The animals were then sacrificed, and renal tissues were further processed for histological examination.
Urinary albumin excretion was more prominent in mice with proximal tubule particular deletion of UNC5B.
Substantial upregulation of Netrin-1 in proximal tubules in the heterozygous diabetic group was noted.
Recombinant Netrin-1 supplementation also considerably decreased in diabetes-brought albuminuria and repressed interstitial and glomerular injuries.
Cross-sectional study of human subjects (obese patients and normal controls)
An overall of sixty-two nonalbuminuric and normotensive obese individuals (with and without insulin insensitivity) and sixty-four normal controls were involved in the study.
Obese subjects exhibited substantially greater Netrin-1 urinary excretion than the control groups.
The level of Netrin-1 and creatinine in urine was also meaningfully amplified in obese patients with insulin resistance than those without insulin resistance.
Samples from urine and blood were collected to analyze the level of creatinine, albumin, and Netrin-1.
But no significant differences were detected between these subjects for the occurrence of microalbuminuria.
Animal models of (5/6 nephrectomized (Nx) and sham-operated rats) chronic renal failure
Animals were randomly assigned into three groups ( rats/group): sham-operated rats received the control adenovirus; 5/6 Nx rats received with the control adenovirus (empty vector); and 5/6 Nx rats were treated with recombinant adenovirus (Ad-Netrin-1).
The 5/6 Nx groups showed markedly reduced Netrin-1 level compared with the sham-operated group
The Ad-Netrin-1-received group displayed markedly increased Netrin-1 expression compared with the 5/6 Nx control group.
Despite that 5/6 Nx rats showed initial elevation, Ad-Netrin-1 treatment exhibited a remarkable reduction in Scr and BUN levels.
The effect of Netrin-1 to attenuate the progression of renal dysfunction might be via inhibiting EndoMT in 5/6 Nx rats.
Observational studies on human subjects having AKI, non-AKI, and control subjects
Patients who were admitted to the ICU were divided into (a) patients who had AKI (49 subjects) and (b) non-AKI (101) patients and (c) fifty subjects as the control group.
The level of Scr began to rise after 24 h of admission while the extent of Netrin-1 has amplified expressively within the 1st h and persistently elevated up to 48 h among AKI patients.
Of which 15 patients with AKI while 26 without AKI and 20 healthy subjects as the control group.
Despite the extent that NGAL and IL-18 were intensified in AKI patients, the levels of Netrin-1 and NHE3 were comparable compared to patients without AKI.
Urine samples were collected on days 1 and 4 after birth from patients with PA and the control group.
For the samples taken on postnatal day 4, only NGAL levels were knowingly raised in AKI compared to non-AKI patients.
Cross-sectional study (on patients who experienced orthotropic liver transplantation (OLT))
Sixty-three patients who underwent OLT were involved.
The urinary levels of Netrin-1, semaphorin 3A, and NGAL were amplified meaningfully and peaked at 2 h while Scr was raised after 48 h of post-transplantation in patients with AKI compared to matched non-AKI individuals.
Among which individuals who had preexisting renal failure were excluded and patients that undertook OLT and had intact renal function were finally recruited.
The prognostic power of Netrin-1, as verified by the AUC for diagnosis of AKI at 2, 6, and 24 h post-transplantation, was 0.66, 0.57, and 0.59, respectively.
Preoperative samples (urine + blood) were obtained from individual patients at various time intervals.
The AUC for diagnosis of AKI was 0.63 and 0.65 for semaphorin 3A and NGAL at 2 h, respectively.
Case-control study (on diabetes and nondiabetes subjects)
In this study, sixty diabetes (30 patients with microalbuminuria and 30 subjects without microalbuminuria) and fifty-six healthy volunteers were enrolled.
Plasma Netrin-1 level was suggestively higher in diabetes compared to the nondiabetic group whereas the level was comparable between the group without diabetes and nonalbuminuric diabetic patients.
The plasma samples were collected after fasting (8–12 h), and urinary albumin level was assessed.
The level of plasma Netrin-1 in microalbuminuric diabetic individuals was meaningfully increased compared with the group without diabetes and the nonalbuminuric diabetic group.
Plasma and urinary creatinine, HbA1c, glucose, and cholesterol expressions were also quantified.
The higher Netrin-1 expression was positively correlated with the duration of diabetes, HbA1c, and microalbuminuria/creatinine ratio while it was inversely correlated with the level GFR ().
