3.1.2. Models of diabetic nephropathy and research outputs
For many years, mouse models have been the primary and most extensively
studied species to investigate kidney pathogenesis and for the
development of novel therapies to limit diabetic nephropathy. As
previously mentioned, the diabetogenic ß-islet cell toxin, STZ, is also
by far the most widely used drug to induce diabetic nephropathy.
However, development of overt diabetic nephropathy is strain dependent.
C57BL/6J and BALB/c strains of mice are highly responsive to developing
STZ-mediated diabetes although C57BL/6J mice are relatively resistant to
developing diabetic nephropathy[107]. Hence, C57Bl/6J mice are
commonly crossbred with genetic mouse models known to have impaired
renal function. One approach is to breed eNOS-/- mice
onto the C57BL/6 background. Deletion of eNOS diminishes NO
availability, thereby phenocopying the well-documented decreased NO
levels and its effects on the vasculature in human diabetic nephropathy.
STZ-induced diabetic eNOS-/- mice on the C57BL/6
background develop significant impairment of renal function in which
clear pathophysiological changes are visible after 24 weeks of
diabetes[108]. On its own, a reduction in eNOS functionality is
sufficient to develop vascular dysfunction, hypertension and
albuminuria, all of which accelerate renal injury, however, when
superimposed on a diabetic background the severity of the albuminuria
appears to be enhanced, and more advanced glomerular lesions and
glomerular endothelial injury are exhibited in the STZ-induced diabetic
eNOS-/- mouse model. However, high dose administration
of STZ should be avoided in this model since this can be highly lethal,
although the extent of albuminuria and renal injury observed is milder
than that of the eNOS-/- db/db (Type2 diabetes) mouse
model[108].
Genetic mouse models, such as the ob/ob and db/db mice, have been widely
studied as models of diabetic nephropathy, although the degree of
severity differs between these two models. In the db/db mice, larger
glomeruli with increased mesangial matrix are visible by 5-6month of
age, becoming more pronounced over time, along with thickening of the
glomerular basement membrane. The extent of renal disease is far less in
ob/ob mice when compared to db/db mice. When on the C57Bl/6 background,
ob/ob mice are insulin resistant but do not develop severe diabetes. The
major renal pathology in ob/ob mice primarily includes diffuse and
nodular lipo-hyaline changes in the glomerulus with a relative paucity
in mesangial expansion[109]. Both ob/ob and db/db mice have been
crossbreed with numerous genetically modified mice. Initially
established by Zhao et al. the eNOS-/-;db/db mouse
model has been identified as the most robust model of Type2
diabetes[110]. These mice display many human pathophysiological
features of diabetic nephropathy including significant albuminuria (more
than 30-fold that of controls), reduction in glomerular filtration rate
and glomerular basement membrane thickening[110].
The importance of performing animal studies in both male and female
mice, as opposed to studies in one gender only (mostly male), is
becoming increasingly apparent to improve the translation of novel
therapies to both males and females. Recent findings showed that there
were no significant differences in renal structural and functional
damage between male and female diabetic eNOS-/-db/db
mice [111]. Both male and female eNOS-/-db/db mice
showed hyperglycaemia and obesity as early as 8 weeks of age. Although
male diabetic mice had a tendency towards greater body weights than
female counterparts, the difference was not significant. Both male and
female diabetic mice showed progressive albuminuria, renal hypertrophy,
and significantly elevated serum creatinine, although this was slightly
greater in female diabetic mice[111]. Notably, both male and female
mice displayed significantly greater glomerular injury compared to their
wild-type counterparts, but no differences were noted between genders.
Hence, the eNOS-/-db/db mouse model represents a
robust model of diabetic nephropathy enabling the study of drug
treatment in both male and female mice without gender impacting
experimental outcomes. However, it should not be assumed that drug
treatments will always necessitate the same outcomes, and for this
reason both genders should be evaluated. However, historically male mice
strains have been used to demonstrate more robust hyperglycaemia and
renal injury.
