagliptin exerted slightly better

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action probably due to its covalent bonds to DPP-4. Unlike in the first phase, switch to vildagliptin
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resulted in UACR worsening during the second phase. UACR differences between two phases were

probably related to a possible diabetic nephropathy progression, small sample size, short follow up.
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Results from this study suggested a possible antialbuminuric effect of DPP-4is, independently from

glycemic control80. Moreover, in another comparative prospective study, the antialbuminuric effects of
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DPP-4is and sulfonylureas in addition to metformin were investigated in 101 T2DM naïve patients,

aiming to evaluate any relationship between renal effects and their glycemic control capabilities. After
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24 weeks of treatment, UACR significantly decreased among patients in treatment with DPP-4is, while

sulfonylureas patients did not show any significant change. Comparable changes in serum HbA1c
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occurred, suggesting potential glucose independent effects of DPP-4is on albuminuria81.

Similarly, another study evaluated potential differences between sitagliptin and sulfonylureas effects on

UACR of T2DM patients with albuminuria. However, after a follow up of 9 months, in which sitagliptin

or sulfonylureas have been co-administered with metformin, results showed comparable changes of

20
HbA1c and UACR between both groups, with a strong correlation between the parameters; in contrast

with previous considerations82.

4. DPP-4 inhibitors in non-diabetic nephropathies.

Considering the ability of DPP-4is to reduce microalbuminuria in some diabetic patients, it is logical to

of
question whether this nephroprotective effect is also valid in non-diabetic patients.

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Higashijima and collaborators evaluated the anti-inflammatory role of DPP-4is in a non-diabetic

glomerular injury model. The Authors observed that alogliptin significantly reduced the number of

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CD68+ macrophages in the inflammatory renal infiltrate without a significant reduction in proteinuria83.
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In another study, Eun Lee J and collaborators demonstrated the benefits of the administration of DA-

1229, a new DPP-4i, in two models of podocyte damage. Although there were no effects on glycemic
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control and insulin resistance, treatment with DA-1229 induced an improvement in lipid profile, control

of albuminuria, and intestinal fibrosis. DA-1229 also allowed a decrease in DPP-4 activity in the kidney
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and a reduction in macrophage infiltration. This suggests that renal DPP-4 activation appears to play a

role in the progression of chronic renal failure84. To confirm these results, another study documented the
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effectiveness of sitagliptin in a mouse model of doxorubicin nephropathy in terms of improved

tubulointerstitial damage, inflammatory infiltration, and interstitial fibrosis. It also showed a suppression
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of inflammasome with reduced expression of NLRP3, caspase-1, ASC, and IL-1β85.

The pleiotropic actions of these drugs are also well reported in studies on obesity. One of these

investigated the effects of incretin-based therapies on early obesity-related glomerulopathy (ORG) and

the involved inflammatory responses in rats. ORG models were induced using a high-fat diet and then

divided into three groups: ORG vehicles, a group treated with vildagliptin (3 mg/kg/day, qd), and a group

21
receiving liraglutide (200 μg/kg, bid). After eight weeks, albuminuria and histological changes were

analyzed. Both treatments determined an improvement in insulin sensitivity and an evident attenuation

of podocytopenia, with a reduction of endothelial and mesangial proliferation. Interestingly, the

evaluation of macrophage concentrations (M1 - pro-inflammatory, and M2 - anti-inflammatory) showed

a reduction of M1/M2 ratio. A down-regulation of TNF-alpha and IL-6 was also observed86.

Obesity is associated with RAAS hyperactivation; Ang II, through the T1 receptor, inhibits the

expression of megaline (a protein involved in the reabsorption of albumin in the proximal tubule),

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probably DPP-4-mediated, according to several hypotheses (infusion of Ang II in murine models

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involves the over-expression of DPP-4 and the reduction of megaline). The use of DPP-4is restores

megaline concentrations in renal proximal tubule87. Furthermore, the analysis of Nistala and colleagues

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confirms the presence of high plasma DPP-4 concentrations and podocyte damage in obese rats and
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documents a reduction of SDF-1 alpha (endothelial stromal-cell-derived factor-1 alpha) and an increase

in oxidative stress factors. DPP-4i therapy leads to an improvement in proteinuria, through the reduction
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of ROS and the successful attempt to restore the filtration barrier40. The same research group came to

similar conclusions in a study on the action of DPP-4is in rats subjected to Western Diet, which is high
na

in fructose and fat. These rats were obese, insulin-resistant, and hypertensive and developed proteinuria

and an increase in plasma DPP-4 activity and uric acid values. They also had high DPP-4 activity and
ur

monocyte chemoattractant protein-1 and IL-12 concentrations and suppressed IL-10 levels in the kidney,

indicating macrophage-mediated inflammation, glomerular and tubulointerstitial damage. Also in this

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study, the administration of DPP-4is resulted in a reduction of hyperuricemia, oxidative stress, and IL-

12 levels with an increase of IL-10 expression25. Evidence also shows as even the simple hypertensive

state in non-diabetic rats, with the administration of large amounts of dietary salt, correlates with high

DPP-4 activity. Therapy with saxagliptin or sitagliptin, without affecting glycemia, reduces DPP-4

activity by more than 95%, with improvement of proteinuria and nephroprotective effect 88.

22
A 4-year prospective study of 664 Chinese women and men aged 18-70 years without diabetes, but with

risk factors for the development of microalbuminuria (high body mass index [BMI], poorly controlled

blood pressure) showed that baseline DPP-4 activity was an independent predictive factor for proteinuria

onset89.

5. Conclusions

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DPP-4is are an important therapeutic innovation among oral hypoglycemic agents. The advantages are

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clear and indisputable in terms of effectiveness in glycemic control without the risk of hypoglycemia and

weight gain, wide tolerance and safety up to the last stages of chronic renal failure, especially with

linagliptin.

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Recent data seem to indicate their potential use as antiproteinuric agents, noting this pleiotropic effect in

several animal studies. DPP-4is behave as antifibrotic drugs, able to restore megaline levels, thus
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intervening on an important progression factor of CKD. The overall cardiovascular safety in patients

with renal function impairment remains to be clarified, due to the very limited follow-up duration of
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performed studies. However, except for increased hospitalization with sitagliptin in a single study, at our

knowledge, it does not appear to be any other negative reported result.

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At present, owing to scarce evidence in humans, the administration of DPP-4is requires caution in non-

diabetic nephropathy. Future studies may lead to new evidence on antiproteinuric action and extend the
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indications of the use of DPP-4is.

Conflict of interest

The authors declare no conflict of interest related to the present work

23
Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or

not-for-profit sectors.

Acknowledgments

We greatly appreciate the support of Dr.ssa Maria Teresa Zicarelli (Renal Unit, Department of Health

Sciences, "Magna Graecia" University, Catanzaro, Italy) in the graphical abstract design.

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