STZ-induced diabetic mouse model (to assess renal proximal tubular epithelial- (RPTE-) cell expression of Netrin)
Diabetes was induced for both Netrin-1 transgenic mice and wild-type (WT) mice using STZ while the control group received vehicle.
The chicken Netrin-1 transgene was prominently expressed in transgenic mouse RPTE cells while minimal endogenous Netrin-1 expression was noticed in WT and transgenic mouse kidney.
After rats were anesthetized, the kidney was removed and a 2-month timed-release pellet of DOCA was embedded. The water consumption of DOCA-treated rats was subsequently followed for 1 month. Blood pressures were assessed weekly and after 4 weeks of DOCA and 10 weeks of STZ, respectively.
Urinary Netrin-1 level was also suggestively raised from the hypertensive group at 4 weeks as compared to controls.
A substantial elevation of albuminuria was noted in diabetic rats (on weeks four and ten) as compared to controls.
Similar to diabetic model in rats, early Netrin-1 excretion also augmented in diabetic mice and the highest expression was associated with disease severity.
A single-center, cross-sectional study (diabetes with and without albuminuria)
A total of eighty-eight diabetes subjects (of which 40 had no albuminuria, 38 had microalbuminuria, and 9 had macroalbuminuria) and 42 nondiabetic individuals without CKD as control were enrolled.
Type 2 normoalbuminuric diabetes had higher Netrin-1 excretion as compared to control than matching type 1 DM patients.
Urinary Netrin-1 levels were substantially elevated in diabetic subjects with normoalbuminuria compared to controls, proposing early tubular damage.
Netrin-1 expression was also elevated in late stages of nephropathy in both micro- and macroalbuminuric patients as compared to controls; however, the level of Netrin-1 between these two groups of albuminuria was insignificant.
A noticeable positive correlation was noted between urine Netrin-1 and occurrence of CV disorders, albumin/creatinine ratio, plasma creatinine, and HbA1c, while there was a negative association with urinary creatinine.
Peritubular capillary (PTC) loss and hypoxia in 5/6 nephrectomized (Nx) in rat models
Male sex rats (Sprague-Dawley) were randomly allocated into 3 groups: (a) sham-operated rats received control adenovirus; (b) 5/6 Nx rats received control adenovirus; and (c) 5/6 Nx rats were administered recombinant adenovirus-facilitated Netrin-1 (Ad-Netrin-1) gene.
The Ad-Netrin-1-received 5/6 Nx group exhibited a marked elevation of Netrin-1 expression compared to the 5/6 Nx group, though still lower than the sham-operated group.
Blood urea nitrogen, serum creatinine, and 24-h urinary albumin excretion extents were quantified.
Ad-Netrin-1 administration induced a significant intensification in renal PTC density, accompanied by a substantial reduction in HIF-1α levels.
Histopathological alterations in kidney tissues were evaluated. The expression of Netrin-1 and hypoxia inducible factor-1α (HIF-1α) was also assessed.
Ad-Netrin-1 administration also attenuated PTC injury, diminished tissue hypoxia, and restored kidney function, which improve renal pathological alteration and interstitial fibrosis in 5/6 Nx rats than the 5/6 Nx control group.
Besides, a 24-h urinary albumin concentration was expressively decreased in the Ad-Netrin-1-received 5/6 Nx group, compared with the 5/6 Nx control group.
Renal ischemia-reperfusion (RIR) induced AKI and CKD in C57BL/6J mice and age-paralleled Netrin-1 transgenic mice
Animal were grouped into treatment (administered recombinant IL-6 (10 ng) and Netrin-1 (250 ng/mL)) and control groups.
Netrin-1 transgenic mouse kidney function improved more quickly compared with wild-type.
Animals were sacrificed after surgery, and renal tissue was taken and processed for protein isolation.
Upregulation of tubular Netrin-1 displayed a remarkable suppression of tubular atrophy and glomerular sclerosis.
Cells were then treated with IL-6, Netrin-1, or a combination of both IL-6 and Netrin-1 for 24 h.
The RIR interstitial fibrogenesis and capillary injury were inhibited by upregulation of Netrin-1, and expressions were also suppressed in Netrin-1 transgenic mice.
IL-6-boosted hypoxic fibrotic response in mouse RPTE cells was notably suppressed by Netrin-1.