Initially characterized by Clee et al., the black and tan, brachyuric
(BTBR) mouse crossed onto the ob/ob background is a widely used mouse
model to investigate Type2 diabetic nephropathy[112]. The BTBR mouse
strain has alleles promoting insulin resistance resulting in natural
hyperinsulinemia. When crossed with the ob/ob mouse, it develops a
severe Type2 diabetic phenotype, maintaining sustained
hyperglycaemia[112] and closely recapitulating the kidney lesions
observed in human diabetic nephropathy. In particular, BTBR ob/ob mice
exhibit glomerular hypertrophy, accumulation of mesangial matrix, loss
of podocytes and glomerular lesions that are similar to that seen in
human T2D kidney disease[113]. BTBR ob/ob mice display
overactivation NF-κß and JAK/STAT pathways, thereby promoting
inflammation and the infiltration of lymphocytes and macrophages
compared to BTBR wild type mice. Indeed, use of the BTBR ob/ob mouse
model has demonstrated the therapeutic potential of targeting the
transcription factor NF-κß in the prevention of diabetic
nephropathy[114].
An additional, albeit invasive approach to develop diabetic nephropathy,
is through the use of unilateral nephrectomy (UNx) that can
significantly shorten the time required to develop the
pathophysiological signs of diabetic nephropathy. This technique
involves removing the left kidney after ligation of the renal artery,
vein and ureter. Initially performed on STZ-induced diabetic
Sprague-Dawley rats, it was later applied to mouse models to develop
enhanced renal injury. Together with STZ, it has a synergistic effect by
accelerating the development of glomerular lesions and impaired renal
function. It has been shown that the combination of STZ and UNx is an
effective way to induce diabetic nephropathy consistent with Type1
diabetes, especially in the resistant C57BL/6J mouse strain[115].
This model includes albuminuria, thickening of the glomerular basement
membrane, mesangial matrix expansion, glomerular hypertrophy and
upregulation of pro-fibrotic markers[115]. However, the use of a
western-diet to induce Type2 diabetes was not effective in inducing
significant albuminuria and glomerular injury when combined with STZ and
UNx, despite the presence of hyperglycaemia. A possible explanation for
this protective effect may have been driven by metabolic changes in the
renal cells of the remaining kidney after UNx, where the
hyperfunctioning nephron is able to avoid hypoxia-induced injury.
Moreover, 15-weeks of a western diet may not have been sufficient to
induce renal injury due to the high cholesterol, potentially providing
protective effectives in the shorter-term (<4 months),
therefore longer HFD-feeding may be required
(~9months)[115].
More recently, the incorporation of proteomics and/or metabolomics
studies is providing novel insights into the molecular pathways of
diabetic nephropathy, as well as identifying mouse models that more
closely phenocopy human diabetic kidney disease. Metabolomics analysis
predominantly involves nuclear magnetic resonance spectroscopy or
chromatography-mass spectrometry, enabling either the detection of known
metabolites or numerous metabolites respectively, the latter technique
without any prior knowledge of the metabolites required[116]. Using
a proteomics and metabolomics approach, Rossi et al showed that a tissue
inhibitor of metalloproteinases (TIMP3) knockout mouse model alone or in
combination with STZ-mediated diabetes, led to significant alterations
in kidney lipid metabolism and peripheral acylcartinine levels, which
have been implicated in Type2 diabetes[117]. Loss of TIMP3 in the
kidney was associated with features of diabetic nephropathy including
higher levels of medium and long chain acylcarnitine in the blood. In
contrast, levels of free carnitine and short chain beta-hydroxy and
dicarboxy-acylcarnitines were lower diabetic in TIMP3 knockout mice as
compared to WT-STZ mice[117]. Similar metabolomics signatures were
found in a study by Lim et al who showed the accumulation of a number of
short acylcarnitines in the plasma of patients who developed diabetic
kidney disease over time, as well as data by Gurley et al who found
elevated levels of short acylcarnitines in the Akita mouse[118],
indicating that this model might closely resemble the human metabolic
profile. Furthermore, the incorporation of flow cytometry allows the
identification of immune cell-specific population in diabetic
nephropathy. Indeed, a significant increase of intrarenal CD3+ T cells
that was associated with augmented production of interferon-γ and TNF-α
in diabetic mice was revealed via flow cytometry[119]. In addition,
transcriptomic profiling using scRNA-seq is identifying factors
underlying the pathophysiology of diabetic nephropathy and is therefore
a useful tool for identifying new therapeutic avenues for research.
Using scRNA-seq, Fu et al demonstrated that the number of glomerular
cell-specific markers that were significantly higher in diabetic mice
compared with controls. These immune cells were predominantly
macrophages and greater number expressed M1 phenotype than M2. Moreover,
regulation of angiogenesis and migration pathways in endothelial cell
from diabetic mice were altered[120]. Hence, these studies, in
addition to adding valuable insights into the diseased kidney, also
highlight the potential to generate a mouse model more prone to diabetic
nephropathy.
Diabetic retinopathy