Prospective study on subjects having renal injury (allograft, ischemic AKI, AKI associated with sepsis, and radiocontrast- and drug-induced AKI) and healthy individuals
A total of 63 patients (22 subjects underwent a renal allograft, eleven with ischemic AKI, thirteen with AKI related with sepsis, 9 with radiocontrast-brought AKI, and 8 with drug-induced AKI) who had kidney injuries and 10 healthy controls were included in this observational study.
The initial urinary Netrin-1 level at 2 h after surgery revealed a very high concentration in renal transplant patients compared to the control.
Evaluation of urinary Netrin-1 level was carried out by sandwich enzyme-linked immunosorbent assay.
These patients were monitored till day 18, and Netrin-1 levels kept decreasing as renal function improved and eventually disappeared from urine.
Netrin-1 levels were dramatically increased in urine from patients with ischemic ATN, sepsis, and radiocontrast-induced and drug-induced acute renal failure.
There was still high level of Netrin-1 found in the urine samples, suggesting the ongoing renal injury.
Ischemia reperfusion in wild-type (WT) and RAG-1 knockout mice induced severe renal injury model
RAG1 knockout mice were subjected to about half an hour of ischemia after that reperfusion.
Both WT and RAG1 knockout mice developed severe IRI while the corresponding sham-operated groups revealed no renal injury.
The expression of inflammatory mediators (neutrophil, and cytokine and chemokine) was significantly suppressed in WT as well as in RAG1 knockout mice, which received Netrin-1 whereas the matching groups which received vehicle displayed a substantial expression of these inflammatory mediators.
IFNγ or LPS induced a high COX-2 expression and PGE2 formation in the macrophage.
The level of COX-2 expression was notably upregulated after reperfusion whereas the level of COX-1 was not changed. Injection of Netrin-1 repressed the level of COX-2 in both WT and RAG1 knockout mice.
Renal reperfusion-induced ischemia exhibited an increment of the formation of PGE2 and its renal excretion, which was remarkably suppressed in groups that received Netrin-1.
Netrin-1-injected mice also displayed significant reduction of IFNγ and LPS-induced COX-2 and PGE2 concentrations.
Ischemia reperfusion damage of the renal tissue and cisplatin-brought kidney toxicity by tissue-specific UNC5B receptor knockout and half-deficient mice
Mice with UNC5B2/flox/GGT-cre and matched mice without cre (UNC5B2/flox) or WT mice were subjected to about half an hour of ischemia then reperfusion.
Both UNC5B2/flox/GGT-cre group and corresponding mice without -cre passed away within 1-day after subjected to ischemia while the WT mice were subsisted.
UNC5B2/flox/GGT-cre and UNC5B2/flox mice were exposed to 22 minutes of ischemia then a 48 h of reperfusion. Renal function was evaluated by determining the extent of Scr at different intervals.
WT mice did not display any elevation of Scr levels with a slight form of ischemia, whereas mice with RPTE cell-specific UNC5B deletion exhibited a substantial intensification in Scr and blood urea nitrogen.
Majority of the mice died by 72 h, which indicated that the UNC5B receptor has a crucial value against ischemic RPTE cells.
Chemical-induced nephrotoxicity in mice and case control study in patients with AKI.
Renal ischemia-reperfusion and cisplatin, lipopolysaccharide (LPS), and folate were given for various groups of C57BL/6J mice to induce AKI.
A significant upregulation of Netrin-1 (by 47-fold) in renal epithelial cells after ischemia reperfusion injury was noticed.
Urine is collected at different periods of time, and kidney function (BUN and Scr) and Netrin-1 levels were assessed.
On the contrary, the Scr levels were considerably increased only after 6 h and peaked at 24 h after reperfusion.
The urinary expression of Netrin-1 was notably amplified in the cisplatin-received group by 10 and 30 folds within 3 and 6 h, respectively, after administration while the Scr level started to rise in the middle of 24 and 48 h post-administration.
In the folate-brought nephrotoxicity group, urinary Netrin-1 excretion was noticed within 3 h after injection before substantial alteration on Scr was noted and the Netrin-1 level persisted for 48 h.
Urinary Netrin-1 excretion was also elevated (by 60 folds) in LPS-brought kidney dysfunction within 1 h and peaked at 6 h after administration.
Urinary Netrin-1 levels were elevated in AKI subjects while 4 patients presented a lower, but still noticeable expression compared with control.
